Protective Effect And Mechanism Study Of Salvianolate Injection In The Treatment Of Acute Ischemic Stroke

Acute Ischemic Stroke(AIS)has the characteristics of high morbidity and high recurrence rate,has been used herbal extracts in the treatment of AIS has always been a hot research topic.The use of herbal therapies is becoming an increasingly attractive approach for the treatment of AIS.Salvianolate injection(SLI)is made of the extraction of Danshen(radix salviae miltiorrhizae).In this study we investigated the effects and mechanism of SLI on the regulation of inflammatory cytokine production,anti-apoptosis,and anti-fibrinolysis.Part 1 Clinicalobservation of the effect of SLI in treating patients with AIS.Objective:To research the protective effect and mechanism of SLI in patients with ACI,we testing the charges of blood biochemistry,coagulation indexes and efficacy were observed before and after treatment.Methods:Collected in January 2016 to August 2018 for the first time of the AIS.Totally 100 AIS patients with AIS were devided into SLI group(50 cases)and control group(50 cases).Compared two groups in baseline,blood biochemical,blood coagulation index,and changes of NHISS scores and m RS scoress.Totally 60 AIS patients were randomly divided into control group(30 cases)and SLI treatment group(30 cases).Compared the changes of ICAM-1,VCAM-1,IL-4,u PA,and PAI-1/t PA.Results:1.To compared two groups with the baseline data had no significant statistical difference(P>0.05).2.PLT level and FIB level in SLI group were lower than that of control group after treatment(P<0.01,P<0.05).PDW level in SLI group were significantly higher than that of control group(P<0.01).Comparison between the control group before and after treatment,level of BUN rised after 7 days,P<0.05;and level of hs CRP and DD reduced after 7 days,P<0.01.3.Both groups had less NHISS scores after 7 days and 1 month,P<0.01.NHISS scores in SLI group reduced more significantly after 7 days,P<0.05.To compared two groups,the NHISS score levels had no significant statistical difference with each other,P>0.05.And m RS scores after 1 month and treatment efficiently after 3 months had no significant statistical difference with each other,P>0.05.4.Levels of ICAM-1,VCAM-1,u PA,PAI-1/t PA in SLI group after treatment were lower than before treatment(P<0.05).Level of IL-4 in SLI group had no significant statistical difference(P>0.05).Levels of ICAM-1,VCAM-1,IL-4,u PA,PAI-1/t PA in control group after treatment were lower than before treatment(P<0.05).Conclusion:1.SLI had no significant effect on liver function and renal function before and after treatment,and no adverse reactions occurred during treatment,suggesting that SLI was safe.2.SLI could protect patients with AIS,by reducing hs CRP?ICAM-1?VCAM-1.SLI was as effective as conventional treatments in inhibiting inflammatory reaction.3.SLI could protect patients with AIS,by reducing the level of PLT,FIB,u PA,PAI-1,t PA,u PA and increasing PDW level.SLI was better in improving the function platelet aggregation fibrinolysisPart 2 The human umbilical vein endothelial cells(HUVECs)injury model induced by H₂O₂ and Effect of SLI on platelet activation rate.Objective:The injury model of HUVECs induced by H₂O₂ was established,and the effects of SLI at different concentrations on HUVECs cell activity were observed.The main components of SLI were analyzed.Observe the changes of platelet activation rate influenced by SLI in vitro.Methods:HUVECs were cultured in vitro.The cell viability and cell growth curve assay was evaluated via CCK8 assay.Detect the protective effects of SLI on damage of HUVECs induced by H₂O₂.HPLC and LC-MS were used to analysis the mainly bioactive compounds of SLI.Cells induced by ADP are used to calculate different concentration SLI on rat platelet activation rate and the effect of inhibition rate.Results:1.The results showed that the HUVECs induced by H₂O₂ were successfully produced,and the cell growth curve assay was evaluated.2.The cell viability of H₂O₂-induced HUVECs was increased by treatment with SLI(0.8,1.6 mmol/L),in dose-dependent manner compared with the control cells.And the cell viability of the H₂O₂-induced HUVECs was increased in a time-dependent manner,P<0.01.3.By HPLC method,the effective components of the SLI were salvianolic acid D(3.70%),rosemary(2.68%),purple oxalic acid(3.86%),and salvianolic acid B(53.81%).By LC-MS method,the effective components of the SLI contains 29 definite chemical composition,including 14 kinds of common known phenolic acids.4.Different concentrations(0.2,0.4,0.8mmol/L)of SLI could inhibition the PLT of rats significantly,P<0.05.And different concentration of SLI had no significant statistical difference,P>0.05.Conclusion:1.SLI could protected the modle of H₂O₂-induced HUVECs H₂O₂time-dependently.2.The composition of SLI was clear and stable.SLI was useful for treatment in clinical for a variety of ingredients we had known.3.SLI could promoting blood circulation to remove blood stasis by inhibiting the platelet aggregation,and protecting the ischemic anoxia injury.Part3 The protective effects of SLI on damage of HUVECs Induced by H₂O₂.Objective:Analysis the protective effect and mechanismof SLI on damage of HUVECs induced by H₂O_2.Methods:Effect of SLI in anti-inflammatory and anti-fibrinolysis was assessed using ELISA assay kit.The changes of apoptosis were measured using JC-1 stainin and hoechst 33258 staining.Western blotting was used to detect the expression of Bcl-2,Bax and Cleaved-caspase-3 protein in the H₂O₂-induced HUVECs.Results:1.SLI could protect the H₂O₂-induced HUVECs by significantly inhibited the level of IL-1??IL-17a,ICAM-1,VCAM-1,TNF-?and IGF-1(P<0.0001).2.The levels of u PA?PAI-1/t PA were inhibited by SLI(0.05,0.1,0.2,0.4,0.8,1.6,3.2mmol/L)in dose-dependent manner compared with the control cells(P<<0.0001,P<0.0001,P<0.01).3.We used JC-1 staining to detect changes in the HUVECs mitochondrial membrane potential.Compared with the H₂O₂ indced group,the proportions of red and green fluorescence intensity in the different dose groups of SLI increase(0.1,0.2,0.4,0.8mmol/L).4.We used hoechst to test the apoptosis of HUVECs induced by H₂O_2.The nuclei of the cells treated with SLI(0.4,0.8,1.6mmol/L)were stained much brighter than that of untreated cells due to chromatin condensation.5.Western blotting showed that levels of Bax and Cleaved-caspase3 were higher in H₂O₂-induced HUVECs,and the level of Bcl-2 was lower(P<0.01).SLI(0.2,0.4,0.8,1.6 mmol/L)could inhibited the apoptosis by increased Bcl-2,and inhibited Bax and Cleaved-caspase-3 protein expression in the H₂O₂-induced HUVECsConclusion:1.Suitable concentration of SLIcould protected the H₂O₂-induced HUVECs by significantly inhibited H₂O₂-induced IL-1??IL-17a,ICAM-1,VCAM-1 and TNF-?production and expression,and increaced the expression of IGF-1.SLI could also protected the H₂O₂-induced HUVECs by significantly inhibited H₂O₂-induced u PA?PAI-1,and increaced the expression of t PA.2.SLI could inhibited the apoptosis of H₂O₂-induced HUVECs in a dose-dependent manner by increased Bcl-2,and inhibited Bax?Cleaved-caspase-3 protein expression in the H₂O₂-induced HUVECs.