CN104473991A - Use of origanum vulgare oil in preparation of diquat toxicity-resistant drugs and origanum vulgare oil oral emulsion - Google Patents
Use of origanum vulgare oil in preparation of diquat toxicity-resistant drugs and origanum vulgare oil oral emulsion Download PDFInfo
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Abstract
The invention discloses a use of origanum vulgare oil in preparation of diquat toxicity-resistant drugs. A drug effect experiment shows that through oral administration, origanum vulgare can substantially improve intestinal tract antioxidant enzyme activity, reduce MDA content, reduce the amount of escherichia coli in chime, improve the amount of lactobacilli, inhibit high expression of an intestinal mucosa inflammatory factor, improve expression of tightly connected Occludin, reduce excess damage caused by diquat to intestinal mucosal cells and effectively protect an intestinal tract mechanical barrier. The invention provides an oral emulsion for resisting diquat-caused toxic reaction. The oral emulsion comprises 0.01-10% of origanum vulgare oil, 5-20% of an emulsifier, 0.1-10% of a physical stabilizer, 0.1-10% of a co-emulsifier, 0.01-1% of a thickening agent and the balance water. The oral emulsion has the characteristics of good drug absorption, fast action and high bioavailability, can mask the odor and poor taste of origanum vulgare oil and can improve drug compliance.
Description
Technical field
The invention belongs to cattle breeding field, be specifically related to the purposes of dysentery relieving grass oil in the anti-diquat dibromide drug toxicity of preparation and a kind of Orally taken emulsion of the toxic reaction caused for anti-diquat dibromide.
Background technology
Diquat dibromide (Diquat) is that a kind of conductivity is tagged steriland herbicide, and by China's pesticide toxicity grading criteria, diquat dibromide belongs to moderate toxicity herbicide.The electron transmission of inhibited photosynthesis after diquat dibromide is absorbed by green plants, the Bipyridine compound of reducing condition is under photoinduction, oxidized very soon when aerobic exists, and forms active hydrogen peroxide, and the accumulation of this material makes the cell membrane of plant be destroyed.Diquat dibromide can be used as phanerogamous desiccant, and what also can be used for the crops such as Rhizoma Solani tuber osi, Cotton Gossypii, Semen sojae atricolor, Semen Maydis, Sorghum vulgare Pers., Caulis et Folium Lini, Helianthi urges withered dose.The daily ration of poultry, mainly from corn and the by-product thereof in field, if once there is drug residue, can cause the pollution of feedstuff.Although the diquat dibromide content trace carried in feedstuff, can not cause clinical symptoms, if search for food the feedstuff polluted for a long time, also can have a negative impact to growth of animal.
Research confirms that Diquat enters after in animal body, and molecular oxygen can be utilized to produce O
2-, and then be transformed into hydroperoxides.These active oxygen species can start lipid peroxidation, the more free radical of further generation, when the free radical of these excessive generations has exceeded the Scavenging activity of body antioxidant system, body redox equilibrium state is broken, cause apoptosis, finally cause damage to body especially gastrointestinal tract, serious causes death.
Modern medicine study finds: the general volatile that extracts from dysentery relieving grass oil---dysentery relieving grass oil has the effects such as antibacterial, antifungal, antioxidation and protozoacide.And a large amount of animal experiment also shows: dysentery relieving grass oil has antibacterial growth promotion, improves the effects such as efficiency of feed utilization, the medicated feed additive of plant source of Ye Shi China approval application, but the relevant report using it for toxic reaction that anti-diquat dibromide causes especially injury of gastrointestinal tract there is not yet.
Summary of the invention
The object of the present invention is to provide the purposes of dysentery relieving grass oil in the anti-diquat dibromide drug toxicity of preparation, the present invention also provides a kind of Orally taken emulsion of the toxic reaction caused for anti-diquat dibromide of determined curative effect.
The present invention is achieved in that
First by rats by intraperitoneal injection diquat dibromide, produce toxic action, thus set up intestinal injury model in induced rat body, then give rat administered by oral gavage dysentery relieving grass oil and the therapeutic effect evaluated intestinal injury, result shows:
1) the dysentery relieving grass oil of high dose can reduce hydrocortisone in rat plasma and noradrenaline levels;
2) dysentery relieving grass oil can significantly improve jejunum SOD enzyme activity, the GSH-Px enzymatic activity of intestine in rats damage model, and reduces MDA content, and its effect is better than common antioxidant vitamin E;
3) dysentery relieving grass oil can reduce the ROS content in rat model jejunal mucous membrane and chyme;
4) dysentery relieving grass oil can make the flora in rat model Jejunal chyme change, and can reduce colibacillary quantity, increases the quantity of lactobacillus, and not this effect of common antioxidant;
5) dysentery relieving grass oil can reduce the mrna expression level of inflammatory factor TNF-α, IL-1 β, IL-6 in rat model jejunal mucous membrane, shows that it has certain antiinflammatory action;
6) dysentery relieving grass oil can significantly improve rat model jejunum compact siro spinning technology associated protein---the protein expression amount of occlusion albumen Occludin;
7) dysentery relieving grass oil can make that rat model jejunum structure is relative with the seriality of enterocyte structure keeps complete.
Because the intestinal injury mechanism of diquat dibromide induction is very complicated, it can not only damage blood plasma and jejunal mucous membrane Antioxidant Enzyme Systems, colibacillary quantity can also be induced significantly to increase simultaneously, and the great expression etc. of inducing immune cells inflammatory cytokine, therefore treatment difficulty is very large.Above-mentioned experimental result shows: administered by oral gavage dysentery relieving grass; significantly can strengthen the activity of intestinal antioxidase; reduce MDA content; reduce escherichia coli in chyme, improve lactobacillus quantity; suppress the great expression of endo-enteritis sex factor; add the expression of compact siro spinning technology occlusion albumen Occludin, thus decrease the excessive damage of diquat dibromide to epithelium of intestinal mucosa, effectively protect the mechanical barrier of intestinal.
In purposes of the present invention, preferred pharmaceutical dosage forms is Orally taken emulsion, because the drug dispersion of Emulsion is good, surface area is large, effect experiment shows, Emulsion advantageously in the dysentery relieving grass absorption of oil component and the performance of drug effect, can improve bioavailability, be better than other dosage form to the therapeutic effect of intestinal injury, bad smell and the mouthfeel of dysentery relieving grass oil can also be covered simultaneously, improve medication compliance.
Preferred further, described Emulsion is made up of the composition of following percentage by weight: dysentery relieving grass oily 0.01-10%, emulsifying agent 5%-20%, stabilizing agent 0.1%-10%, co-emulsifier 0.1%-10%, thickening agent 0.01%-1%, water is surplus.
Preferred further again, described emulsifying agent is castor oil hydrogenated; Described stabilizing agent is one or more in ethylene glycol, propylene glycol, starch; Described co-emulsifier is one or more in dehydrated alcohol, n-butyl alcohol, glycerol, n-amyl alcohol; Described thickening agent is one or more in arabic gum, sodium carboxymethyl cellulose, xanthan gum, gellan gum.
Accompanying drawing explanation
Fig. 1 is the microphotograph of jejunum in rats mucous epithelium morphosis before and after injection diquat dibromide.
Fig. 2 is the impact of dysentery relieving grass oil on active oxygen ROS content in jejunum in rats mucosa and chyme.
Fig. 3 is the impact (A be enterococcus faecalis, B be escherichia coli, C be lactobacillus) of dysentery relieving grass oil on flora in jejunum in rats chyme.
Fig. 4 is dysentery relieving grass oil on the impact of the mrna expression level of inflammatory factor TNF-α, IL-1 β, IL-6 in jejunum in rats mucosa (A is TNF-α, B be IL1-β, C be IL-6).
