| ObjectiveTo explore the establish method of animal model of focal cerebral ischemia-reperfusion with phlegm-blood stasis syndrome.And then taking the successfully established animal model as the research object,the relationship between signal pathway of microRNA-155 and the mechanism of inflammatory reaction in ischemic stroke with phlegm-blood stasis syndrome was revealed by using Q-PCR,Western blot and ELISA detection techniques.Method1.Establishment and evaluation of animal model of cerebral ischemia-reperfusion with phlegm-blood stasis syndromeFifty SD male rats were randomly divided into normal group(10 rats)and high-fat group(40 rats),and they were fed with normal diet and high-fat diet for 4 weeks respectively.Then 40 rats in the high-fat group were renumbered and divided into cerebral ischemia-reperfusion model group(30 rats)and sham-operation group(10 rats).In cerebral ischemia-reperfusion model group,the right middle cerebral artery was occluded by ligating the carotid artery and inserting fishline(MCAO).In sham-operation group,the blood vessels were separated without ligation blood vessels and insertion of fishline.Zea Longa score was performed 24 hours after the completion of the model in the cerebral ischemia-reperfusion model group,the changes of physical signs were observed,the blood lipid and coagulation function were detected to evaluate the model.2.Inflammatory mechanism of ischemic stroke with phlegm-blood stasis syndromeRats in cerebral ischemia-reperfusion model group were randomly divided into atorvastatin calcium tablet group,Danlou tablet group and model group.After one week of drug intervention according to the corresponding regimen,brain tissue was taken to calculate the brain coefficient and make pathological section to observe the pathological changes in infarcted area.Blood lipid levels and coagulation function were measured again.The expression of microRNA-155,SOCS1 and SIRT1 mRNA in brain tissue was measured by Q-PCR.The expression of SOCS1,SIRT1 and NF-K B protein in brain tissue was detected by Western blot.The levels of TNF-a and IL-6 in serum were detected by ELISA.To further study the relationship between the signal pathway of microRNA-155 and the mechanism of inflammatory response in ischemic stroke with phlegm-blood stasis syndrome.Result1.Establishment of animal model of focal cerebral ischemia-reperfusion with phlegm-stasis syndromeDuring 4 weeks of hyperlipidemia modeling,the daily average diet and water intake in the high-fat group were lower than that in normal group,and the difference was significant(P<0.05).After 10 days of feeding,the weight of rats in the high-fat group were lower than that in the normal group,and the difference was significant(P<0.05).But after 20 days and 4 weeks of feeding,the weight of rats in the high-fat group was no obviously different from that in normal group(P>0.05).After 4 weeks of feeding,the rats in the high-fat group were slow in reaction,slow in action,mousefur dark yellow and lose luster.The levels of blood lipid in the high-fat group were higher than that in the normal group,and the difference were significant(P<0.05).The above results indicate that the hyperlipidemia animal model was successfully replicated.After establishing the model of focal cerebral ischemia reperfusion,the left limb hemiplegia,right facial paralysis,eyelid droop,eye crack reduction,walking was counterclockwise tail-like,even tilted to the healthy side,mental malaise,body shrinkage,reduced activity,purple tongue,and even blood stasis spots.The levels of PT in the cerebral ischemia-reperfusion model group were lower than that in the sham-operated group and normal group,and the levels of APTT were lower than that in the normal group,the difference were significant(P<0.05).But there was no obviously difference in FIB among groups(P>0.05).A week after the completion of the model,the brain coefficient of the model group was higher than that of the normal group and the sham-operated group,and the difference were significant(P<0.05).The brain cells in the model group were enlarged,deformed,blurred in structure,with the nucleus on one side,and the neurons shrinked or even died.In conclusion,the animal model of local cerebral ischemia-reperfusion with phlegm-blood stasis syndrome was successfully established.2.Effect of drug intervention(1)Blood lipid levelDrug intervention for one week,the levels of TC,TG and LDL-C in sham-operated group,model group,atorvastatin calcium tablet group and Danlou tablet group were still higher than that in normal group(P<0.05).The levels of TC in atorvastatin calcium tablet group were lower than that in model group(P<0.05),while the levels of HDL-C were higher than that in model group(P<0.05).The levels of blood lipid in Danlou tablet group were no obviously different from that in model group(P>0.05).