CN101485648A - Application of 3,4-dihydroxy-phenyl-lactic acid in preparing medicament for treating microcirculatory disturbance - Google Patents
Application of 3,4-dihydroxy-phenyl-lactic acid in preparing medicament for treating microcirculatory disturbance Download PDFInfo
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Abstract
The invention discloses application of 3,4-dihydroxyl-phenyl lactic acid in preparing a medicine treating microcirculatory disturbance. Salvia Miltrorrhiza (SM) is contained in a great number of the prior Chinese medicines used to treat blood vessel diseases, and 3,4-dihydroxyl-phenyl lactic acid(DLA) is one of main active ingredients in the Salvia Miltrorrhiza. The microcirculatory disturbance is mesentery microcirculatory disturbance and is caused by ischemia/reperfusion. The invention also discloses application of the 3,4-dihydroxyl-phenyl lactic acid in preparing a medicine for preventing adhesion of white blood cells caused by the microcirculatory disturbance, a medicine for restoring adhesional white blood cells caused by the microcirculatory disturbance, a medicine for inhibiting exudation of albumin from small veins caused by the microcirculatory disturbance, a medicine for inhibiting degranulation of mast cells caused by the microcirculatory disturbance, a medicine for inhibiting expressions of adersional molecules CD11b and CD18 on neutrophilic granulocytes caused by the microcirculatory disturbance and a medicine for inhibiting generation of peroxides caused by the microcirculatory disturbance.
Description
Technical field
The present invention relates to field of medicaments, relate in particular to 3, the application of 4-dihydroxy-phenyl-lactic acid in the medicine of the microcirculation disturbance that preparation treatment ischemia/reperfusion causes.
Background technology
Ischemia/reperfusion (I/R) causes a large amount of microcirculatory injury reactions, as increasing the oxygen-derived free radicals product that produces from epithelial cell, L-on the leukocyte increasing selects the E-on plain and the endotheliocyte to select plain expression, cause leukocytic fluctuation, the expression of CD11b/CD18 on the leukocyte increasing and the ICAIU on the endotheliocyte (ICAM-1).These molecular energies make thin vein (postcapillary venules) behind the leukocyte adhesion blood capillary, thereby the release that causes oxygen-derived free radicals causes the damage of blood vessel endothelium.In addition, thin vein also discharges response I/R with mast cell degranulation and vaso-active substance behind the blood capillary, and it involves leukocyte increasing and adheres to thin vein and albumin outside leakage behind the blood capillary.The release and the mast cell degranulation of the perfusion leukocytic adhesion in back again, oxygen-derived free radicals are considered to closely related with the process of microvascular lesions.
It is reported, carry out adhesion and the inductive oxygen-derived free radicals of I/R-that pretreatment can suppress leukocyte thin vein behind blood capillary that I/R causes with the antibody of antagonism adhesion molecule and generate.Also have report, carry out pretreatment with antioxidant or mast cell degranulation inhibitor and can weaken that the oxygen-derived free radicals that I/R-promotes generates, the adhesion and the mast cell degranulation of leukocyte thin vein behind blood capillary.Yet that does not see whether relevant antioxidant can recover that I/R causes adheres to leukocytic report on the thin vein wall.
(Salvia miltiorrhiza SM) is used to treat various angiopathys in China, Korea S, Japan and other Asian countries to Radix Salviae Miltiorrhizae.
The microcirculation disturbance that Radix Salviae Miltiorrhizae causes ischemia-reperfusion has the effect of many target treatments: Radix Salviae Miltiorrhizae is used for the treatment of the cardiovascular and cerebrovascular vessel obstacle disease clinically.At microcirculation disturbance the back Radix Salviae Miltiorrhizae having taken place has still had therapeutical effect preferably.In order to observe the therapeutical effect of the microcirculation disturbance that Radix Salviae Miltiorrhizae causes ischemia-reperfusion, existing research begins to give continuously Radix Salviae Miltiorrhizae afterwards at ischemia-reperfusion 10 minutes (being after microcirculation disturbance takes place).Ischemia-reperfusion is in the time of 10 minutes, and microcirculation disturbance obviously takes place, and blood vessel wall DHR fluorescence intensity strengthens, leukocyte adhesion occurs in blood vessel wall, albumin outside leakage etc.When superoxide dismutase SOD gave before ischemia-reperfusion, can prevent microcirculation disturbance, but when ischemia-reperfusion began to give SOD again in the time of 10 minutes, fail to remove peroxide effectively, failing to dissociate has sticked to leukocyte on the thin vein endotheliocyte, has failed to suppress mast cell degranulation, has also failed to safeguard vascular permeability.And Radix Salviae Miltiorrhizae is when ischemia-reperfusion gave in the time of 10 minutes, can remove the leukocyte that sticks on the thin vein, removes peroxide, safeguards vascular permeability, suppress mast cell degranulation.As seen, Radix Salviae Miltiorrhizae has the preventive effect identical with SOD when preventative giving.After microcirculation disturbance took place, Radix Salviae Miltiorrhizae had the multiple peroxide of the not available removing of SOD, dissociate with the adherent leukocyte of blood vessel, suppress effects such as mast cell degranulation.The microcirculation disturbance that SOD prevents ischemia-reperfusion to cause by the disproportionation superoxide anion, its action target spot, the effect link single.After microcirculation disturbance exceeded this link, the effect of SOD just was difficult to show.And the target spot of Radix Salviae Miltiorrhizae microcirculation improvement obstacle is more, even also can bring into play therapeutical effect after microcirculation disturbance takes place.