Fig. 5 is dysentery relieving grass oil on the impact of mrna expression level of occlusion albumen Occludin (A) and banded Occludin ZO-1 (B).
Fig. 6 is that dysentery relieving grass oil is on the impact being engaged albumen Occludin (A) and banded Occludin ZO-1 (B) expressing quantity.
Fig. 7 is that dysentery relieving grass oil is on the microphotograph of jejunum in rats mucous epithelium form impact.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.
The foundation of embodiment 1 diquat dibromide (Diquat) damage model
1 test method
1.2 animal grouping and feeding methods
Select the 7-8 female Wistar rats 12 in age in week of cleaning grade, be divided into matched group and diquat dibromide induced oxidation stress processed group (calling processed group in the following text) at random.Often organize 6 repetitions, often repeat 1, raise with the single cage in room, natural lighting, free choice feeding and drinking-water.Wistar rat basal diet formula is in table 1.
Mus basal diet tested by table 1
| Components | g/kg |
| Semen Maydis | 570g |
| Bean cake | 180g |
| Testa Tritici | 170g |
| Fish flour | 50g |
| Yolk powder | 10g |
| Defatted milk powder | 15g |
| Sal | 5g |
| Vitamin premix a | 1g |
| Microelement pre-mix material b | 1.6g |
aevery kilogram of daily ration adds: 7500IU vitamin A, 1200IU vitamin D3,7.77mg vitamin E, 1.12mg vitamin K3,0.71mg vitamin B1,2.58mg vitamin B2,0.35mg vitamin B6,0.04mg vitamin B12,12.57mg vitamin B3,0.24mg vitamin B5,0.01mg vitamin C, 150mg choline chloride.
bevery kilogram of daily ration adds: 501.5mgZn, 500mg Mn, 400mg Fe, 75.24mg Cu, 2.58mg Co, 15.13mgI, 1.57mg Se.
1.3 test process
Experimental mouse, after one week laundering period, enters the formal test phase, and the formal test phase is 15 days.Test the 15th day, matched group injected isopyknic normal saline, and processed group rat is pressed 0.1mmol/kg.bw and injects diquat.Inject after 6 hours, abdomen femoral artery gets blood, puts to death rat rapidly, gets liver and intestinal jejunal segment sample.
1.4 sample collecting
The pretreatment of sample: be placed in ice immediately after the blood sampling of abdomen femoral artery and preserve, 3000r/min prepares blood plasma in centrifugal 10 minutes, and-20 DEG C save backup.With normal saline flushing liver and the jejunum of pre-cooling, the liver of same rat and jejunum load in a hospital gauze bag, immerse in liquid nitrogen freezing, after proceed to-80 DEG C of Refrigerator stores.
The collection of intestinal tissue sample: aseptic clip whole section of jejunum.Get about 1cm jejunal tissue be placed in 4% formalin fix, for the preparation of paraffin section.
1.5 blood plasma cortisols and norepinephrine detect
Blood plasma cortisol and norepinephrine, illustrate by ELISA kit (R & D System) and measure.In blood plasma, cortisol levels represents with ng/ml, and noradrenaline levels represents with pg/ml.
1.6 blood plasma and organize Antioxidant Indexes to detect
Antioxidant Indexes detection kit and standard protein build up Bioengineering Research Institute purchased from Nanjing.Detect index malonaldehyde (MDA) content of active oxygen (ROS) and instruction level of lipid peroxidation in the superoxide dismutase (SOD) in blood plasma, liver and jejunal tissue homogenate supernatant, glutathion peroxidase (GSH-Px) activity, mucosa and chyme respectively.
Xanthine oxidase is used to measure SOD active.Xanthine oxidase can produce superoxide ion, and reduction nitroblue tetrazolium is blue methyl hydrazone, and the SOD in sample can remove superoxide ion, suppresses the reduction reaction of nitroblue tetrazolium.Use 5,5 '-two sulfur Nitrodracylic acid method to detect GSH-Px active, (note: the response speed that GSH-Px vigor is oxidized with catalysis GSH, namely in the unit interval, the amount of GSH minimizing represents to be determined at the absorbance at 412nm place.GSH and 5,5 '-two sulfur Nitrodracylic acid reaction can generate yellow 5-sulfo-2-nitrobenzoyl acid anion under GSH-Px catalysis, has maximum absorption band, measures this ion concentration, can calculate the amount that GSH reduces in 412nm wavelength).In tissue, antioxidase SOD Activity Results represents with U/mg prot, and in blood plasma, antioxidase SOD activity represents with U/ml, and GSH-Px activity represents with U.
Use 2-thiobarbituricacidα-method to measure MDA content, 532nm place measures absorbance.In blood plasma, MDA content represents with nmol/ml, and the MDA content in tissue represents with nmol/mgprot.
ROS adopts chemiluminescence determination, with luminol (amino-2, the 3-dihydros-Isosorbide-5-Nitrae-benzodiazine diketone of 5-, Sigma) as probe.Method reference Du etc. (2010) have also done some adjustment, and instrument adopts the multi-functional microplate reader of Mithras LB 940 (BERTHOLD company, Germany).Every hole adds 150 μ l Krebs-HEPES buffer (118mM NaCl, 4.7mM KCl, 1.3mM CaCl
2, 1.2mM MgCl
2, 1.2mM KH
2pO
4, 25mM NaHCO
3, 10mM HEPES, 10mM glucose; PH=7.4), 50 μ l samples, 20 μ l horseradish peroxidase (12.4U; 310U/mg, Sigma), finally in mixed system, automatically add the luminol solution of 50 μ l 1.25mM by machine and record luminous value until it returns to base value, with Origin 9.0 software, matching being carried out to area under curve.The ROS result RLU/mgprot of tissue homogenate sample represents.
1.7 jejunal tissue morphologys
With reference to Zhang Pengfei (2012) method, key step is as follows:
Draw materials with fixing: with normal saline by clean for jejunum (after pylorus end, 10cm starts one section of intestinal of long 15cm) crosscut sample wash, drop in the 10% formalin fixative for preparing in advance and make tissue, the protein denaturation of cell solidifies, to prevent cell self-dissolving after death or the decomposition of antibacterial, thus the morphosis keeping cell original.
Dewater transparent: make dehydrant with low concentration to alcohol in high concentration, slough the moisture content in piece of tissue gradually.Again piece of tissue is placed in clarifier dimethylbenzene transparent, replaces out the ethanol in piece of tissue with dimethylbenzene.
Waxdip embeds: transparent piece of tissue is placed in the paraffin dissolved, and puts into wax-dissolving box insulation.Immerse completely after piece of tissue until paraffin and embed: be first ready to embed frame, pour the paraffin dissolved into, the piece of tissue that paraffin has been soaked in rapid gripping is put into wherein, cooled and solidified in bulk.
Film-making: embedded wax stone is fixed on microtome, thinly slices, be generally about 5 μm thick.The thin slice cut often fold, be put into (45-50 DEG C) pressing in the water of heating and be attached on microscope slide again, roasting sheet machine is dried.
HE dyes: section dimethylbenzene dewaxes, the ethanol extremely washing through variable concentrations: dimethylbenzene (I) 5-10min → dimethylbenzene (II) 5-10min → 95% ethanol 5min → 80% ethanol 5min → 75% ethanol 5min → distillation washing 1min.Brazilwood extract dyeing 5-10min → flowing water slightly washes away Lignum Sappan seminal fluid 1min → 1% acidic alcohol and washes 1-3s → slightly wash 10-30s → 1% ammonia and return blue 10-30s → running water 10-15min → Yihong liquid dyeing 1-3min → distilled water and slightly wash 1-2s → 80% ethanol and slightly wash 1-2s → 95% ethanol, (I) 3-5min → 95% ethanol, (II) 3-5min → dehydrated alcohol, (I) 5-10min → dehydrated alcohol, (II) 5-10min → dimethylbenzene, (I) 3-5min → dimethylbenzene, (II) 3-5min → dimethylbenzene, (III) 3-5min → neutral gum sealing.
