CN115105492B - New use of petroselinic acid - Google Patents
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- CN115105492B CN115105492B CN202110284764.8A CN202110284764A CN115105492B CN 115105492 B CN115105492 B CN 115105492B CN 202110284764 A CN202110284764 A CN 202110284764A CN 115105492 B CN115105492 B CN 115105492B
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Abstract
The invention relates to a new application of petroselinic acid, in particular to an application of petroselinic acid in preparing a medicament for preventing or/and treating intestinal ischemia reperfusion injury. Compared with the prior art, the invention has the following beneficial effects: the invention uses petroselinic acid for preventing and/or treating intestinal ischemia reperfusion injury, and the effect of the medicine is verified on a constructed classical intestinal ischemia reperfusion model, and the verification result shows that the petroselinic acid obviously improves intestinal tissue injury induced by intestinal ischemia reperfusion of mice, improves the survival rate of the mice, inhibits the expression of inflammatory factors, has obvious effect, is safe and nontoxic, and has small side effect.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a new application of petroselinic acid, and in particular relates to an application of petroselinic acid in preparing medicines for preventing or/and treating intestinal ischemia reperfusion injury.
Background
Petroselinic acid (Petroselinic acid, PSA) is a rare fatty acid found mainly in the seeds of the family umbelliferae. The school name of petroselinic acid is octadeca-6-enoic acid, and the molecular formula is C 13 H 24 O 2 The molecular weight is 282.468, and the chemical structure is shown as the following formula:
petroselinic acid can inhibit the expression of the mucilaginous Serratia biological membrane and virulence genes of environmental pathogenic bacteria. However, the role of petroselinic acid in Ischemia Reperfusion (I/R) has not been reported. Intestinal ischemia reperfusion (intestinal I/R) injury is one of the tissue and organ injuries commonly seen in surgical practice, and plays an important role in pathophysiological evolution of operations such as severe infection, trauma, shock and intestinal obstruction, abdominal aortic aneurysm artificial vascular replacement, extracorporeal circulation and small intestine transplantation. The intestinal I/R not only can cause damage to the intestines themselves, but also can cause intestinal endotoxin and bacterial translocation due to the damage of intestinal mucosa barriers, thereby causing systemic inflammatory response syndrome (Systemic Inflammatory Response Syndrome, SIRS) and causing damage to distant organs such as lung, liver, kidney and the like, and finally can cause multiple organ dysfunction syndrome (Multiple Organ Dysfunction Syndrome, MODS). In the last twenty years, despite the substantial improvement in surgical techniques and perioperative management levels, patients who experience intestinal I/R injury once they develop MODS, still have mortality rates as high as 67% -80%. However, no effective drug has been developed for targeted treatment of intestinal ischemia reperfusion injury. Therefore, the exploration of an effective control strategy for intestinal ischemia reperfusion injury is a technical problem to be solved in clinic at present.
Disclosure of Invention
Based on the above, the invention provides a new application of petroselinic acid, in particular to an application of petroselinic acid in preparing a medicament for preventing or/and treating intestinal ischemia reperfusion injury.
The technical scheme of the invention is as follows:
use of petroselinic acid for the preparation of a medicament for the prevention or/and treatment of intestinal ischemia reperfusion injury.
In one embodiment, the medicament comprises petroselinic acid and pharmaceutically acceptable excipients.
In one embodiment, the petroselinic acid content of the medicament is > 99% by weight.
In one embodiment, the pharmaceutical dosage form is a tablet.
In one embodiment, the tablet is a coated tablet.
In one embodiment, the pharmaceutical dosage form is a capsule.
In one embodiment, the pharmaceutical dosage form is an oral liquid.
In one embodiment, the pharmaceutical dosage form is an oral granule.
In one embodiment, the pharmaceutical dosage form is an oral powder.
In one embodiment, the pharmaceutical is in the form of an injection.
In one embodiment, the injection is a lyophilized powder for injection.