(2)Coagulation functionDrug intervention for one week,the APTT levels in sham-operated group,model group and atorvastatin calcium tablet group were still lower than that in normal group(P<0.05).The APTT and PT levels in Danlou tablet group were higher than that in model group(P<0.05).The APTT and PT levels in atorvastatin calcium tablet group were no obviously different from that in model group(P>0.05).The FIB levels in each group were no obviously different(P>0.05).3.The relationship between microRNA-155 and inflammatory response of ischemic stroke with phlegm-blood stasis syndrome(1)Expression of inflammatory factors(TNF-?,IL-6)in rat serumThe levels of TNF-a and IL-6 in sham-operated group and model group were higher than that in normal group,the difference were significant(P<0.05).The levels of TNF-a,IL-6 in model group were higher than that in sham-operated group,the difference were significant(P<0.05).The levels of TNF-a and IL-6 in atorvastatin calcium tablet group were lower than that in model group,and the levels of TNF-? in Danlou tablet group were also lower than that in model group,the difference were significant(P<0.05).(2)Expression of NF-kappa B protein in ischemic brain tissueThe levels of NF-kappa B protein in model group were higher than that in sham-operation group and normal group,the difference were significant(P<0.05).The levels of NF-kappa B protein in Danlou tablet group were lower than that in model group,the difference was significant(P<0.05).But there was no obvious difference in the expression of NF-kappa B protein between atorvastatin calcium tablet group and normal group(P>0.05).(3)Expression of microRNA-155,SOCS1 and SIRT1 mRNA in ischemic brain tissueThe levels of microRNA-155 in sham-operated group were higher than that in normal group,while the levels of SOCS1 mRNA were lower than that in normal group,the difference were significant(P<0.05).The levels of microRNA-155 in model group were higher than that in normal group and sham-operated group,while the levels of SOCS1 and SIRT1 mRNA were lower than that in normal group and sham-operated group,the difference were significant(P<0.05).The levels of microRNA-155 in atorvastatin calcium tablet group and Danlou tablet group were lower than that in model group,while the levels of SOCS1 mRNA were higher than that in model group,the difference were significant(P<0.05).The levels of SIRT1 mRNA in atorvastatin calcium tablet group were also higher than that in model group,the difference was significant(P<0.05).(4)Expression of SOCS1 and SIRT1 proteins in ischemic brain tissueThe levels of SOCS1 and SIRT1 proteins in sham-operated group and model group were lower than that in normal group,the difference were significant(P<0.05).The levels of SIRT1 proteins in model group were lower than that in sham-operated group,the difference was significant(P<0.05).The levels of SOCS1 and SIRT1 proteins in atorvastatin calcium tablet group and Danlou tablet group were higher than that in model group,the difference were significant(P<0.05).The trend of protein change was basically consistent with that of gene change.conclusion1.The animal model of focal cerebral ischemia-reperfusion with phlegm-blood stasis syndrome can be successfully constructed in SD male rats by feeding high-fat diet combined with middle cerebral artery occlusion.2.There are obviously inflammation after cerebral ischemia-reperfusion occurs on the basis of hyperlipidemia,accompanied by increased expression of microRNA-155 and activation of NF-kappa B,as well as decreased expression of SOCS1?SIRT1 mRNA and proteins.The possible mechanism is that when microRNA-155 is activated rapidly after cerebral ischemia injury,it can target and inhibit the expression of its downstream genes SOCS1 and SIRT1 mRNA,thereby regulating the inflammatory signaling pathway mediated by NF-kappa B,promoting the production and release of inflammatory factors in vivo,and aggravating brain tissue damage.3.Inflammation is one of the biological basis of "phlegm" and "blood stasis"in Chinese medicine.Lipid-lowering drugs and Blood-activating and stasis-removing drugs can effectively reduce the levels of inflammatory factors after cerebral ischemia,and the pathway may be related to the signal pathway of microRNA-155. |
PDF Full Text Request