Chemical compound main in the Radix Salviae Miltiorrhizae water extract comprises 3, and 4-dihydroxy-phenyl-lactic acid (DLA, Fig. 1), protocatechualdehyde (PAl), protocatechuic acid (PA), caffeic acid (CA, single phenolic acid), salvianolic acid A-G, salvianolic acid (lithospermate acid, LsA, polyphenol acid).DLA in them is the chemical compound of confirming, it also constitutes the part of salvianolic acid A-G and salvianolic acid (LsA, polyphenol acid) structure.It is reported that DLA and caffeic acid detect in the experimenter's of oral water solublity total salvianolic acid blood plasma.
In the past decade, DLA and the biological agent that comprises the chemical compound of DLA have attracted many concerns, and the spectrogram of the pharmaceutically active of these reagent is also revealed comes out.Experimental result proves in the body, and the organ injury that I/R causes can and comprise the chemical compound of DLA from DLA and is improved.As, the water soluble compound of SM (WSC) it is reported can increase survival rate, reduce the ratio that blocks size and left ventricle size, finds that LsB can reduce the myocardial damage of cleft lip blood heart, observes SalA and SalB and can protect brain to avoid damage.Observe pills for curing heart disease (CP, its main component is DLA and SalB) and can improve hepatic injury, and LSB and MLB it is reported and can protect lung to avoid damage.
In addition, find that also DLA can protect mitochondrial membrane to avoid damage and lipid peroxidation (LPO) that I/R causes.On the other hand, also carried out many in vitro studies, to the mechanism by the protective effect of the organ of I/R damage, and a large amount of evidence has confirmed the antioxidant activity of these chemical compounds with the chemical compound that discloses DLA and comprise DLA.As, it is reported that DLA can remove the superoxide anion (O by the reaction system generation of xanthine and xanthine oxidase
2-), found that SalA can be at LPO and the removing hydroxyl (OH) of vitro inhibition by the brain of I/R generation, find that SalB can potential removing 1.1-biphenyl-2 picryl phenylhydrazine (DPPH), suppress LPO and the active chalcogen of removing in primary rat hepatocyte and liver spider cell (HSCs) (ROS), the ROS that suppresses to be caused by 4 amyloid A is at PCL
2Generation in the cell is at external removing hydrogen peroxide (H
2O
2), and the ROS product that neoplasm necrosis alpha factor (TNF-α) causes in the inhibition human aortic smooth muscle cell (HASMCs) and the activity of nadph oxidase, LsB it is reported and can remove peroxynitrite (ONOO
-).
In addition, in vitro study proves that the WSC of the SM that comprises DLA can suppress the expression of blood vessel adhesion molecule.Next be the relevant example of many announcements: the water solubility extract of SM (WSE) can suppress the expression by adhesion molecule ICAM-1 on the inductive human umbilical vein's endotheliocyte of TNF-α (HUVECs) and vascular cell adhesion molecule 1 (VCAM-1), and suppress the inductive nuclear factor κ of TNF-α B (nuclear factor kappa B, NF-κ B) from Cytoplasm to nuclear migration, and reduce glutathion (GSH) in the endotheliocyte.
PAl among the SM, SalB and WSE suppress the expression of the VCAM-1 of TNF-α-inductive ICAM-1 and endotheliocyte, VCAM-1 among PAl inhibition ICAM-1 and VCAM-1 and the HUVECs and the mRNA of ICAM-1 express, and it is active by the combination of TNF-α inductive NF-κ B and activator protein-1 (AP-1) DNA in the mode of dose dependent, SalB weakens the expression of VCAM-1 and ICAM-1, suppress the activity in the plain expression of NF-κ B E-selection in the endotheliocyte that causes by TNF-α, CP suppresses TNF-α with VCAM-1 among the inductive HUVECs of the mode of dose dependent and the expression of ICAM-1, suppresses the synthetic and cell proliferation of DNA in platelet derived growth factor BB (platelet-derivedgrowth factor BB) the inductive vascular smooth muscle cell (VSMCs).
Previous reported DLA can suppress endotaxin induction neutrophil adhesion molecule CD11b/CD18 expression and in the adhesion of leukocyte to rat mesentery thin vein wall.In addition, the Radix Salviae Miltiorrhizae Injection that comprises the DLA chemical compound is proved to be and can suppresses the oxygen superoxide that LPS-causes and generate from rat mesentery thin vein wall.For example, it is reported that DLA can remove the superoxide anion (O that is produced by xanthine and xanthine oxidase reaction system
2-), SalA removes OH, and SalB removes hydrogen peroxide and suppresses the activity of nadph oxidase, and LsB removes ONOO
-Water solubility extract that it should be noted that all SM has common structure DLA, and its two oh groups are considered to and elimination activity chalcogen (reactive oxygen species) and ONOO
-Relevant.Yet not clear DLA removes the potentiality of the peroxide that response I/R generates in the body, does not know also whether DLA can work to the peroxide that is caused by I/R that has occurred.