In one embodiment, the injection is an emulsion for injection.
Compared with the prior art, the invention has the following beneficial effects:
the invention uses petroselinic acid for preventing and/or treating intestinal ischemia reperfusion injury, and the effect of the medicine is verified on a constructed classical intestinal ischemia reperfusion model, and the verification result shows that the petroselinic acid obviously improves intestinal tissue injury induced by intestinal ischemia reperfusion of mice, improves the survival rate of the mice, inhibits the expression of inflammatory factors, has obvious effect, is safe and nontoxic, and has small side effect.
Drawings
FIG. 1 is a graph of the results of petroselinic acid increasing the survival rate of ischemia reperfusion in mice intestine; in fig. 1, the meaning of the reference symbols is: the data adopts a Log-rank (Mantel-Cox) test;
FIG. 2 is a graph showing pathological results of petroselinic acid in improving intestinal tissue damage induced by ischemia reperfusion in mice; in FIG. 2, (A) is a graph of HE staining for morphological changes in intestinal tissue of each group, and (B) is a quantitative scoring result of intestinal tissue injury of each group, with a scale of 100 μm; the meaning of the reference symbols in the figures is: data were analyzed using T-test, which indicates that differences compared to group I/R have statistical significance p <0.05;
FIG. 3 is a bar graph of petroselinic acid reducing the level of intestinal tissue inflammatory factor mRNA expression induced by ischemia reperfusion in mice; in fig. 3, the meaning of the reference symbol is: data were analyzed using the T-test, which indicates that differences compared to the I/R group were statistically significant p <0.05.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
In order to facilitate an understanding of the present application, the meaning of some terms and expressions in the text of the present invention will be explained below.
As used herein, the term "ischemia" relates to a condition that may occur in any organ or tissue that lacks an oxygen supply and/or a metabolite supply. Ischemia occurs when there is an imbalance between oxygen supply and demand due to inadequate perfusion (i.e., blood supply). The lack of oxygen supply may be caused by thrombosis, the presence of stenotic atherosclerosis, restenosis, anemia, stroke, arterial clotting, vasoconstriction and/or endothelial dysfunction of the microvascular system (Taku Su Bo syndrome).
The term "ischemia reperfusion injury" refers to an injury to an organ or tissue due to insufficient blood supply to the organ or tissue during ischemia prior to the onset of reperfusion (i.e., an ischemic injury is an injury caused by ischemia during the time between the onset of ischemia and the onset of reperfusion).
A typical and pathological manifestation of ischemic injury is the ischemic area becoming pale. In contrast, in reperfusion, non-necrotic ischemic tissue regains its physiological color.
From a biochemical point of view, ischemic injury is characterized by local and systemic changes in pH (acidification) in blood (leukocytes, preferably PBMCs) in ischemic tissue, changes in ATP concentration, increased susceptibility of platelets to activation, and enhanced inflammatory responses in both ischemic tissue localization and the blood system.
Ischemic injury may be caused, for example, by atherosclerosis, thrombosis, thromboembolism, lipid embolism, hemorrhage, stents, surgery, angioplasty, intraoperative bypass surgery, organ transplantation, total ischemia, myocardial infarction, vasoconstriction, microvascular dysfunction, and/or combinations of two or more thereof.
Ischemic injury may involve cell death of muscle cells (preferably by necrosis and/or apoptosis, more preferably by necrosis), injury due to acidification of intracellular pH caused by ischemia, and/or injury due to inflammatory reactions initiated by ischemia and further amplified during reperfusion.