Take a picture and statistics: utilize Nikon E-CLIPSE80i type advanced studies with just putting biological digital photography microscope observed slice under low power lens, choosing three typical visuals field from often opening section, utilizing Nikon NIS-Elements image processing software to measure five most long wool staple lengths and the darkest Crypt depth in each visual field.
1.8 data statistics
Independent sample significance T tast is carried out with SAS software (v8.2, SAS company).P<0.05 represents significant difference, and P<0.01 represents that difference is extremely remarkable.
2 results and analysis
2.1 Stress Hormone Levels of Plasma
Hydrocortisone and noradrenaline levels in table 2 blood plasma
| Project | C | T | SEM | P |
| Hydrocortisone (ng/ml) | 12.15 b | 12.95 a | 0.18 | 0.014 |
| Norepinephrine (pg/ml) | 218.80 | 235.82 | 8.78 | 0.363 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Table 2 is depicted as injection Diquat to the impact of stress hormone hydrocortisone, noradrenaline levels in rat plasma.Compared with matched group, in injection Diquat post processing group rat plasma, cortisol levels significantly raises (P < 0.05), but noradrenaline levels difference is not significantly (P > 0.05)
2.2 blood plasma activities of antioxidant enzymes and level of lipid peroxidation
Activities of antioxidant enzymes and MDA content in table 3 blood plasma
| Project | C | T | SEM | P |
| SOD(U/ml) | 92.91 a | 48.88 b | 8.12 | ＜0.01 |
| GSH-Px(U) | 3356.12 a | 1452.09 b | 304.48 | ＜0.01 |
| MDA(nmol/ml) | 4.45 b | 5.85 a | 0.28 | ＜0.01 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Table 3 is depicted as injection Diquat to the impact of activities of antioxidant enzymes in rat plasma and MDA content.Compared with matched group, in injection Diquat post processing group blood plasma, two kinds of main antioxidase SOD and GSH-Px activity extremely significantly reduce (P<0.01), indicate the MDA content then extremely significantly rising (P<0.01) of level of lipid peroxidation.
2.3 hepatic antioxidant activity and level of lipid peroxidation
Activities of antioxidant enzymes and MDA content in table 4 liver
| Project | C | T | SEM | P |
| SOD(U/ml) | 63.11 a | 44.06 b | 3.89 | ＜0.01 |
| GSH-Px(U) | 998.71 a | 784.66 b | 38.30 | ＜0.01 |
| MDA(nmol/ml) | 1.93 b | 3.69 a | 0.34 | ＜0.01 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Table 4 is depicted as injection Diquat to the impact of activities of antioxidant enzymes and MDA content in rat liver tissue homogenate.Change similar to the index in blood plasma, compared with matched group, injection Diquat post processing group hepatic antioxidant SOD and GSH-Px activity extremely significantly reduce (P<0.01), and indicate that the MDA content of level of lipid peroxidation extremely significantly raises (P<0.01).
2.4 jejunal mucous membrane activities of antioxidant enzymes and level of lipid peroxidation
Activities of antioxidant enzymes and MDA content in table 5 Jejunal mucosa
| Project | C | T | SEM | P |
| SOD(U/ml) | 41.55 a | 32.37 b | 2.39 | 0.046 |
| GSH-Px(U) | 46.38 a | 28.67 b | 3.96 | 0.015 |
| MDA(nmol/ml) | 0.67 b | 1.06 a | 0.08 | ＜0.01 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Table 5 is depicted as injection Diquat to the impact of activities of antioxidant enzymes and MDA content in jejunum in rats tissue homogenate.After injection Diquat, in jejunal mucous membrane, antioxidase SOD and GSH-Px is active significantly reduces (P<0.05), and MDA content extremely significantly raises (P<0.01).
2.6 jejunal tissue morphological observations
Be illustrated in figure 1 injection Diquat to the impact of jejunum in rats mucous epithelium morphosis.Therefrom can see that, compared with matched group (C group), processed group (T group) Intestinal Mucosa epithelium sustains damage, and intestinal villus has obvious atrophy, Fragmentation Phenomena.
3 brief summaries
The poisonous effect of 3.1 rats by intraperitoneal injection diquat dibromide
Diquat is 1,1'-ethylene-2, and the English common name of 2'-bipyridyl dibromo salt, Universal Chinese character is called diquat dibromide.It is a kind of conventional moderately toxic herbicide.After lumbar injection, Diquat can be absorbed by mesentery rapidly and enter in animal body, utilizes molecular oxygen to produce oxygen-derived free radicals and hydrogen peroxide.These active oxygen species can start lipid peroxidation, thus produce more free radical further, when the free radical of these excessive generations has exceeded the Scavenging activity of body antioxidant system, body redox equilibrium state is broken, thus generation oxidative stress, cause apoptosis, finally oxidative damage is caused to body.This result of the test shows: the Diquat of rat disposable celiac injection 0.1mmol/kg.bw, can effectively produce toxic action in induced rat body and can not cause test rats death.
After the Diquat of lumbar injection 0.1mmol/kg.bw, in the blood plasma of rat, liver, extremely significantly reducing appears in SOD and GSH-Px enzymatic activity, shows that antioxidant ability of organism is subject to major injury.Meanwhile, after injection Diquat, in the blood plasma of rat, liver, MDA content but extremely significantly raises, and shows that body suffers that free radical is attacked, there occurs serious lipid peroxidation phenomenon.Therefore, this test shows that lumbar injection concentration is the Diquat of 0.1mmol/kg.bw, can realize significant damage effect in rat body.
3.2 diquat dibromide damage intestinals
Both at home and abroad about Diquat induced rat damage model, principal concern concentrates on blood, liver aspect.Because lumbar injection is the mode that Diquat induced rat the most often adopts, the position contacting Diquat in this mode is at first mesentery, and therefore intestinal may also be the important place of the damage generation of Diquat induction.Research finds, gastrointestinal tract to stress with stress medium very responsive, be one of first target organs that ROS attacks.This result of the test shows, after injection Diquat, really there occurs significant damage effect in intestinal, showing as ROS in jejunal mucous membrane significantly increases, and the activity of jejunal tissue antioxidase SOD and GSH-Px all significantly reduces, but lipid peroxidation product MDA content raises extremely significantly.Histomorphological shows, and injection Diquat causes jejunum in rats epithelium Villus atrophy, breakage.These results show, the damage model of Diquat induction, not only at whole body, and forms serious damage effect at intestinal.
The oily Orally taken emulsion of embodiment 2 dysentery relieving grass is to the prevention and therapy effect of Diquat induced damage
1 test material
1.1 experimental animals and medicine
7 week age Healthy female Wistar rats 30, body weight 200 ± 5g, purchased from Disease Control and Prevention Center of Hubei Province.The oily Orally taken emulsion of dysentery relieving grass is provided by Guangzhou Mei Ruitaike biotechnology company limited.
1.2 main agents
Diquat dibromide (Diquat): Chem Service, CAS6385-62-2; PCR mixed system (PCR Master Mix): Bio-Rad company; Taq archaeal dna polymerase, dNTPs, DL2000Marker, DL15000Marker: the precious biotech firm (TAKALA) in Dalian; M-MLV reverse transcription, reverse transcription primer Oligo-(dT) 20n, RNAse inhibitor, 10mMdNTPs, 10 × TransFast Taq Buffer: company (Toyobo) is spun by Japanese Japan; TRIzol reagent (Invitrogin), chloroform, isopropyl alcohol, dehydrated alcohol (traditional Chinese medicines group); DEPC, bromophenol blue (Promega), EB (ethidium bromide, Sigma), Tris alkali (Amersham), agarose (Biowest), tryptone, yeast extract (Oxoid), sodium chloride (traditional Chinese medicines), agar powder, ammonia benzyl mycin (Ling Fei science and technology).