Summary of the invention
An object of the present invention is to provide 3, the application of 4-dihydroxy-phenyl-lactic acid in the medicine of preparation treatment microcirculation disturbance.
Wherein said microcirculation disturbance is the mesentery microcirculation disturbance.
Described microcirculation disturbance is caused by ischemia/reperfusion.
Another object of the present invention provides 3, and 4-dihydroxy-phenyl-lactic acid prevents application in the medicine of the leukocyte adhesion that microcirculation disturbance causes in preparation.
A further object of the invention provides 3, the application of 4-dihydroxy-phenyl-lactic acid in the caused adherent leukocytic medicine of preparation recovery microcirculation disturbance.
A further object of the invention provides 3, and 4-dihydroxy-phenyl-lactic acid suppresses the application of albumin from the medicine that thin vein oozes out that microcirculation disturbance causes in preparation.
A further object of the invention provides 3, the application of 4-dihydroxy-phenyl-lactic acid in the medicine of the mast cell degranulation that preparation inhibition microcirculation disturbance causes.
A further object of the invention provides 3, and 4-dihydroxy-phenyl-lactic acid suppresses application in adhesion molecule CD11b and CD18 express on the neutrophilic granulocyte that microcirculation disturbance causes the medicine in preparation.
A further object of the invention provides 3, the application in the medicine that the peroxide that 4-dihydroxy-phenyl-lactic acid causes at preparation inhibition microcirculation disturbance generates.
In order to reach the foregoing invention purpose, the experimental program that the present invention adopts is as follows, under pentobarbital sodium anesthesia, observe the mesentery microcirculation of the ileocecus of male West (Wistar) rat by live body inverted microscope, the effect of the rat mesentery microcirculatory injury that research DLA causes ischemia/reperfusion (I/R) with CCD or SIT photographic system.
Mesenteric artery and vein ligation were unclamped and are poured into after 10 minutes.Measurement ischemia preceding ten minutes inject or do not inject incessantly the thin vein of DLA (5mg/kg/hr) or superoxide dismutase (SOD12000units/kg/hr) diameter, adhere to the oozing out of leukocytic amount, Fluorescein isothiocyanate (FITC) tagged albumin, dihydro rhodamine 123 (dihydrorhodamine, fluorescence DHR) and mastocyte take off granule.In another group experiment, injected DLA or SOD in back ten minutes incessantly in perfusion beginning again.Measure the expression of neutrophil adhesion molecule CD11b and CD18 by experiment in vitro.
The result shows generation, leukocyte albuminous oozing out in the rat mesentery that adhesion, mast cell degranulation and the I/R of thin vein wall cause of adopting DLA pretreatment obviously minimizing peroxide on the thin vein wall.The DLA post processing improves the rat mesentery disorder that I/R causes, and the generation of peroxide on the thin vein wall and albuminous leaching rate are remained unchanged by the injection of DLA, also recover attached to the leukocyte on the thin vein wall, be not subjected to the influence of DLA post processing except mast cell degranulation by DLA.
The fluorescence intensity of CD11b and CD18 is passed through H
2O
2Stimulation obviously increase, and this increase handled significantly in the concentration dependent mode by DLA and weakens, improving the ability that DLA suppresses leukocyte adhesion may the probability relevant with the potentiality of its inhibition adhesion molecule expression.For the parameter that great majority are measured, the abated effect that DLA plays is simulated by SOD, means that the rat mesentery microcirculation disturbance that the antioxidant activity of DLA causes I/R has potential multiple improvement effect.
Among the present invention, observe I/R and cause a series of malfunction of thin vein behind the blood capillary, as leucocyte adherence, peroxide the generation of thin vein tube wall, albumin from the thin vein tube wall ooze out with body in mast cell degranulation.More outstanding, the inductive phenomenon of the I/R of all observations, can handle by DLA and obviously reduce or complete obiteration, no matter this processing is implemented after before ischemia still being, the situation of exception is can only be passed through to adopt the DLA pretreatment to suppress by the increase of the inductive mast cell degranulation of I/R.
In addition, in vitro study shows and uses H
2O
2Stimulation can strengthen the fluorescence intensity of the CD11b/CD18 of neutrophilic granulocyte, and this enhancing can obviously be weakened by the DLA processing.
This proves vascular endothelial cell response I/R well, generates O by xanthine and xanthine oxidase explosively
2-, because reaction xanthine and the xanthine oxidase of SOD all change into H
2O
2Gained H
2O
2Perhaps be converted into H by catalase
2O, perhaps and O
2 -Generate hydroxy radical through the Haber-Weiss reaction.Nitric oxide and superoxide radical rapid-action result form peroxide nitroso-group (peroxynitrite), a kind of Cytotoxic medium important in response to oxidative stress.