During ischemia, anaerobic metabolism dominates, resulting in a decrease in cell pH. To buffer this accumulation of hydrogen ions, na + /H + The exchanger discharges excess hydrogen ions, which creates a large influx of sodium ions. Calogris (Kalogeris) et al, int Rev Cell Mol biol.2012;298:229-317, which summarize the major pathological events of ischemia and reperfusion components contributing to tissue damage. Ischemia also depletes cellular ATP, inactivating atpase (e.g., na+/k+ atpase), reducing active Ca 2+ Outflow and limiting the re-uptake of calcium by the Endoplasmic Reticulum (ER), thereby producing an intracellular calcium overload. These changes are accompanied by mitochondrial permeability transition (mitochondrial permeability transition, MPT) pore opening that dissipates mitochondrial membrane potential and further impairs ATP production. These changes and thus the extent of tissue damage vary to some extent with the magnitude of the blood supply reduction and the duration of the ischemic period.
Ischemic injury may involve the following symptoms: chest discomfort, shortness of breath, other areas of the upper body, nausea and/or anxiety.
As used herein, the term "reperfusion" relates to restoration of blood flow to ischemic tissue. Although there are clear benefits to reperfusion of blood to ischemic tissue, it is well known that reperfusion itself can lead to a series of adverse reactions that paradoxically harm tissue.
As used herein, the term "reperfusion injury" refers to an injury to an organ or tissue that results when blood supply returns to the organ or tissue after an ischemic period. Thus, reperfusion injury is an injury that is caused during the time between the start of reperfusion and the end of reperfusion (typically, a substantial portion of the injury will be caused during the first few minutes of reperfusion). The underlying mechanism of reperfusion injury is complex, multifactorial, and involves (1) the reintroduction of molecular oxygen upon blood flow remodeling to promote the production of reactive oxygen species (reactiveoxygen species, ROS), (2) calcium overload, (3) the opening of MPT pores, (4) endothelial dysfunction, (5) the appearance of a pre-thrombotic phenotype, and (6) a significant inflammatory response. The lack of oxygen and nutrients in the blood during the ischemia period creates a situation in which the recovery of circulation leads to inflammation and oxidative damage by inducing oxidative stress rather than restoring normal function. Oxidative stress associated with reperfusion may cause damage to the affected tissue or organ. The biochemistry of reperfusion injury is characterized by oxygen depletion during an ischemic event followed by reoxygenation during reperfusion with concomitant production of active oxygen. The damage that occurs with reperfusion is the result of interactions between substances accumulated during ischemia and substances delivered upon reperfusion. The basis of these events is oxidative stress, which is defined as an imbalance between oxygen radicals and endogenous scavenging systems. The result is cell damage and death, which is initially localized, but eventually becomes systemic if the inflammatory response is not examined.
Reperfusion injury is primarily characterized by oxygen bursts and inflammatory response reperfusion injury and consequent tissue damage may occur after revascularization of infarcted (ischemic) tissue. This is associated with an impaired mitochondrial membrane potential, further with the progression of apoptosis, reperfusion-related arrhythmias, cardiac arrest and an overall increase in infarct size caused by ischemia. Thus, the final infarct size (tissue damage) depends on the ischemic damage (tissue damage caused by itself during ischemia) and to a lesser extent on the tissue damage caused by reperfusion.
Reperfusion injury may be caused, for example, by a mechanical event, or by one or more surgical procedures or other therapeutic interventions that restore blood flow to a tissue or organ that has undergone reduced blood flow supply. Such surgical procedures include, for example, coronary artery bypass grafting, coronary angioplasty, and organ grafting. In particular embodiments, reperfusion injury results from treatment of an ischemic process resulting from rupture/erosion of an atherosclerotic plaque and superimposition with a thrombus, thromboembolism, lipid embolism, hemorrhage, stent, surgery, angioplasty, end of a bypass during surgery, organ transplantation, total ischemia, vasoconstriction or microvascular dysfunction, or a combination thereof.
Reperfusion injury may involve oxidative damage, and injury and/or cardiomyocyte death due to an inflammatory response that, although weaker, becomes evident upon reperfusion, that is initiated during ischemia. Preferably, reperfusion injury involves oxidative damage, injury due to inflammatory response and cardiomyocyte death. More preferably, reperfusion injury involves oxidative damage, injury due to inflammatory reactions, and cardiomyocyte death, rather than by intracellular pH acidification.