DNA glue reclaims test kit purchased from Axygen company; RNA purification kit (TURBO DNA-free) is purchased from Ambion company; Rapid ligation kit (pGEM-T Easy) is purchased from promega company.
SOD, GSH-Px, MDA test kit builds up purchased from Nanjing.Luminol (Sigma company), horseradish peroxidase (Sigma company).
1.3 instrument and equipment
Ultraviolet spectrophotometer (UV-2102C, Shanghai Shun's space perseverance is flat), trace ultraviolet spectrophotometer (Thermo company, the U.S.), just putting biological digital photography microscope (E-CLIPSE80i, U.S.'s Nikon), microplate reader (MK3, Thermo company, the U.S.), the multi-functional microplate reader of Mithras LB 940 (BERTHOLD company, Germany), double one side superclean bench (FLC-3, east, Harbin connection), regular-PCR instrument (Bio-Rad company, the U.S.), quantitative real time PCR Instrument (Bio-Rad company, the U.S.), gel imaging system (Syngene company, Britain), microwave oven (Haier), agarose gel electrophoresis groove (Beijing 6 1), small-sized high speed centrifugal machine (Sigma company, Germany), ultrapure water system (ELGA company, Britain).
1.4 animal grouping and feeding methods
Employing single factor experiment designs.Select the 7-8 female Wistar rats in age in week 30 of cleaning grade, after one week laundering period, entered experimental period, experimental mouse is divided into 5 groups at random, often organize 6.Raise with the single cage in room, natural lighting, free choice feeding and drinking-water.The basal diet (formula is with embodiment 1) that all experimental mouse are fed identical, morning every day, 10:00 carried out an administered by oral gavage process (see table 6) by syringe to experimental mouse.
Table 6 tests grouping and feeding method
| Blank group (C) | Administered by oral gavage normal saline |
| Negative matched group (NC) | Administered by oral gavage normal saline |
| Positive control group (PC) | Administered by oral gavage 20mg/t.bw vitamin E |
| Process 1 group (LO) | Administered by oral gavage 5mg/t.bw dysentery relieving grass oil |
| Process 2 groups (HO) | Administered by oral gavage 10mg/t.bw dysentery relieving grass oil |
1.5 test process
Blank group administered by oral gavage every day normal saline, and test the 15th day by 0.1mmol/kg.bw injecting normal saline (calling C group in the following text); Negative matched group administered by oral gavage every day normal saline, and within the 15th day, inject Diquat (calling NC group in the following text) by 0.1mmol/kg.bw in test; Positive control is administered by oral gavage 20mg/t.bw vitamin E every day, and within the 15th day, injects the VE group (calling PC group in the following text) of Diquat by 0.1mmol/kg.bw in test; Process 1 is administered by oral gavage low dosage (5mg/t.bw) dysentery relieving grass oil every day, and within the 15th day, injects the group (calling LO group in the following text) of Diquat by 0.1mmol/kg.bw in test; Process 2 is administered by oral gavage high dose (10mg/t.bw) dysentery relieving grass oil every day, and within the 15th day, injects the group (calling HO group in the following text) of Diquat by 0.1mmol/kg.bw in test.Diquat is after 6 hours in injection, and abdomen femoral artery gets blood, puts to death rat rapidly, gets intestinal jejunal segment sample.
1.6 test specimen collections
The pretreatment of sample, the acquisition method of intestinal tissue sample and intestinal contents is with embodiment 1.
In 1.7 blood plasma, hydrocortisone and norepinephrine detect
With embodiment 1.
In 1.8 jejunal mucous membrane Antioxidant Indexes and chyme, active o content detects
With embodiment 1.
Microbial DNA abstraction and quantification in 1.9 Jejunal chyme
The method of reference Zeng Zhiguang etc. (2009) and Steed et al. (2011).Key step is as follows:
The extraction of bacterial genomes DNA:
(1) get appropriate amount of sample to add 2mL PBS solution vortex wherein in centrifuge tube A, shake up, be placed in 800rpm/min in centrifuge, centrifugal 4min.
(2) get centrifugal after supernatant in new centrifuge tube B.
(3) precipitation after centrifugal with PBS solution washing, vortex, to shake up, is placed in 800rpm/min in centrifuge, centrifugal 4min.
(4) get centrifugal after supernatant in new centrifuge tube B.
(5) centrifuge tube B is placed in 800rpm/min in centrifuge, centrifugal 4min.
(6) supernatant is got in centrifuge tube B in new centrifuge tube C.
(7) precipitation PBS solution in centrifuge tube A is washed, vortex, be placed in 800rpm/min in centrifuge, centrifugal 4min, get supernatant in centrifuge tube B, repeat this step 1 time.
(8) supernatant in centrifuge tube B is placed in 800rpm/min in centrifuge, centrifugal 4min.
(9) supernatant after centrifugal is taken out be placed in centrifuge tube C, centrifuge tube C is placed in 7000g in centrifuge, from 10min.
(10) abandon supernatant, rinse precipitation with PBS, continue 7000g, centrifugal 10min, this process repeats till supernatant is limpid.
(11) in the precipitation after centrifugal, add 2mL lysate (0.15mol/L Nacl, 0.1mol/L EDTA, PH8.0) 2mL lysozyme (0.3g/2mL), vortex, be placed in 37 DEG C of shaking table 3h.
(12) in centrifuge tube, add 2mL (0.15mol/L Nacl, 0.5mol/L Tris-Hcl, PH8.0) 10%SDS solution again, be placed in 37 DEG C of shaking tables, 30min.
(13), after taking out centrifuge tube, add the saturated phenol of 2mL wherein, vortex, centrifugal 7000g, 10min, leave the supernatant after the heart in new centrifuge tube D, abandon precipitation.
(14) supernatant in D is repeated step 13, supernatant is forwarded in new centrifuge tube E, abandon precipitation.
(15) add equal-volume phenol, chloroform/isoamyl alcohol (24:1) mixing to supernatant in E, centrifugal 11000rpm/min, 15min, turn supernatant in new centrifuge tube F, abandon precipitation.
(16) supernatant in F is repeated step 15, supernatant is forwarded in new centrifuge tube G, abandon precipitation.
(17) equal-volume chloroform (isoamyl alcohol) is added to supernatant in G, mixing, centrifugal 11000rpm/min, 15min.
(18) get centrifugal after supernatant in new centrifuge tube H, and add equal-volume isopropyl alcohol, mixing ,-20 DEG C of overnight precipitation.
(19) centrifuge tube H is taken out, 12000rpm/min, centrifugal 15min.
(20) abandon supernatant, precipitation uses 75% washing with alcohol, 12000rpm/min, and centrifugal 10min, repeats this step 1 time.
(21) precipitation of having washed is put into 37 DEG C of baking ovens dry, or naturally dry.
(22) in dry precipitation, add 50uL distilled water, centrifugal 11000rpm/min, 10min, get supernatant in EP pipe.
(23) gained supernatant adopts DNA purification kit to carry out purification.Sample after purification is as-20 DEG C of preservations.
The purity testing of intestinal bacteria STb gene:
With micro-ultraviolet spectrophotometer NANODROP2000 (U.S. Thermo), the DNA extracted is detected, first extracted DNA stock solution is carried out 10 times of dilutions, then DNA content is wherein measured, DNA concentration according to each sample adds ultra-pure water, ensures that the DNA concentration of each sample during working sample is consistent.
