| ObjectiveStroke is characterized by high prevalence rate,high disability rate and high mortality rate.Ischemic stroke is one of the main types of stroke,and traditional Chinese medicine(TCM)has unique advantages in the treatment of ischemic stroke.According to previous clinical observation,it was found that bezoar,bile arisaema,Huafengdan and other prescriptions fermented by cattle bile(CB)are often used in the treatment of ischemic stroke.As one of the representative prescriptions for stroke treatment,Yaomu,which is the sovereign drug of Huafengdan has a unique fermentation method and a high content of CB.The natural formation process of bezoar is closely related to the fermentation of CB.However,there is still lacks of systematic understanding on the changes of efficacy and drug system of CB during fermentation.In this study,CB will be fermented separately by using the fermentation method of Yaomu.The study will explore the influence of fermentation on the drug composition,efficacy and related efficacy mechanism of CB by in vitro and in vivo experiments,in order to expand the clinical application of CB fermentation and provide new ideas for the clinical treatment of ischemic stroke.Methods1.This study used rat model of middle cerebral artery occlusion(MCAO).Rats were administered by gastric tube with the same dosage of Yaomu,according to the dose reported in the literature.First,we observed the weight change,neurological deficit score and cerebral infarction volume of rat to determine the effect of the Yaomu.Next,we fermented CB independently using the fermentation method of Yaomu,and proved the safety of fermented cattle bile(FCB)by acute toxicity experiment.Then MCAO rats were given different doses of FCB to observe the efficacy of FCB in the treatment of ischemic stroke and determine the best dose.2.The UHPLC-MS/MS high-throughput target quantification technology was used to make the qualitative and quantitative analysis of the main active ingredient of CB.The difference of bile acid composition before and after CB fermentation was studied.3.Based on the difference composition in bile acid before and after CB fermentation,we compared the efficacy of CB and FCB in treating ischemic stroke by rat weight changes,neurological deficit score,cerebral infarction volume and pathological feature.The contents of TNF-α and IL-1β and the expression of Iba-1 positive microglia and NLRP3 in brain tissue were examined to explore the regulation of inflammatory response on ischemic stroke before and after CB fermentation.Thereafter,an in vitro neuroinflammation model of microglia was established,the effect of CB and FCB on microglial viability was determined by CCK8.Next,nontoxic concentration to the cells were selected for detecting the contents of NO,TNF-α and IL-1β to determine the effects of inflammatory mediators and inflammatory factors before and after CB fermentation.Finally,we used WB for detecting the expression of iNOS,TNF-α and IL-1β at the best administered dose,to further compare the intervention effect of CB and FCB on microglial neuroinflammation.4.Combined with the changes in bile acid composition before and after CB fermentation,we used WB to detect TGR5-NLRP3 pathway to clarify the specific mechanism of CB on the anti-inflammatory response in ischemic stroke before and after fermentation.Results1.In this study,we used steady and mature rat model of MCAO to detect the efficacy of the Yaomu.We observed obvious weight loss,increased neurological deficit score and cerebral infarction volume(P<0.001)in the rat model.After the treatment of the Yaomu,the weight loss of rats decreased(P<0.05),the neurological deficit score and the cerebral infarction volume were significantly reduced(P<0.01);The CB was fermented separately according to the fermentation method of Yaomu,then it was administered to a group of ICR mice of half male and half female.Seven days later,both blank group and administration group were in good mental state,having normal activity,drinking and eating normally.None of the animals died within 7 days.Compared with the blank group,mice in the administration group had little change in the weight and the organ index of heart,liver,spleen,lung and kidneys.Next,we administered different doses of FCB(2 ml/kg,4 ml/kg,8 ml/kg)to MCAO rats,we found that compared with the model group,the decrease in body weight was significantly reduced in the FCB group,as well as neurological deficit scores and cerebral infarction volume were decreased with dose-dependent(P<0.05).2.A total of 41 bile acids were detected by UHPLC-MS/MS.After the pre-processing,we eventually got 28 bile acids.Among them,dehydrolithocholic acid,isolithocholic acid,7-ketolithocholic acid,12-ketolithocholic acid,ursodeoxycholic acid,7-ketodeoxycholic acid,12-dehydrocholic acid,and 3-dehydrocholic acid were significantly increased after fermentation(P<0.05),glycolithocholic acid,glycochenodeoxycholic acid,glycodeoxycholic acid,glycocholic acid,taurochenodeoxycholic