Reperfusion injury may involve symptoms of palpitations, acute respiratory distress, fatigue, and/or edema.
The embodiment of the invention relates to the following steps:
the embodiment of the invention relates to application of petroselinic acid in preparing a medicament for preventing or/and treating intestinal ischemia reperfusion injury.
Preferably, the medicament comprises petroselinic acid and pharmaceutically acceptable excipients.
Preferably, the petroselinic acid content of the medicament is > 99wt%.
It will be appreciated that the medicament of the embodiments of the present invention may be formulated with different pharmaceutically acceptable excipients to produce suitable clinical dosage forms including, but not limited to, the following: tablets (including but not limited to coated tablets), capsules, oral liquids, oral granules, oral powders, injections (including but not limited to lyophilized powder injection or emulsion for injection). Such pharmaceutically acceptable excipients include, but are not limited to, diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modulators, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, topical analgesics, pH modifying agents, isotonic or isotonic agents and the like. Further: diluents such as starches, sucrose, celluloses, inorganic salts and the like; wetting agents such as water, ethanol, and the like; binders such as starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, and the like; disintegrants such as starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium dicarboxymethyl cellulose, crospovidone, surfactants, running disintegrants, etc.; lubricants such as talc, calcium stearate, magnesium lauryl sulfate, silica gel micropowder, polyethylene glycol, etc.; color, flavor, taste, and smell modifiers such as coloring matter, perfume, sweetener, mucilage, and corrigent, specifically such as fuchsin and xylitol; solvents such as water, oil, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, etc.; solubilizers such as tweens, sellers, polyoxyethylene fatty alcohol ethers, soaps, sulphates, sulphonates and the like; cosolvents such as organic acids (e.g., citric acid) and salts thereof, amides and amines, inorganic salts, polyethylene glycol, povidone, glycerin, and the like; emulsifying agents such as span, tween, herba Euphorbiae Helioscopiae, benzyl, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silica, bentonite, etc.; suspending agents such as glycerin, syrup, acacia, tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, thixotrope and the like; antioxidants such as sulfite, metabisulfite, bisulfite, ascorbic acid, gallic acid, esters thereof, and the like; metal complexing agents such as disodium edetate, polycarboxylic acid compounds, and the like; inert gases such as nitrogen, carbon dioxide, and the like; preservatives, such as nipagins, organic acids and salts thereof (e.g., sodium benzoate), quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, volatile oils, and the like; local analgesics such as benzyl alcohol, chlorobutanol, lidocaine, procaine and the like; pH adjusting agents such as hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, citric acid, citrate, etc.; isotonic or isotonic agents, such as glucose, sodium chloride, sodium citrate, sorbitol, xylitol, and the like. It will be appreciated that the diluents described in the examples of the present invention may also be referred to as fillers, which may also function the same in pharmaceutical formulations; the water in the embodiment of the invention is water meeting the requirements of medicaments, such as water for injection, purified water and the like, and the oil is oil for injection; the preservative provided by the embodiment of the invention can also be called an antibacterial agent, and plays roles in inhibiting the growth of microorganisms, prolonging the shelf life and the like in the preparation; the lubricant of the embodiment of the invention contains a glidant, an anti-sticking agent and the like; the sugar in the embodiment of the invention can be powdered sugar or syrup, and the type of the sugar is not limited to glucose; perfumes according to embodiments of the present invention include, but are not limited to, fragrances.
It will be appreciated that the medicaments according to the embodiments of the present invention may be formulated in different dosage forms based on different excipients, and accordingly, the mode of administration may also be varied.
Example 1: petroselinic acid can improve survival rate of ischemia reperfusion injury of mouse intestine
1. Experimental materials
1.1 Experimental animal
45 male C57BL/6J mice with the age of 6 weeks to 8 weeks are selected in the experiment, the weight is 18g to 22g, the mice are purchased in the animal center of the south hospital, the raising place is the SPF-class animal experiment department of the south hospital of the university of south medical science, the operation involved in the animal raising process is approved by the ethical committee, and the requirements of animal ethics are met.