Reference culture genome extracts:
(1) join in the centrifuge tube of the 1.5ml of sterilizing by the bacterium liquid 1ml of various different bacterium, vortex concussion mixing makes thalline fully suspend;
(2) bacterial suspension mixed is in 4 DEG C, and the centrifugal 5min of 600r/min, removing coarse granule, collects supernatant in the centrifuge tube of another 1.5ml sterilizing, the centrifugal 5min of 10000r/min, remove supernatant, collect bacterial precipitation.
(3) in precipitation, add the PBS of 1ml, cleaning mixing antibacterial, 4 DEG C, the centrifugal 5min of 10000r/min, removes supernatant, collects bacterial precipitation.Repeat 2-3 time.
(4) in bacterial sediment, add the sterilizing ultra-pure water of 500 μ l, boil 10min in boiling water, after cooling, 4 DEG C, the centrifugal 10min of 10000r/min, collects supernatant in 1.5ml sterile centrifugation tube.As reference culture DNA profiling.
The design of primer:
With reference to (Fierer et al., 2005) such as Fierer.In table 7.
Table 7 object flora real-time fluorescence quantitative PCR primer
The preparation of standard substance and qualification:
(1) amplification of genes of interest: respectively with the genome of three reference cultures for template, reaction system is 10 × Buffer2.5 μ l, dNTP2 μ l, the each 0.5 μ l of upstream and downstream primer of 3 Pseudomonas preparation standard product, Taq DNA polymerase 0.5 μ l, template 1 μ l, adds water to 25 μ l.Reaction condition is as follows:
Lactobacillus reaction condition: denaturation 94 DEG C of 4min, degeneration 94 DEG C of 30s, anneal 56 DEG C of 30s, extends 72 DEG C of 20s, 36 circulations, last 72 DEG C of 7min.
Colibacter reaction condition: denaturation 94 DEG C of 4min, degeneration 94 DEG C of 30s, anneal 56 DEG C of 30s, extends 72 DEG C of 20s, 36 circulations, last 72 DEG C of 7min.
Enterococcus faecalis belongs to reaction condition: denaturation 94 DEG C of 4min, degeneration 94 DEG C of 30s, and anneal 56 DEG C of 30s, extends 72 DEG C of 20s, 36 circulations, last 72 DEG C of 7min.
The AT clone of genes of interest:
(1) according to the explanation that Axygen company agarose gel recovery test kit provides, the above-mentioned PCR primer of purification is reclaimed.Reclaim product to be connected with pMD18T carrier.Linked system: pMD18T carrier 0.3 μ L, reclaim product 1.5 μ L, solution I (Solution I) 1.2 μ l, 5 μ L, flick mixing, are placed in 4 DEG C of refrigerator overnight.
(2) conversion of product is connected.Whole link products is joined in competence, with pipettor featheriness tip-tap several times, ice bath 30min; Centrifuge tube is placed in 42 DEG C of water-baths, heat shock 90s, keeps resting state; Be placed on rapidly on ice, cold shock 2min; In super-clean bench, add 500 μ L LB liquid culture mediums, in 37 DEG C of shaking tables, 180-200r/min, 45-60min, make thalline recover; The 100 μ L bacterium liquid stayed are coated on the solid medium containing Amp by the centrifugal 4min of 4000r/min, honest and upright and thrifty 400 μ L in absorption uniformly; Just be placed in 37 DEG C of incubator 15min, treating that flat board fully absorbs liquid, be inverted, continuing to cultivate 10-12h, observe and produce with or without bacterium colony.
(3) bacterium liquid positive colony detects and order-checking.On super-clean bench, the sterilizing 1.5mL centrifuge tube needed for preparation, and add the LB fluid medium containing Amp, from the single bacterium colony of flat board sterilizing toothpick picking, be inoculated into respectively in centrifuge tube, each picking 5 single bacterium colonies; In 37 DEG C of shaking tables, 180-200r/min, 4-6h, make thalline amplification culture.After end, with this bacterium liquid for template, the primer corresponding to object fragment, for detecting primer, carries out pcr amplification.Through electrophoresis detection, the bacterium liquid of positive colony serves the order-checking of marine growth engineering company.
(4) extraction of plasmid: the explanation provided according to Axygen company mini-scale plasmid extraction agent box, extract the standard substance plasmid of preparation, consumption ultraviolet spectrophotometer (NANODROP2000) detects its concentration, and calculates its copy number.Formula: plasmid concentration × N (Avogadro's number: 6.02 × 10
23molecule/mole)/amplification base number × 660 (base pair mean molecule quantity).
(5) enzyme action qualification: choose two the efficient restriction enzyme site property entered double digestions on pMD18T carrier.Enzyme action system is Hinc II 0.5 μ l, Ecor I 0.5 μ l, 10 × M 2 μ l, plasmid 4 μ l.In 37 DEG C of constant incubators, place 4h, be then placed in PCR instrument 65 DEG C of 10min and make enzyme deactivation, then put into rapidly ice chest and cool, carry out electrophoresis detection afterwards.
The foundation of quantitative fluorescent PCR standard curve and the determination of the range of linearity:
Each Pseudomonas standard substance of preparation, with 10 times of gradient dilutions, are got and are no less than 5 dilution factors as template, and each dilution factor does three pipes and repeats: the range of linearity of getting the standard substance confirmed standard curve of 10 times of gradient dilutions.
The fluorescence quantitative PCR detection of jejunum object flora adopts the change of LightCycle quantitative real time PCR Instrument and the different object bacterium of supporting analysis software rat cecal chyme.Adopt Bio-RAD PCR kit for fluorescence quantitative, reaction system is 25 μ l, and comprise SYBR Premix Ex TaqTM 9 μ l, each 0.5 μ l of upstream and downstream primer, template DNA 4 μ l, add sterilizing distilled water to 25 μ l.
1.10 jejunitis sex factors and compact siro spinning technology associated protein mrna expression horizontal detection
According to test kit operation instruction, extract test kit (Invitrogen company) with TRIzol and extract liver and jejunum total serum IgE, the micro-ultraviolet spectrophotometer Nano of the total serum IgE after extraction
nD-1000 (NanoDrop scientific & technical corporation) detectable concentration and purity, the integrity of the RNA that Denaturing Agarose Gel electrophoresis detection is extracted.Use Prime ScriptTM Reverse Transcription box (TaKaRa company) to carry out reverse transcription, by specification operates, and reverse transcription RNA consumption is 2 μ g.Adopt quantitative real time PCR Instrument (Roche LC480) to carry out real time fluorescent quantitative detection to the cDNA that reverse transcription obtains, fluorescence quantitative kit is Bole iTaq Universal SYBR Green Supermix.
Specific primer sequence is in table 8.
Table 8 real-time quantitative PCR primer
Quantitative fluorescent PCR reaction system (20 μ l) is as follows:
PCR reaction condition is as follows: 95 DEG C, denaturation 5min; 95 DEG C, 20s; Annealing temperature (annealing temperature according to each gene is determined) and extension 30s, 70 DEG C keep 1min, totally 40 circulations.Use 2
-Δ Δ Ctmethod calculates the expression of each gene.