acid,taurodeoxycholic acid,taurocholic acid was significantly reduced after fermentation(P<0.05).3.Administering MCAO rats with CB and FCB,72 h after reperfusion,in the MCAO group,weight loss,neurological deficit scores and cerebral infarction volume were significantly increased(P<0.001);compared with the MCAO group,the weight loss and cerebral infarction volume were decreased in CB group(P<0.05).For FCB group,there were significant improvement in weight change,neurological deficit score and cerebral infarction volume(P<0.01,P<0.05,P<0.001).Among them,the cerebral infarction volume in the FCB group was significantly reduced compared with the CB group(P<0.01).HE and Nissl staining indicated that,significant pathological changes appeared in the MCAO group,and Nissl bodies were significantly reduced(P<0.001).Compared with the MCAO group,the pathological changes were reduced in the CB group,the number of Nissl bodies increased(P<0.05).The pathological manifestations of brain tissue were significantly reduced in the FCB group.The increase in the number of Nissl bodies was even more significant(P<0.001).The number of Nissl bodies increased significantly in the FCB group compared with the CB group(P<0.001).4.The TNF-α and IL-1β levels were significantly increased in the MCAO group of rats(P<0.001).After the treatment with CB,there were no significant changes in TNF-α and IL-1β contents in rat brain tissue.After treatment with FCB,the TNF-α and IL-1β contents were significantly decreased in the rat brain tissue(P<0.001,P<0.01).Moreover,the contents of TNF-α and IL-1β in the FCB group were lower than those in the CB group(P<0.001,P<0.05).Immunofluorescence of brain tissue revealed hyperactivation of Iba-1 positive microglia in the brain ischemic region of rats in the model group,and the NLRP3 protein levels were also significantly increased(P<0.001).After treatment with CB and FCB,the expression of Iba-1 positive microglia and NLRP3 decreased(P<0.05),which were more pronounced in the FCB group(P<0.001).Next,we established a model of microglial neuroinflammation in vitro.By CCK8 detection,three non-toxic concentrations(15.63 μg/ml,31.25 μg/ml,62.5 μg/ml)were determined,then LPS-stimulated microglia was intervened with CB and FCB.The results showed that the FCB was significantly decreased contents of NO,TNF-α,IL-1β and iNOS(P<0.05).However,there were no significant changes in the CB group.Fermentation significantly reduced the contents of NO,TNF-α,IL-1β and iNOS in CB(P<0.001,P<0.01,P<0.01,P<0.01).Cell immunofluorescence showed the NLRP3 expression was increased significantly in the LPS-induced microglia(P<0.001).Both CB and FCB pretreatment reduced the expression of NLRP3(P<0.05,P<0.001).Compared with CB group,NLRP3 expression in FCB group was significantly decreased(P<0.001).5.By the detection of the TGR5-NLRP3 pathway,we found that TGR5,cAMP,NLRP3,Cleaved caspase-1 and ASC were all elevated in MCAO rats.After the treatment with CB and FCB,TGR5 and cAMP were up-regulated in the CB group(P<0.01,P<0.05),and up-regulation was more significant in the FCB group(P<0.001).For the cAMP expression,the FCB group was more significantly than the CB group(P<0.001).NLRP3 expression decreased in CB group(P<0.01),there were decreasing trend of Cleaved caspase-1 and ASC protein expression but with no statistical difference in CB group.The expression of the NLRP3,Cleaved caspase-1 and ASC protein were all decreased in the FCB group with significant difference(P<0.001,P<0.01,P<0.01).Fermentation reduced the expression of NLRP3 inflammasome in CB(P<0.05).In the neuroinflammation model of microglia,both CB and FCB can reverse the decrease in TGR5 and cAMP caused by LPS-induced cells.FCB significantly increased the expression of cAMP(P<0.01).The expression of NLRP3 inflammasome in the model group was significantly increased(P<0.001).After the administration of CB,there was a decreasing trend of NLRP3,but with no significant statistical difference.Both Cleaved caspase-1 and ASC protein expression were decreased(P<0.05).After the administration of FCB,the NLRP3,Cleaved caspase-1,and ASC expression were all significantly decreased(P<0.01,P<0.001,P<0.01).In comparison with the CB group,NLRP3 inflammasome expression difference decreased in FCB group(P<0.05).Conclusions1.There is an exact curative effect of Yaomu of Huafengdan in the treatment of ischemic stroke.The CB fermented by the fermentation method of Yaomu is safe and non-toxic,which has obvious therapeutic effect on ischemic stroke.2.After fermentation,the bile acid composition of CB has an overall trend of change from conjugated bile acid to unconjugated bile acid and primary bile acid to secondary bile acid.3.The efficacy of CB after fermentation is significantly improved,which has an obvious inhibitory effect on the inflammatory response after ischemic stroke.4.FCB has regulating effect on the inflammatory response after ischemic stroke via the TGR5-NLRP3 pathway. |
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