The Effect And Mechanism Of Chuanzhitongluo Capsule On Alleviating Brain Injury In Photochemical Stroke Mice

Objective:Chuanzhitongluo capsule(CZTL)is a mixed preparation of traditional Chinese medicine,and its effective ingredients include Leech,Chuanxiong,Salvia and Astragalus.Clinically,CZTL is currently used in the recovery period of patients with acute ischemic stroke(AIS),which can effectively reduce the neurological deficit of patients with ischemic stroke.In this study,our purpose is to evaluate whether CZTL can regulate the microglial polarization and inflammatory response to reduce brain damage in the acute phase of AIS,and providing experimental data and theoretical basis for CZTL treatment of AIS patients.Methods:The mouse model of AIS was prepared by the photochemical method.After the photochemical stroke mice were successfully prepared,the stroke mice were randomly divided into CZTL group and control group.The CZTL group mice were treated with different drug concentrations(30mg/Kg,50 mg/Kg,70 mg/Kg,90 mg/Kg,110 mg/Kg,130 mg/Kg).The control group mice were treated with 0.9%sodium chloride solution of the same volume orally,with the intervention frequency of twice a day for three consecutive days.After three days of CZTL drug intervention,the neurological function of mice was evaluated by the Modified Neurological Severity Scores(m NSS).2,3,5-triphenyltetrazolium chloride(TTC)staining was used to analyze the change trend of cerebral infarction volume in photochemical stroke mice.Terminal deoxynucleotidyl transfer d UTP nick end labeling(TUNEL)method was used to evaluate neuronal apoptosis.The changes of gene expression profile in stroke mice were explored by gene chip technology.Use protein chip technology to further analyze the trend of inflammatory factors.The activity of superoxide dismutase(SOD)and the level of malondialdehyde(MDA)were measured with the kit.The expression level of NLRP3 inflammatory body was evaluated by Western blotting.The co-expression of CD16~+/Iba1~+and CD206~+/Iba1~+in the penumbra of cerebral ischemia was detected by immunofluorescence double labeling method.The polarization of mouse microglia cell lines(BV2 cells)was further evaluated by in vitro experiments.Results:Compared with the control group,CZTL treatment reduced the neurological damage(6.5±0.62 vs.3.93±0.5;P<0.01),alleviated the volume of cerebral infarction(77.03±6.45 vs.51.5±2.78;P<0.01),and inhibited the apoptosis of neurons(69.29±3.11 vs.51.72±1.73;P<0.01).Differences in the expression of inflammation related genes at the RNA level in stroke mice treated with CZTL,which suggests that CZTL may participated in the regulation of neuroinflammatory response.CZTL inhibited neuroinflammatory reaction by down-regulating the expression of pro-inflammatory cytokines and enhancing the level of anti-inflammatory cytokines.CZTL decreased the expression level of MDA(2.67±0.14 vs.2.26±0.14;P<0.01)and increased the activity of SOD(45.6±0.51 vs.60.01±2.29;P<0.01),which showed that CZTL could inhibited the oxidative stress damage in AIS mice.Further experimental studies showed that CZTL inhibited the activation of NLRP3 inflammatory body(0.85±0.05 vs.0.64±0.07;P<0.05),and further alleviated the inflammatory reaction.On the other hand,CZTL also promoted the transformation of microglia from M1phenotype of pro-inflammatory effect to M2 phenotype of anti-inflammatory effect.Conclusion:CZTL may play a protective role in brain by adjusting the polarization of microglia and restraining neuroinflammatory reaction.