1.2 reagents and instruments
Petroselinic acid (Shanghai Ala Biochemical technologies Co., ltd.); isoflurane (ravode life technologies limited); microvascular arterial clip (north american biotech limited); sterile silk (Ningbo medical needle limited); normal saline (Shijizhuang four-medicine limited); phosphate buffered saline (phosphate buffer saline, PBS) pH7.4 buffer (Gibco).
2. Experimental methods and results
2.1 Animal experiment
(1) Establishment of a mouse superior mesenteric artery I/R model (an animal model of intestinal ischemia reperfusion is a model of perioperative intestinal injury constructed by classical superior mesenteric artery occlusion):
the mice were anesthetized by free feeding, free drinking, and isoflurane inhalation before surgery, and the superior mesenteric artery was occluded with a non-invasive microvascular arterial clip, blocking blood flow.
After the intestinal ischemia lasted 45min, the arterial clamp was loosened to restore blood supply, intestinal reperfusion was performed, and after no bleeding was detected in the abdominal cavity, the peritoneum, muscle and skin were sutured layer by layer with sterile silk.
After interruption and during reperfusion, 0.5ml of warm normal saline at 37 ℃ is subcutaneously injected for liquid resuscitation, and survival and perfusion time of mice are observed and recorded.
(2) Experimental grouping
24C 57BL/6 mice from 6 weeks to 8 weeks were randomly assigned to Sham, intestinal I/R (I/R) and intestinal I/R+petroselinic acid (I/R+PSA).
1) Sham surgery group (Sham): performing abdominal opening, separating upper mesenteric artery without occlusion, and then performing intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution treatment;
2) Intestinal group I/R (I/R): establishing an intestinal I/R model, and performing treatment by using an intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution during reperfusion;
3) Intestinal group I/R + petroselinic acid (I/R + PSA): an intestinal I/R model was established and 100mg/kg of petroselinic acid was administered intraperitoneally during reperfusion.
2.2 experimental results
The experimental results are shown in fig. 1, and the results in fig. 1 show that the time for 45min ischemia reperfusion survival of mice can be improved and the survival rate of mice can be improved after petroselinic acid treatment is given.
Example 2: petroselinic acid slows down ischemia reperfusion-induced intestinal tissue pathological damage of mice
1. Experimental materials
1.1 Experimental animal
24 male C57BL/6J mice with the age of 6 weeks to 8 weeks are selected in the experiment, the weight is 18g to 22g, the mice are purchased in the animal center of the south hospital, the raising place is the SPF-class animal experiment department of the south hospital of the university of south medical science, the operation involved in the animal raising process is approved by the ethical committee, and the requirements of animal ethics are met.
1.2 reagents and instruments
Petroselinic acid (Shanghai Ala Biochemical technologies Co., ltd.); isoflurane (ravode life technologies limited); microvascular arterial clip (north american biotech limited); sterile silk (Ningbo medical needle limited); normal saline (Shijizhuang four-medicine limited); phosphate buffered saline (phosphate buffer saline, PBS) ph7.4 buffer (Gibco); hematoxylin-eosin staining (Beijing Lei Gen Biol); absolute ethanol (Guangdong Guanghua technology Co., ltd.); xylene (Guangdong Guanghua technology Co., ltd.); paraffin wax (lycra); 4% paraformaldehyde (Beijing Soy Bao technology Co., ltd.); neutral gums (Solarbio); full-automatic fluorescence microscope (olympus).
2 experimental methods and results
2.1 animal experiments:
(1) Establishment of a mouse superior mesenteric artery I/R model (an animal model of intestinal ischemia reperfusion is a model of perioperative intestinal injury constructed by classical superior mesenteric artery occlusion):
the mice were anesthetized by free feeding, free drinking, and isoflurane inhalation before surgery, and the superior mesenteric artery was occluded with a non-invasive microvascular arterial clip, blocking blood flow.