In addition, adhere to the release that the leukocyte of thin vein behind the blood capillary causes oxygen-derived free radicals.Hydroxy radical (OH) and peroxide nitroso-group (ONOO
-) bring out the damage of lipid peroxidation and DNA, thereby cause the damage of epithelial cell and basement membrane.Therefore, eliminating peroxide is the important countermeasure of improving the microcirculation disturbance that is caused by I/R.Increasing experiment in vitro evidence has proved the non-oxidizability potentiality of SM and derivant thereof.
Result of study proof DLA of the present invention can remove the interior mesentery thin vein wall of rat body stimulates the ability of the peroxide generation that causes by I/R, and shows, is not only administration in advance, and administration after the test, and DLA can bring into play its scavenging action in this system.
It is the key results that is stimulated by I/R that leukocyte recovers, and it is from the release of proinflammatory medium and the activation by the NF-κ B enhancing to the expression of adhesion molecule.Adhesion molecule CD11b/CD18 on the leukocyte causes leukocyte adhesion to epithelial cell in conjunction with they ligand i CAM-1 on endothelium, causes producing peroxide and protease, therefore, and damage epithelial cell and basement membrane.
Therefore, microcirculation disturbance can improve from the weakening of leukocyte adhesion, and wherein the prevention or the adherent leukocytic recovery that weaken by leukocyte adhesion of leukocyte adhesion realize.
Dynamic observe in the body of the present invention and disclosed the DLA pretreatment and can suppress by of the adhesion of the inductive leukocyte of I/R to rat mesentery thin vein tube wall, its effect to adopt the SOD pretreatment similar.In addition, experiment in vitro proof DLA is to by H
2O
2The expression of CD11b/CD18 on the leukocyte that stimulates is inhibited.These results can weaken by the adhesion of the inductive leukocyte of I/R to rat mesentery thin vein tube wall in conjunction with the presentation of results DLA that has announced before, and it is undertaken by adhesion molecule CD11b/CD18 on the inhibition leukocyte and the expression of the ICAM-1 on the epithelial tissue.A meaningful discovery of this research is, if I/R administration when leukocytic adhesion has taken place after ten minutes, DLA can promote to adhere to leukocytic recovery.
Above-mentioned result of the test and the SOD performance under experiment situation of the same race forms a sharp contrast, and SOD can prevent that leukocyte from further adhering to, but can not recover to have adhered to the leukocyte of thin vein tube wall.
The granule that takes off of mastocyte is one of particular event with the I/R generation, and it causes a series of consequence, as the release of proinflammatory medium, and the permeability of thin vein and blood capillary and albuminous increase of oozing out.Another of this research has the result to be, the taking off granule and can weaken of the mastocyte that localizes around the blood vessel that causes by I/R by DLA or SOD pretreatment, and post processing can not weaken.Mast cell degranulation can excite by the bonded multiple stimulation that comprises oxidative stress and IgE.
DLA and SOD pretreatment weaken the similarity explanation of mast cell degranulation, and the antioxidant activity of DLA is the reason that prevents mast cell degranulation in this case.The post processing of DLA or SOD does not have influence to the rat mesentery mast cell degranulation that is caused by I/R, illustrate that mast cell degranulation triggers before DLA or the SOD giving, and this process is irreversible, so this process Once you begin, be can not be interferential by using antioxidant.
Select albuminous oozing out as an index that is stimulated the blood vessel injury that causes by I/R in the research of the present invention for use, it is the result of oxidative stress, and increases the weight of by leukocyte adhesion and mast cell degranulation subsequently.Presentation of results DLA of this research and SOD pretreatment can both obviously weaken the albumin that is caused by I/R and ooze out.
Adopt DLA and SOD post processing also can obviously weaken albuminous the oozing out that is caused by I/R, but different with the mode of pre-treatment, albumin oozes out constantly in DLA and SOD infusion process, shows and can prevent the further increase that albumin oozes out by this reagent.It should be noted that, oxidant responsive type fluorescent probe DHR on the thin vein wall responds the DHR fluorescence that DLA and SOD:I/R cause in a similar manner can be suppressed by using DLA or SOD pretreatment, yet under the situation of post processing, as long as DLA or SOD occur, it remains unchanged.
The similarity that the albumin of response DLA and SOD post processing oozes out between kinetics and DHR fluorescence shows that effectively observed blood vessel injury and oxidative stress are closely connected under this condition.
In a word, of the present invention studies have shown that the DLA pretreatment obviously reduce by the inductive rat mesentery peroxide of I/R generate, leukocyte is to adhesion, mast cell degranulation and the albuminous rate that spills of wall of micrangium.The DLA post processing of pouring into beginning after 10 minutes more also can improve the inductive rat mesentery obstacle by I/R, but it carries out with unique mode, injection by DLA remains unchanged the generation of peroxide and albuminous oozing out, the leukocyte that has adhered to blood vessel wall can recover by DLA, and it is different with the effect of SOD, SOD can prevent that in this case leukocyte from further adhering to, but can not recover adherent leukocyte, what make an exception is that mast cell degranulation is not subjected to the influence of DLA or SOD post processing.