1.11 Diagnosis of Sghistosomiasis notations detect expressing quantity
The Western Immuno marking is analyzed (Western blot) and is adopted Kansagra (2003) method improved.Get that 500mg is finely divided ice freezes tissue sample, add the lysis buffer of 2ml pre-cooling, mixing, lysate is containing 2mMTris-HCL (three (methylol) aminomethane-hydrochloric acid), 100mM Phenylmethanesulfonyl fluoride, 0.5mM dodecyl sodium sulfate and 1mM dithiothreitol dithio.Detect protein concentration in supernatant according to two Kui Lin formic acid methods (BCA) of standard, determine applied sample amount, add isocyatic protein sample, electrophoresis in SDS-PAGE gel, the albumen transferring film of separation is on nitrocellulose filter.Close with the TBS containing 3% defatted milk powder, hatch primary antibodie, 4 DEG C are spent the night.The antibody used in experiment has: (1:400 dilutes Occludin Claudin-1 rabbit polyclonal antibody, Santa Cruz company), occlusion albumen Occludin rabbit polyclonal antibody (1:400, Santa Cruz company), banded Occludin ZO-1 rabbit polyclonal antibody (1:400, Santa Cruz company).Clean 3 times with the TBS liquid containing 0.1% polysorbas20, hatch 60 minutes with two anti-(anti-rabbit IgG albumen, Santa-Cruz companies) of HRP (horseradish peroxidase conjugate) labelling, clean 3 times.Follow procedure description, uses ECL PlusTM immunoblotting analysis Chemiluminescence Apparatus to detect.React as internal reference with actin β-actin antibody simultaneously.
1.12 jejunal tissue morphology
Sample treatment and Method of Morphological Observation are with embodiment 1.
2 data statisticss
Variance analysis is carried out with the GLM model in SAS software (v 8.2, SAS company).Carry out multiple comparisons by LSD method, analyze difference between each process.P<0.05 represents significant difference, and P<0.01 represents that difference is extremely remarkable.
3 results and analysis
3.1 Stress Hormone Levels of Plasma
Table 9 blood plasma cortisol and noradrenaline levels
| Project | CT | NC | VE | LO | HO | SEM | P |
| Hydrocortisone (ng/ml) | 12.04 b | 12.87 a | 12.90 a | 12.67 a | 12.11 b | 0.12 | 0.034 |
| Norepinephrine (ng/ml) | 217.93 ab | 236.98 a | 229.07 a | 217.51 ab | 205.80 b | 3.49 | 0.038 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Dysentery relieving grass is oily and the impact of VE on rat plasma hydrocortisone, noradrenaline levels is as shown in table 9.Compared with the blank group of not injecting Diquat, the cortisol levels in NC group, PC and LO group rat plasma that causes of bringing out after injection Diquat significantly raises (P < 0.05); But cortisol levels is significantly lower than NC group (P < 0.05) in HO group rat plasma.The NC group of injection Diquat, Plasma Norepinephrine Level is difference remarkable (P > 0.05) compared with C group; PC group and LO group Plasma Norepinephrine Level, with C group still with NC group than difference not significantly (P > 0.05); But HO group Plasma Norepinephrine Level compared with NC group significantly reduces (P < 0.05).
3.2 jejunum activities of antioxidant enzymes and MDA content
Table 10 jejunum activities of antioxidant enzymes and MDA content
| Project | CT | NC | VE | LO | HO | SEM | P |
| SOD(U/mL) | 44.51 b | 31.31 a | 37.29 ab | 41.83 a | 42.16 a | 1.39 | 0.02 |
| GSH-Px(U/mg) | 46.88 a | 27.21 c | 36.39 bc | 38.15 ab | 42.19 ab | 1.59 | ＜0.01 |
| MDA(nmol/ml) | 1.05 a | 0.67 b | 0.80 b | 0.80 b | 0.72 b | 0.03 | ＜0.01 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Dysentery relieving grass is oily and the impact of VE on jejunum in rats activities of antioxidant enzymes and MDA content is as shown in table 10.Compared with C group, after injection Diquat, jejunum SOD enzyme activity is caused significantly to reduce (P < 0.05); The PC group jejunum SOD enzyme activity gavaging VE between NC group and C group, with the two difference all not significantly (P > 0.05); Gavage the LO group of dysentery relieving grass oil, HO group, can significantly improve jejunum SOD enzyme activity (P < 0.05) compared with NC group, makes it to return to the level (P > 0.05) suitable with C group.
Compared with C group, NC group jejunum GSH-Px enzymatic activity extremely significantly reduces (P < 0.01); Compared with NC group, PC group GSH-Px enzymatic activity increases, but increase rate is not significantly (P > 0.05), and the LO group, the HO group that gavage dysentery relieving grass oil can significantly improve GSH-Px enzymatic activity (P < 0.05), make it to return to the level (P > 0.05) close with C group.
Compared with C group, NC group jejunum MDA content extremely significantly raises (P<0.01); Compared with NC group, the PC group jejunum MDA content gavaging VE significantly reduces (P < 0.05), makes it to return to the level (P > 0.05) close with C group; Similar with PC group, gavage the LO group of low dosage dysentery relieving grass oil, jejunum MDA content remarkable reduction compared with NC group (P < 0.05), reaches the level (P > 0.05) close with C group; But gavage the HO group of high dose dysentery relieving grass oil, jejunum MDA content can extremely significantly reduce (P < 0.01) compared with NC group, reaches the level (P > 0.05) suitable with C group.
ROS content in 3.3 jejunal mucous membranes and chyme
Dysentery relieving grass oil and VE are on the impact of active oxygen ROS content in jejunum in rats mucosa and chyme as shown in Figure 2.In jejunal mucous membrane, after injection Diquat, NC group rat is compared with C group, and ROS content significantly extremely significantly raises (P < 0.01); Compared with NC group, gavage ROS content in the PC group jejunal mucous membrane of VE and extremely significantly reduce (P < 0.01), reach the level (P > 0.05) suitable with C group; Similar with it, in HO group jejunal mucous membrane, ROS content also can extremely significantly reduce (P < 0.01), reach the level (P > 0.05) suitable with C group, but ROS content only can return to the level close with C group in LO group jejunal mucous membrane, still all difference is not significantly (P > 0.05) with C group with NC group.
In Jejunal chyme, NC group ROS content compared with C group does not have significant difference (P > 0.05); In PC group Jejunal chyme, ROS content and C group are quite (P > 0.05), with NC group difference also remarkable (P > 0.05); In LO group Jejunal chyme, ROS content is between C group, between PC group and NC group, with three's difference remarkable (P > 0.05); Compared with other each group, in HO group Jejunal chyme, ROS content significantly reduces (P < 0.05).
Flora dynamics in 3.4 Jejunal chyme
Dysentery relieving grass oil and VE are on the impact of flora in jejunum in rats chyme as shown in Figure 3.C group, NC group, PC group, LO group and HO group, all do not have significant difference (P > 0.05) when the quantity of enterococcus faecalis compares mutually in Jejunal chyme.
Compared with C group, in other each group Jejunal chyme of injection Diquat, colibacillary quantity all increases, wherein NC group, PC group and C group significant difference (P < 0.05); The colibacillary quantity of LO group, between C group and NC group, with the two difference all remarkable (P > 0.05); HO group colibacillary number ratio C group is lower slightly, but with C group difference not significantly (P > 0.05), but significantly lower than NC group and PC group (P < 0.05).
Different from colibacillary change, injection Diquat after NC group compared with C group in Jejunal chyme the quantity of lactobacillus significantly reduce (P < 0.05); The quantity gavaging lactobacillus in the PC group Jejunal chyme of VE significantly improves (P < 0.05) compared with NC group, also a little more than C group (P > 0.05); LO group rat, in Jejunal chyme, the quantity of lactobacillus is significantly higher than NC group, C group (P < 0.05), also a little more than PC group (P > 0.05); In HO group Jejunal chyme, the quantity of lactobacillus is significantly higher than C group, NC group and PC group (P < 0.05), but with LO group difference not significantly (P > 0.05).