After the intestinal ischemia lasts for 45min, the arterial clamp is loosened to restore blood supply, the intestinal blood flow reperfusion is carried out, and after no bleeding is detected in the abdominal cavity, the peritoneum, the muscle and the skin are sutured layer by using sterile silk threads.
After blocking and during reperfusion, 0.5ml of warm physiological saline at 37 ℃ is injected subcutaneously to carry out liquid resuscitation, and after 2 hours of perfusion, the intestinal tissue of the mice is taken for examination.
(2) Experimental grouping:
24C 57BL/6 mice from 6 weeks to 8 weeks were randomly assigned to Sham, intestinal I/R (I/R) and intestinal I/R+petroselinic acid (I/R+PSA).
1) Sham surgery group (Sham): performing abdominal opening, separating upper mesenteric artery without occlusion, and then performing intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution treatment;
2) Intestinal group I/R (I/R): establishing an intestinal I/R model, and performing treatment by using an intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution during reperfusion;
3) Intestinal group I/R + petroselinic acid group (I/R + PSA): an intestinal I/R model was established and 100mg/kg of petroselinic acid was administered intraperitoneally during reperfusion.
2.2 detection of pathological changes in intestinal tissue
Fresh intestinal tissue was put into 4% paraformaldehyde for fixation for 24 hours, then dehydrated, embedded, sectioned, then hematoxylin-eosin stained, sealed with neutral gum, observed for pathological changes of intestinal tissue under a fully automatic fluorescence microscope, and then graded scoring of intestinal mucosal lesions was performed by the modified Chiu method.
2.3 experimental results
The experimental results are shown in fig. 2, and the results of the HE staining and scoring of the intestinal tissue according to the (a) and (B) show that the intestinal villi at the top of the I/R model group falls off, telangiectasia, and congestion, and the above lesions of the intestinal tissue of the mice are significantly improved after petroselinic acid treatment. The above data demonstrate that petroselinic acid is capable of slowing down the pathological changes in intestinal tissue of the ischemia reperfusion mice.
Example 3: petroselinic acid inhibits expression of inflammatory factors in intestinal tissue of mice in model of intestinal ischemia reperfusion
1. Experimental materials
1.1 Experimental animal
The experiment selects 24 male C57BL/6J mice with the age of 6 weeks to 8 weeks, the weight is 18 to 22g, the mice are purchased in the animal center of the south hospital, the raising place is the SPF-class animal experiment department of the south hospital of the university of south medical science, the operation involved in the animal raising process is approved by the ethical committee, and the animal ethical requirements are met.
1.2 reagents and instruments
Petroselinic acid (Shanghai Ala Biochemical technologies Co., ltd.); isoflurane (ravode life technologies limited); microvascular arterial clip (north american biotech limited); sterile silk (Ningbo medical needle limited); normal saline (Shijizhuang four-medicine limited); phosphate buffered saline (phosphate buffer saline, PBS) ph7.4 buffer (Gibco); TRIZOL lysate (Invitrogen); chloroform (Guangdong optical bloom); isopropyl alcohol (Guangdong optical bloom); absolute ethanol (guangdong guanghua); DEPC water (Sigma); SYBR Green fluorescent dye (eastern spinning); reverTra Ace qPCR RT Kit (Toyo-spun); PCR apparatus (Eppendorf, germany); fluorescent quantitative PCR apparatus (applied biosystems AB company in the United states).
2 experimental methods and results
2.1 animal experiments:
(1) Establishment of a mouse superior mesenteric artery I/R model (an animal model of intestinal ischemia reperfusion is a model of perioperative intestinal injury constructed by classical superior mesenteric artery occlusion):
the mice were anesthetized by free feeding, free drinking, and isoflurane inhalation before surgery, and the superior mesenteric artery was occluded with a non-invasive microvascular arterial clip, blocking blood flow.