Experiment in vitro shows, H
2O
2The increase of neutrophilic granulocyte CD11b that causes and the fluorescence intensity of CD18 can be handled by the DLA that concentration relies on mode and obviously weakens, and improves DLA and suppresses the leukocytic adhesion probability relevant with the potentiality of its inhibition adhesion molecule expression.DLA produces for most of test parameters attenuation effect and SOD are similar, this fact illustrates that effectively antioxygenic activity and DLA are its bases to the improvement effect of the inductive rat mesentery microcirculation disturbance of I/R to the inhibition of adhesion molecule CD11b/CD18 on the leukocyte.3,4-dihydroxy-phenyl-lactic acid can be used as the ideal medicament of the microcirculatory injury that preparation treatment ischemia/reperfusion causes.
State with other purpose, feature and advantage and can become apparent on the present invention for allowing, preferred embodiment cited below particularly, and conjunction with figs. are described in detail below.
Description of drawings
Fig. 1 is 3, the chemical structural formula of 4-dihydroxy-phenyl-lactic acid (DLA).
Fig. 2 A and Fig. 2 B represent to adopt DLA or SOD pretreatment or post processing to by leukocyte adhesion on the inductive rat mesentery thin vein of the I/R wall through the time influence.Wherein Fig. 2 A represents to adopt DLA or SOD to the pretreatment by the inductive leukocyte adhesion of I/R.The DLA+I/R:DLA pretreated group; The SOD+I/R:SOD pretreated group.Fig. 2 B represents to adopt DLA or SOD to the post processing by the inductive leukocyte adhesion of I/R.I/R+DLA:DLA post processing group; I/R+SOD:SOD post processing group; Data are meansigma methods ± SD of six rats.
*P＜0.05 pair baseline, #P＜0.05 pair I/R group,
P＜0.05 pair SOD group.
Fig. 3 A and Fig. 3 B represent on the rat mesentery thin vein wall DHR fluorescence ratio through the time change.Wherein Fig. 3 A represents to adopt DLA or the SOD pretreatment influence to thin vein tube wall DHR fluorescence ratio, DLA+I/R:DLA pretreated group; The SOD+I/R:SOD pretreated group.Fig. 3 B represents to adopt DLA or the SOD post processing influence to thin vein tube wall DHR fluorescence ratio, I/R+DLA:DLA post processing group; I/R+SOD:SOD post processing group, data are meansigma methods ± SD of six rats.
*P＜0.05 pair baseline, #P＜0.05 pair I/R group.
Fig. 4 A and Fig. 4 B represent that rat mesentery thin vein wall albumin oozes out through the time change.Wherein Fig. 4 A represents the influence of adopting DLA or SOD pretreatment that thin vein tube wall albumin is oozed out, DLA+I/R:DLA pretreated group; The SOD+I/R:SOD pretreated group.Fig. 4 B represents the influence of adopting DLA or SOD post processing that thin vein tube wall albumin is oozed out, I/R+DLA:DLA post processing group; I/R+SOD:SOD post processing group, data are meansigma methods ± SD of six rats.
*P＜0.05 pair baseline, #P＜0.05 pair I/R group.
Fig. 5 A and Fig. 5 B represent DLA and SOD in matched group, I/R group, DLA+I/R group, SOD+I/R group, I/R+DLA group and the I/R+SOD group by the influence of the mast cell degranulation of the inductive rat mesentery thin vein of I/R wall, data are meansigma methods ± SD of six rats.
*P＜0.05 pair baseline, #P＜0.05 pair I/R group.
Fig. 6 represents that DLA is to H
2O
2The influence of the expression of inductive rat mesentery adhesion molecule CD11b and CD18.Being expressed on the vertical coordinate of adhesion molecule represented with fluorescence intensity.H
2O
2: H
2O
2Group; H
2O
2+ DLA0.2:H
2O
2Add the DLA0.2mg/ml group; H
2O
2+ DLA0.5:H
2O
2Add the DLA0.5mg/ml group; H
2O
2+ DLA1.0:H
2O
2Add the DLA1.0mg/ml group.Data are meansigma methods ± SD of six rats.
*P＜0.05 pair matched group, #P＜0.05 pair LPS group.
Fig. 7 represents that DLA alleviates by the ischemia and the sketch map of the microcirculation disturbance that causes of perfusion again.I/R, expression ischemia and perfusion again; XO, the expression xanthine oxidase; CD11b/CD18, expression adhesion molecule CD11b and CD18; ICAM-1, the expression intercellular adhesion molecule-1; ⊥, expression suppresses.
The specific embodiment
The preparation of embodiment one experiment material
Animal
All researchs all are given the ratification, and the processing of all laboratory animals all is to answer the guide of zooscopy committee of university of private school charging no tuition to carry out according to celebrating.The male West rat of body weight 200g-250g (Saitama beautiful county laboratory animal provide company limited, the Saitama County, Japan) fasting 12 hours before test, but allow freely to drink water.