The expression of inflammatory factor mRNA in 3.5 jejunal mucous membranes
Dysentery relieving grass oil and VE are on the impact of the mrna expression level of inflammatory factor TNF-α, IL-1 β, IL-6 in jejunum in rats mucosa as shown in Figure 4.C group, NC group, PC group, LO group and HO group, the mrna expression level of jejunal mucous membrane IL-1 β does not all have significant difference (P > 0.05) each other.Compared with C group, the mrna expression level of NC group TNF-α significantly raises (P < 0.01); The mrna expression level of PC group TNF-α between NC group and C group, with C group, the equal significant difference of NC group (P < 0.05); After gavaging dysentery relieving grass oil, mrna expression level remarkable reduction compared with NC group (P < 0.05) of HO group TNF-α, between C group and PC group, with the two difference all remarkable (P < 0.05); And the mrna expression level of LO group TNF-α is not only remarkable in NC group (P < 0.05), and significantly lower than C group and other each group (P < 0.05).The mrna expression level of NC group IL-6, compared with C group, significantly raises (P < 0.05); Gavage mrna expression level remarkable reduction compared with NC group (P < 0.05) of the PC group IL-6 of VE, return to the level (P > 0.05) suitable with C group; Compared with NC group, gavage the LO group of dysentery relieving grass oil, HO group IL-6 mrna expression level between NC group and C group, with C group, the equal difference of NC group remarkable (P > 0.05).
The m RNA of 3.6 jejunum compact siro spinning technology associated protein and protein expression level
For research dysentery relieving grass oil and VE are on close-connected impact, have detected the mrna expression level of occlusion albumen Occludin and banded Occludin ZO-1, as shown in Figure 5.After injection Diquat, the mrna expression level of NC group Occludin is significantly lower than C group (P < 0.05); Gavage the PC group of VE, significantly improve the mrna expression level (P < 0.05) of Occludin compared with NC group, even only slight beyond C group (P > 0.05); Gavage the LO group of dysentery relieving grass oil, HO group compared with NC group, also significantly increase the mrna expression level (P < 0.05) of Occludin, make it to reach the level (P > 0.05) a little more than C group.The mrna expression level of the NC group ZO-1 after injection Diquat is significantly lower than C group (P < 0.05); Gavaging VE can make the mrna expression level of ZO-1 be increased between NC group, C group, but with the two difference all not significantly (P > 0.05); Gavage the LO group of dysentery relieving grass oil, the mrna expression level of HO group ZO-1, with NC group difference all remarkable (P < 0.05), do not improve the effect of the mrna expression level of ZO-1.
Dysentery relieving grass oil and VE on the impact of Occludin and ZO-1 protein expression amount, as shown in Figure 6.NC group Occludin protein expression amount after injection Diquat is significantly lower than C group (P < 0.05); Gavage the PC group of VE, significantly improve Occludin protein expression amount (P < 0.05) compared with NC group, even only slight beyond C group (P > 0.05); Gavage the LO group of OEO, HO group compared with NC group, also significantly increase Occludin protein expression amount (P < 0.05), make it to reach the level (P > 0.05) a little more than C group.NC group ZO-1 protein expression amount remarkable reduction compared with C group (P < 0.05) after injection Diquat; The PC group ZO-1 protein expression amount after VE that gavages is difference remarkable (P > 0.05) compared with NC group; Gavage LO group, the HO group ZO-1 protein expression amount of dysentery relieving grass oil, difference is not all significantly (P < 0.05) compared with NC group.
3.7 jejunal tissue morphological observations
Dysentery relieving grass oil and VE are on the impact of jejunum in rats mucous epithelium form as shown in Figure 7.Compared with C group, there is Villus atrophy, Fragmentation Phenomena in NC group.And the PC group gavaging VE, LO, HO group jejunum in rats structure of gavaging dysentery relieving grass oil keep complete relatively with the seriality of enterocyte structure.
Table 11 jejunal mucous membrane Epithelial morphology index
| Project | CT | NC | VE | LO | HO | SEM | P |
| Height of naps (μm) | 924.11 a | 759.58 b | 853.27 ab | 889.19 a | 877.64 a | 18.40 | 0.04 |
| Crypt depth (μm) | 326.88 | 356.46 | 335.15 | 324.80 | 321.15 | 6.02 | ＞0.05 |
| Fine hair width (μm) | 303.54 b | 392.00 a | 300.89 b | 286.39 b | 293.02 b | 12.50 | 0.03 |
| Height of naps/Crypt depth (μm) | 2.86a | 2.17 b | 2.58 ab | 2.84 a | 2.80 a | 0.08 | ＜0.01 |
Colleague's numerical value shoulder mark lower case difference person difference extremely significantly (P < 0.05).
Dysentery relieving grass is oily and the impact of VE on jejunum in rats mucous epithelium morphological indexes is as shown in table 11.All each group does not all have significant difference (P > 0.05) on Crypt depth; Compared with C group, injection Diquat significantly reduces the jejunum villi height of NC group rat and the ratio (P < 0.05) of height of naps/Crypt depth, significantly increases fine hair width (P < 0.05); Compared with NC group, the PC group height of naps, the height of naps/Crypt depth ratio that gavage VE increase, between NC group, C group but with the two difference all not significantly (P > 0.05), the PC group fine hair width compared with NC group gavaging VE significantly reduces (P < 0.05), reaches and C group suitable (P > 0.05); Compared with NC group, LO group, HO group and NC group jejunum villi height, height of naps/Crypt depth ratio significantly improve (P < 0.05), and fine hair width significantly reduces (P < 0.05).
4 brief summaries
The mechanism of 4.1 diquat dibromide induction intestinal injury
Existing large quantity research evidence shows that Diquat produces a large amount of ROS by chemical reaction.This research finds, in the rat model set up by lumbar injection Diquat, while producing in a large number along with ROS, blood plasma and jejunal mucous membrane Antioxidant Enzyme Systems suffer remarkable infringement (see table 9, table 10 and Fig. 1) simultaneously.The display of research evidence, jejunal mucous membrane and chyme flora all can produce ROS.It should be noted that Diquat is induction of the remarkable increase (Fig. 3) of producing ROS bacterium (as escherichia coli) quantity.Although compared with blank group, in NC group chyme, the raising of ROS is not significantly (P>0.05), but these ROS may after entering intestinal mucosa layer, become the signaling molecule activating the signal paths such as NF-κ B and MAPKs, the great expression of inducing immune cells inflammatory cytokine, thus the ROS producing excess.In fact, we have also discovered in the negative control rats jejunal mucous membrane of injection Diquat, and the gene expression dose of inflammatory cytokine and the level of ROS are significantly higher than non-stress group (see Fig. 4 and Fig. 2).This means under the effect of mucomembranous immune system, the trace producing enteric cavity (chyme) ROS that ROS bacterium causes changes, and may be cascaded amplification in mucosa.Therefore, not only directly to produce ROS with it relevant for the intestinal injury of Diquat induction, and may with its destruction Antioxidant Enzyme Systems, the quantity of change intestinal product ROS bacterium is relevant.
4.2 dysentery relieving grass oil alleviate the effects anb Mechanism of intestinal oxidative stress
Result of the test shows, gavage the dysentery relieving grass oil of antioxidant VE and varying level, all effectively reduce the intestinal ROS and MDA content that are induced by Diquat, and significantly improve the activity (Fig. 2 and table 10) of SOD, thus maintain intestinal structure relatively complete (Fig. 7).Wherein, the effect on that a time high concentration (10mg/t.bw) dysentery relieving grass oil is alleviating the ROS accumulation of being induced by Diquat is gavaged every day optimum.Research display, the main component in dysentery relieving grass oil is thymol and carvacrol, all has the function directly removing superoxide radical and hydrogen peroxide.In addition, dysentery relieving grass oil controls the effect of intestine in rats damage, also shows that it significantly improves antioxidase SOD activity (table 10).After it should be noted that dysentery relieving grass oil (HO group) gavaging high dose, also significantly suppress the escherichia coli bacterium quantity that intestinal produces ROS, significantly improve lactobacillus quantity (Fig. 3).Therefore, dysentery relieving grass oil, compared with VE, more effectively can alleviate the effect of the intestine in rats damage of being induced by injection Diquat.