After the intestinal ischemia lasted 45min, the arterial clamp was loosened to restore blood supply, intestinal reperfusion was performed, and after no bleeding was detected in the abdominal cavity, the peritoneum, muscle and skin were sutured layer by layer with sterile silk.
After blocking and during reperfusion, 0.5ml of warm physiological saline at 37 ℃ is injected subcutaneously to carry out liquid resuscitation, and after 2 hours of perfusion, the intestinal tissue of the mice is taken for examination.
(2) Experimental grouping
24C 57BL/6 mice from 6 weeks to 8 weeks were randomly assigned to Sham, intestinal I/R (I/R) and intestinal I/R+petroselinic acid (I/R+PSA).
1) Sham surgery group (Sham): performing abdominal opening, separating upper mesenteric artery without occlusion, and then performing intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution treatment;
2) Intestinal group I/R (I/R): establishing an intestinal I/R model, and performing treatment by using an intraperitoneal injection solvent (1% DMSO+40% PEG300+5% Tween80+54% physiological saline) solution during reperfusion;
3) Intestinal group I/R + petroselinic acid group (I/R + PSA): an intestinal I/R model was established and 100mg/kg of petroselinic acid was administered intraperitoneally during reperfusion.
2.2 expression of inflammatory factor mRNA
(1) The RNA extraction method comprises the following steps:
1) 20mg to 50mg of intestinal tissue was placed in 1.5ml RNase-free EP tube, and 500. Mu.l TRIZOL lysate was added thereto for homogenization.
2) 100 mu l of chloroform is added into the homogenate, the homogenate is shaken for 15 to 20 times and kept stand for 1 to 2 minutes at room temperature.
3) The upper aqueous phase was aspirated into a fresh RNase-free 1.5ml EP tube by centrifugation at 12,000rpm at 4℃for 15 min.
4) Adding isopropyl alcohol with the same volume, shaking for 15-20 times, and standing for 10min at room temperature.
5) Centrifugation at 12,000rpm at 4℃for 10min, removal of supernatant, addition of 1ml 80% ethanol (prepared with DEPC water), shaking and shaking, and centrifugation at 7500rpm at 4℃for 5min.
6) The supernatant is discarded, centrifuged at 7500rpm and 4 ℃ for 1min, the residual ethanol is sucked off, the mixture is left at room temperature for 5min to 10min, and 50 mu l to 100 mu l of DEPC water is added for resuspension. The RNA product is put in a refrigerator at the temperature of minus 80 ℃ for standby.
(2) The extracted RNA is subjected to reverse transcription reaction:
1) Mu.l of the extracted RNA was added to 6. Mu.l of nucleic-frewater and denatured at 65℃for 5min on a PCR amplification unit.
2) Immediately after denaturation, the mixture was taken out and cooled on ice, and then, 2. Mu.l of 5 Xreaction buffer, 0.5. Mu.l of RT Enzyme mix and 0.5. Mu.l of Prime mix were added to the reaction system, followed by slight shaking and centrifugation for 3s to 5s.
3) The reaction system was placed on a PCR amplification apparatus, reverse transcribed at 37℃for 15min and inactivated at 98℃for 5min.
4) After completion of the reaction, 190. Mu.l of sterile water was added to obtain a cDNA solution.
(3) Real-time PCR reaction:
1) 6. Mu.l SYBR Green, 1. Mu.l inflammatory factor primer and 5. Mu.l cDNA solution were added to the reaction system and placed in a fluorescent quantitative PCR instrument.
2) Real-time PCR reaction conditions: and (3) Cycling: stage is 15s at 95 ℃; 1min at 60 ℃ for 40 cycles; melt stage:95 ℃ for 15s;60 ℃ for 1min;95 ℃ for 30s; and at 60℃for 15s.