Reagent
DLA (chemical formula is seen Fig. 1) is available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute (BeiJing, China).Superoxide dismutase (SOD), toluidine blue, the fluorescent labeling bovine serum albumin available from sigma chemistry reagent company (Sigma chemical.co) (Missour, USA), dihydro rhodamine 123 (DHR) available from molecular probe (Molecular Probes) (Oregon, USA).Mouse anti CD11b monoclonal antibody (FITC-conjugated mouse anti-rat CD11b monoclonalantibody) in conjunction with FITC, mouse anti CD18 monoclonal antibody (FITC-conjugated mouseanti-rat CD18 monoclonal antibody) in conjunction with FITC, in conjunction with mice IgA, the κ of FITC and in conjunction with the mice IgG1 of FITC, κ is available from BD Biosciences Pharmingen (San Diego, CA), hemolysin available from BD Biosciences Immunocytometer Systems (San Jose, CA).
Single-poly-dissolve medium is available from Dainippon Pharmaceutical Co., Ltd (Dainippon PharmaceuticalCorporation) (Osaka, Japan).(Logan, UT), other all chemical reagent all adopts commercially available highest ranking available from Hyclone for RPMI 1640 and hyclone.
The preparation of ischemia/reperfusion rat
Injection pentobarbital sodium (30mg/kg body weight) anesthesia in the rat peritoneum.Right external jugular vein polyethylene catheter intubate.Open abdominal part by the midline incision that 20-30mm is long.Beat easily the ileocecus of mesentery tail end 20cm, be fixed on the transparent plastic platform that is exclusively used in rat.
By on mesentery, coming holding temperature and humidity with KShi buffer (Krebs-Ringer bicarbonatebuffered solution) continuous surface perfusion at 37 ℃.By the mesenteric mesaraic microcirculatory hemodynamics of inverted microscope (Diaphot TMD-2S, Nikon, Tokyo) observation.Mesentery is by 12V100W DC stabilization light source transillumination.The video camera that is placed on the microscope is transferred to image on the color video monitor, and image is by video tape videocorder record.Video time-time date generator is incident upon time and chronometer on the monitor.Select for use diameter range between 25 to 40 μ m and length experimentize greater than the independent unbranched thin vein of 200 μ m.
After 10 minutes bases of the basic hematodinamics of rat mesentery microvasculature were observed, supply branch by the snare ligation simultaneously anterior mesenteric artery made by polyethylene tube and corresponding thin vein 10 minutes discharged blood flow subsequently again and cause I/R.Because the RBC speed in whole ischemia process medium vessels is not 0, so may have the indirect perfusion to viewing area.Therefore,, tremulous pulse and vein are all carried out ligation, to stop blood supply and to induce thin vein hyperemia in order to promote ischemia.Previously studies show that 10 minutes ischemia then pours into again is enough to induce the mesentery microcirculation disturbance, and the intestinal tissue damage is minimum.The control rats of not carrying out I/R in contrast.
Embodiment two experimental programs
In I/R group (I/R), began in preceding 10 minutes constantly to inject saline solution (6ml/kg/hr) at ischemia, and remain to the observation end always through the jugular vein conduit.Control animals (Sham) is accepted and the identical processing of I/R group.
In DLA pretreated group (DLA+I/R) or SOD pretreated group (SOD+I/R), began in preceding 10 minutes constantly to inject DLA (5mg/kg/hr) or SOD (12000u/kg/hr) at ischemia, and remain to the observation end always through jugular vein.
In DLA post processing group (I/R+DLA) or SOD post processing group (I/R+SOD), began in back 10 minutes constantly to inject DLA (5mg/kg/hr) or SOD (12000u/kg/hr) through jugular vein pouring into again, and remain to observe always and finish.
The mensuration of embodiment three experiment parameters
The measurement of thin vein parameter
The image of thin vein obtains by charge-coupled device (CCD) colour phhotograpy system (CC-090, Flovel, Tokyo).The diameter of mesentery thin vein is by video measuring chi (video measuringgauge) (IV-560; Hoei, the Tokyo) measure.In the replayed section of video-tape image, the leukocytic quantity that determined off-line adheres to and moves.From the video recording image of playback, judge, will surpass 30 seconds leukocyte attached to same position and be defined as adhering to adhesion leukocyte on the thin vein wall.
Along the thin vein of from the video-tape image of record, selecting at random (diameter 25-40 μ m, length 200 μ m) adherent leukocytic quantity is counted, and be expressed as the quantity of every 200um thin vein length.The leukocytic amount that is expressed as around each visual field of thin vein that moves.
For quantitative albumin passes oozing out of mesenteric mesaraic thin vein, as described before, at the bovine serum albumin of preceding 10 minutes clockwise animal subject intravenous injection 50mg/kg FITC labellings of each experiment.Fluorescence intensity (excitation wavelength 420 to 490nm, emission wavelength is at 520nm) is by the photographing unit (C-2400-08 that adopts silicon intensified target; Hamamatsu Photonics, Hamamatsu, Japan) measure.The albuminous fluorescence intensity of FITC-on the thin vein wall is monitored and is measured with image processor.
In another group experiment, as previously described, with the fluorescent probe dihydro rhodamine 123 (DHR of oxidant sensitivity; Molecular Probes, Ore.) joins the oxidant stress reaction of judging in the mesenteric mesaraic perfusate (10 μ mol/L) in the thin vein wall.Observe the DHR fluorescence intensity on the thin vein wall, and measure by image processor (13).The fluorescence intensity of thin vein wall and outdoor matter (extravenular interstitium) is measured by Image-Pro Plus 5.0 softwares.As baseline value, the difference of the fluorescence intensity between the thin vein tube wall by a time point and outdoor the matter and the ratio of baseline value are calculated the ratio of DHR fluorescence intensity with the difference of the fluorescence intensity between thin vein wall before the I/R and outdoor the matter.