4.3 dysentery relieving grass oil alleviate the effect of Intestinal Tract Morphology damage
This result of the test shows, the oxidative stress of Diquat induction can cause Intestinal Mucosa to damage; And dysentery relieving grass oil improves oxidative stress intestine in rats height of naps, fine hair width, height of naps: Crypt depth ratio (Fig. 7, table 11).Increasing research shows, inflammatory factor can affect expression and the distribution of the multiple tight junction protein of enterocyte.The expression of this analysis of experiments two tight junction proteins played a crucial role (intestinal occlusion albumen Occludin and the banded Occludin ZO-1 of band shape).Result shows, and dysentery relieving grass oil significantly improves mRNA and the protein expression of oxidative stress condition lower intestinal tract occlusion albumen Occludin, and does not make significant difference (Fig. 5, Fig. 6) to banded Occludin ZO-1.In fact, the change expressed of Occludin is along with the change (Fig. 4) of intestinal mucosa inflammatory cytokine TNF-a and IL-6mRNA expression.Therefore, the damage mitigation effect of the oily anti-Diquat induction of dysentery relieving grass, may be by inhibit jejunal mucous membrane TNF α (A) and IL-6 (C) mrna expression level, reducing the impact on tight junction protein, and finally alleviate the damage of the intestinal of Diquat induction.
In sum; the oily Orally taken emulsion of administered by oral gavage dysentery relieving grass; significantly can strengthen the activity of intestinal antioxidase; reduce MDA content; reduce escherichia coli quantity in chyme, improve lactobacillus quantity, suppress the great expression of endo-enteritis sex factor, add the expression of compact siro spinning technology occlusion albumen Occludin; thus decrease the excessive damage of diquat dibromide to epithelium of intestinal mucosa, the available protecting mechanical barrier of intestinal.
Embodiment 3
For an Orally taken emulsion for the toxic reaction that anti-diquat dibromide causes, it is made up of the composition of following percentage by weight: dysentery relieving grass oil 2%, castor oil hydrogenated 10%, starch 0.5%, glycerol 0.5%, sodium carboxymethyl cellulose 0.1%, deionized water are surplus.
By dysentery relieving grass oil and castor oil hydrogenated mixing, stirring at low speed 5 minutes, obtains oil phase; Starch and sodium carboxymethyl cellulose are joined in deionized water, utilizes ultrasonic wave concussion to make both dissolve completely, obtain aqueous phase; Slowly poured in oil phase by aqueous phase, then add glycerol, system breast 10 minutes under 20 DEG C of conditions, obtains the oily Orally taken emulsion of dysentery relieving grass.
Prevention and therapy method is: gavage the oily Orally taken emulsion of above-mentioned dysentery relieving grass to poultry, dose is 50 ~ 100ml.
Embodiment 4
For an Orally taken emulsion for the toxic reaction that anti-diquat dibromide causes, it is made up of following composition: dysentery relieving grass oil 10%, castor oil hydrogenated 20%, propylene glycol 0.1%, glycerol 0.05%, n-butyl alcohol 0.05%, arabic gum 0.02%, deionized water are surplus.
By dysentery relieving grass oil and castor oil hydrogenated mixing, stirring at low speed 15 minutes, obtains oil phase; Propylene glycol and arabic gum are joined in deionized water, utilizes ultrasonic wave concussion to make both dissolve completely, obtain aqueous phase; Slowly poured in oil phase by aqueous phase, then add glycerol and n-butyl alcohol, system breast 20 minutes under 30 DEG C of conditions, obtains the oily Orally taken emulsion of dysentery relieving grass.
Embodiment 5
For an Orally taken emulsion for the toxic reaction that anti-diquat dibromide causes, it is made up of following composition: dysentery relieving grass oil 0.01%, castor oil hydrogenated 5%, ethylene glycol 10%, glycerol 5%, n-amyl alcohol 5%, xanthan gum 0.5%, gellan gum 0.5%, deionized water are surplus.
By dysentery relieving grass oil and castor oil hydrogenated mixing, stirring at low speed 20 minutes, obtains oil phase; Ethylene glycol and xanthan gum, gellan gum are joined in deionized water, utilizes ultrasonic wave concussion to make both dissolve completely, obtain aqueous phase; Slowly poured in oil phase by aqueous phase, then add glycerol and n-amyl alcohol, system breast 15 minutes under 25 DEG C of conditions, obtains the oily Orally taken emulsion of dysentery relieving grass.
Embodiment 6
For an Orally taken emulsion for the toxic reaction that anti-diquat dibromide causes, it is made up of following composition: dysentery relieving grass oil 0.1%, castor oil hydrogenated 15%, propylene glycol 3%, starch 0.2%, dehydrated alcohol 0.5%, n-butyl alcohol 1%, xanthan gum 0.5%, sodium carboxymethyl cellulose 1%, deionized water are surplus.
By dysentery relieving grass oil and castor oil hydrogenated mixing, stirring at low speed 8 minutes, obtains oil phase; Propylene glycol, starch and xanthan gum, sodium carboxymethyl cellulose are joined in deionized water, utilizes ultrasonic wave concussion to make both dissolve completely, obtain aqueous phase; Slowly poured in oil phase by aqueous phase, then add dehydrated alcohol and n-butyl alcohol, system breast 30 minutes under 22 DEG C of conditions, obtains the oily Orally taken emulsion of dysentery relieving grass.
Claims (5)
1. the purposes of dysentery relieving grass oil in the toxic reaction medicine of the anti-diquat dibromide initiation of preparation.
2. purposes according to claim 1, is characterized in that: described medicine is Orally taken emulsion.
3. purposes according to claim 2, is characterized in that: described Orally taken emulsion is made up of the composition of following percentage by weight: dysentery relieving grass oily 0.01-10%, emulsifying agent 5%-20%, stabilizing agent 0.1%-10%, co-emulsifier 0.1%-10%, thickening agent 0.01%-1%, water is surplus.
4. purposes according to claim 3, is characterized in that: described emulsifying agent is castor oil hydrogenated; Described stabilizing agent is one or more in ethylene glycol, propylene glycol, starch; Described co-emulsifier is one or more in dehydrated alcohol, n-butyl alcohol, glycerol, n-amyl alcohol; Described thickening agent is one or more in arabic gum, sodium carboxymethyl cellulose, xanthan gum, gellan gum.
5. an Orally taken emulsion for the toxic reaction caused for anti-diquat dibromide, is characterized in that being made up of the composition of following percentage by weight: dysentery relieving grass oily 0.01-10%, emulsifying agent 5%-20%, stabilizing agent 0.1%-10%, co-emulsifier 0.1%-10%, thickening agent 0.01%-1%, water is surplus;
Described emulsifying agent is castor oil hydrogenated; Described stabilizing agent is one or more in ethylene glycol, propylene glycol, starch; Described co-emulsifier is one or more in dehydrated alcohol, n-butyl alcohol, glycerol, n-amyl alcohol; Described thickening agent is one or more in arabic gum, sodium carboxymethyl cellulose, xanthan gum, gellan gum.
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| WO2008033443A2 (en) * | 2006-09-13 | 2008-03-20 | Larry Lancaster | Composition for increasing soil fertility |
| CN103750033A (en) * | 2014-01-03 | 2014-04-30 | 宁波大学 | Prawn feed additive with intestinal tract protection effect as well as preparation method and application thereof |
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| WO2008033443A2 (en) * | 2006-09-13 | 2008-03-20 | Larry Lancaster | Composition for increasing soil fertility |
| CN103750033A (en) * | 2014-01-03 | 2014-04-30 | 宁波大学 | Prawn feed additive with intestinal tract protection effect as well as preparation method and application thereof |
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