2.3 experimental results
The experimental results are shown in FIG. 3, and the data in FIG. 3 show that the expression levels of inflammatory factors TNF-alpha, IL-1 beta, IL-6, iNOS, CXCL-1, CXCL-2, CCL-3 and CCL-5mRNA in the intestinal tissues of the PSA group are obviously reduced compared with those of the I/R (p is less than 0.05), and the experimental results show that petroselinic acid inhibits the expression of inflammatory factors in the intestinal ischemia-reperfusion intestinal tissues of the mice.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (11)
1. Use of petroselinic acid as sole active ingredient for the preparation of a medicament for the prevention and/or treatment of intestinal ischemia reperfusion injury.
2. The use according to claim 1, wherein the medicament comprises petroselinic acid and pharmaceutically acceptable excipients.
3. Use according to claim 1 or 2, characterized in that the petroselinic acid content of the medicament is > 99% by weight.
4. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is a tablet.
5. The use according to claim 4, wherein the tablet is a coated tablet.
6. The use according to claim 1 or 2, wherein the medicament is in the form of a capsule.
7. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is an oral liquid.
8. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is an oral granule.
9. The use according to claim 1 or 2, wherein the pharmaceutical dosage form is an oral powder.
10. The use according to claim 1 or 2, wherein the medicament is in the form of an injection.
11. The use according to claim 10, wherein the injection is a lyophilized powder for injection.
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Citations (4)
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|---|---|---|---|---|
| EP0888773A1 (en) * | 1997-07-05 | 1999-01-07 | Societe Des Produits Nestle S.A. | Use of petroselinic acid for the treatment of inflammations of superficial tissues |
| EP1013178A1 (en) * | 1998-12-22 | 2000-06-28 | Loders Croklaan B.V. | Petroselinic acid and its use in food, fat compositions containing petroselinic acid |
| WO2009000149A1 (en) * | 2007-06-21 | 2008-12-31 | Tianjin Tasly Pharmaceutical Co. Ltd. | Use of notoginsenoside r1 in the preparation of the medicament for treating hepatic injuries |
| WO2017222912A1 (en) * | 2016-06-20 | 2017-12-28 | Vivacelle Bio, Inc. | Compositions and methods for reducing reperfusion injury |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6919088B2 (en) * | 2002-06-05 | 2005-07-19 | Rolland F. Hebert | Water-soluble stable salts of petroselinic acid |
| FR3005418B1 (en) * | 2013-05-07 | 2015-05-22 | Nestec Sa | PETROSELINIC ACID OR ASSOCIATED ASSETS COMPRISING AT LEAST PETROSELINIC ACID TO PROMOTE WEIGHT LOSS AND / OR WEIGHT RETENTION |
| FR3005409B1 (en) * | 2013-05-07 | 2016-08-19 | Oreal | USE OF PETROSELINIC ACID TO COMBAT AESTHETIC DISORDERS OF SILHOUETTE |
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2021
- 2021-03-17 CN CN202110284764.8A patent/CN115105492B/en active Active
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|---|---|---|---|---|
| EP0888773A1 (en) * | 1997-07-05 | 1999-01-07 | Societe Des Produits Nestle S.A. | Use of petroselinic acid for the treatment of inflammations of superficial tissues |
| EP1013178A1 (en) * | 1998-12-22 | 2000-06-28 | Loders Croklaan B.V. | Petroselinic acid and its use in food, fat compositions containing petroselinic acid |
| US6365175B1 (en) * | 1998-12-22 | 2002-04-02 | Unilever Patent Holdings | Petroselinic acid and its use in food |
| WO2009000149A1 (en) * | 2007-06-21 | 2008-12-31 | Tianjin Tasly Pharmaceutical Co. Ltd. | Use of notoginsenoside r1 in the preparation of the medicament for treating hepatic injuries |
| WO2017222912A1 (en) * | 2016-06-20 | 2017-12-28 | Vivacelle Bio, Inc. | Compositions and methods for reducing reperfusion injury |
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