Mastocyte by confirming in mesentery topical application 0.1% toluidine blue vital staining (vital staining) method after the I/R in 30 minutes.The quantity of not degranulated mastocyte and degranulated mastocyte is measured from the CCD video image, and the quantity of calculating degranulated mastocyte accounts for the ratio of the quantity of whole test and appraisal mastocytes, and is expressed as the ratio of degranulated mastocyte.
The mensuration of the expression of adhesion molecule CD11b and CD18 on the neutrophilic granulocyte
Blood is taken from the abdominal aortic of normal rat and with anticoagulant heparin.Each experimental group is got whole part of blood plasma of 200 μ l.At matched group (n=6), sample does not add any additives, at H
2O
2Add H in group (n=6) sample
2O
2(100mM).In the DLA processed group, add H in the sample
2O
2(100mM) and DLA (0.2mg/ml, 0.5mg/ml or1.0mg/ml).
All preparations kept two hours at 37 ℃, adopt haemolysis usually to make erythrocytolysis subsequently with hatching under mice isoreagent (the 5 μ g/ml) room temperature of the antibody of anti--CD18 (5 μ g/ml) of the anti--CD11b (5 μ g/ml) of FITC labelling or FITC labelling or corresponding FITC labelling 20 minutes, and according to manufacturer's explanation.Cell washes twice with PBS, and average fluorescent strength adopts flow cytometer (FACS Calibur; BD company, the U.S.) test and assess.Neutrophilic granulocyte is according to report (Sun K, Wang CS, Guo J, Liu YY, Wang F, Liu LY, He JG, Fan JY, and Han JY.Effect ofPanax notoginseng saponins on lipopolysaccharide-induced adhesion ofleukocytes in rat mesenteric venules.Clin Hemorheol Microcirc) describe preceding-/characteristic of side-scattering is classified, and 5,000 neutrophilic granulocytes of each sample determination.
Embodiment four statistical analysiss
Check (Fishe ' s post hoctest) that data are analyzed by one factor analysis of variance (ANOVA) and Fischer afterwards.All values represents that with average ± standard error establishing P＜0.05 has statistical significance.
The analysis of embodiment five experimental results
The thin vein vary in diameter
In whole experiment, the I/R group is not observed the tangible change of thin vein diameter, and no matter with DLA or SOD pretreatment or post processing, situation all remains unchanged.(data are not write out)
Adhere to the change of the quantity of leucocyte of thin vein wall
Fig. 2 A and Fig. 2 B represent with DLA or SOD carry out pretreatment or post processing to the inductive leukocyte of I/R to the adherent influence of rat mesentery thin vein wall.Adherent quantity of leucocyte increases sharply when perfusion begins again, and quantity further increase in time, up to pouring into 30 minutes again.Can reduce the inductive adhesion leukocyte by I/R with the DLA pretreatment, it obviously reduces when perfusion begins again and further reduces in time in whole refilling process.Can reduce the inductive adhesion quantity of leucocyte by I/R in a similar fashion with the SOD pretreatment, but reduce to littler scope, it reduces obviously (Fig. 2 A) when pouring into 10 minutes again.
With the further increase of SOD post processing inhibition leukocyte adhesion, but invalid to adherent leukocyte.On the contrary, significantly reduce adherent leukocytic amount, show the leukocytic separation (Fig. 2 B) that sticks on the thin vein tube wall with the DLA post processing.
The change of thin vein tube wall DHR fluorescence intensity
Thin vein tube wall DHR fluorescence intensity ratio is along with the change of time course is shown in Fig. 3 A and Fig. 3 B.At matched group, thin vein tube wall DHR fluorescence intensity ratio does not significantly change in whole observation process.In the I/R group, thin vein tube wall DHR fluorescence intensity finishes by obviously linear increasing up to pouring into again.Obviously weaken inductive DHR fluorescence enhancing with the DLA pre-treatment by I/R.Also obviously weaken increase by the inductive thin vein tube wall of I/R DHR fluorescence ratio with the SOD pre-treatment, comparing with the I/R group has significantly different (Fig. 3 A) when pouring into 10 and 30 minutes again.
Suppressing the inductive DHR fluorescence of I/R ratio in a similar fashion with DLA and SOD post processing increases, thin vein wall DHR fluorescence intensity almost keeps constant during DLA or SOD infusion, and compare with I/R group and when pouring into 30 minutes again, to reach significant difference, it mainly organizes the increase of DHR fluorescence intensity owing to I/R, shown in Fig. 3 B.
The change that albumin oozes out from the thin vein tube wall
Rat mesentery thin vein tube wall albuminous oozed out in Fig. 4 A and Fig. 4 B in time quantification and represents.Before I/R, in all groups, all do not find albuminous oozing out, and this situation remains in the whole observation of matched group.In the I/R group, in case perfusion beginning again, then the albumin that oozes out from thin vein increases immediately, and when pouring into 30 minutes again, oozes out further increase.Obviously weaken by of the increase of oozing out (Fig. 4 A) of the inductive albumin of I/R with DLA or SOD pretreatment from the thin vein tube wall.
Also the inductive albumin of I/R is produced tangible abated effect from oozing out of thin vein tube wall with DLA or SOD post processing, shown in Fig. 4 B.Experiment shows that as long as beginning infusion DLA or SOD, then thin vein tube wall albumin oozes out and remains unchanged, and shows that adopting DLA or SOD to handle can prevent the albuminous further increase of oozing out.
Mast cell degranulation around the blood capillary
I/R causes that mast cell degranulation obviously increases (Fig. 5 A and Fig. 5 B).Quantitative analysis shows by DLA and SOD pretreatment has tangible abated effect to the mast cell degranulation increase that I/R brings out, but invalid by DLA and SOD post processing.
Adhesion molecule CD11b on the neutrophilic granulocyte and the fluorescence intensity of CD18
Carry out in vitro study and measure adhesion molecule CD11b on the neutrophilic granulocyte and the fluorescence intensity of CD18, the result is illustrated among Fig. 6.Compare H with matched group
2O
2Stimulate the fluorescence intensity that obviously strengthens CD11b and CD18, and this enhancing can obviously be weakened by mode with concentration dependent with DLA (0.2mg/ml, 0.5mg/ml or 1.0mg/ml) processing.
DLA alleviate by ischemia and again the microcirculation disturbance that causes of perfusion sketch map as shown in Figure 7, I/R, expression ischemia and pouring into again; XO, the expression xanthine oxidase; CD11b/CD18, expression adhesion molecule CD11b and CD18; ICAM-1, the expression intercellular adhesion molecule-1; ⊥, expression suppresses.
Though the present invention discloses as above with preferred embodiment; right its is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the present invention; when can doing a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
Claims (9)
1. 3, the application of 4-dihydroxy-phenyl-lactic acid in the medicine of preparation treatment microcirculation disturbance.
2. application according to claim 1 is characterized in that described microcirculation disturbance is the mesentery microcirculation disturbance.
3. application according to claim 1 and 2 is characterized in that described microcirculation disturbance is caused by ischemia/reperfusion.
4. 3,4-dihydroxy-phenyl-lactic acid prevents application in the medicine of the leukocyte adhesion that microcirculation disturbance causes in preparation.
5. 3,4-dihydroxy-phenyl-lactic acid recovers application in the caused adherent leukocytic medicine of microcirculation disturbance in preparation.
6. 3,4-dihydroxy-phenyl-lactic acid suppresses the application of albumin from the medicine that thin vein oozes out that microcirculation disturbance causes in preparation.
7. 3,4-dihydroxy-phenyl-lactic acid suppresses application in the medicine of the mast cell degranulation that microcirculation disturbance causes in preparation.
8. 3,4-dihydroxy-phenyl-lactic acid suppresses application in adhesion molecule CD11b and CD18 express on the neutrophilic granulocyte that microcirculation disturbance causes the medicine in preparation.
9. 3,4-dihydroxy-phenyl-lactic acid suppresses application in the medicine that peroxide that microcirculation disturbance causes generates in preparation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048818A (en) * | 2009-11-05 | 2011-05-11 | 天津天士力制药股份有限公司 | Total salvianolic acid, panax notoginseng saponins, and compatibility thereof in prevention and treatment of diseases caused by microcirculatory disturbance |
| EP2497469A1 (en) * | 2009-11-05 | 2012-09-12 | Tasly Pharmaceutical Group Co., Ltd. | Use of danshensu, notoginsenoside r1 or their combination in preparation of medicaments for preventing and treating diseases caused by microcirculation disorder |
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2008
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102048818A (en) * | 2009-11-05 | 2011-05-11 | 天津天士力制药股份有限公司 | Total salvianolic acid, panax notoginseng saponins, and compatibility thereof in prevention and treatment of diseases caused by microcirculatory disturbance |
| EP2497469A1 (en) * | 2009-11-05 | 2012-09-12 | Tasly Pharmaceutical Group Co., Ltd. | Use of danshensu, notoginsenoside r1 or their combination in preparation of medicaments for preventing and treating diseases caused by microcirculation disorder |
| JP2013510099A (en) * | 2009-11-05 | 2013-03-21 | テースリー ファーマシューティカル グループ カンパニー リミテッド | Use of danshensu, notoginsenoside R1 or a combination thereof in the preparation of a medicament for preventing and treating diseases caused by microcirculatory disturbances |
| EP2497469A4 (en) * | 2009-11-05 | 2013-08-07 | Tasly Pharmaceutical Group Co | Use of danshensu, notoginsenoside r1 or their combination in preparation of medicaments for preventing and treating diseases caused by microcirculation disorder |
| US9101640B2 (en) | 2009-11-05 | 2015-08-11 | Tasly Pharmaceutical Group Co., Ltd. | Use of Danshensu, Notoginsenoside R1 or their combination in preparation of medicaments for preventing and treating diseases caused by microcirculation disorder |
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