| Stroke is a disease that seriously harming human health and life all over the world with the characteristic of high incidence,high disability and high mortality.Ischemic stroke(IS)accounts for the majority of stroke and has been hot in research.Cerebral ischemia reperfusion injury(CIRI)is a severe state caused by the recovering of blood after cerebral ischemia.Increasing studies showed that inflammation plays a critical role in CIRI.Unfornately,the potential mechanism has not been expounded clearly.Recently,NLRP3 inflammasome,a protein complex,has attracted much attention for its effect on IS.NLRP3 inflammasome can provide a molecular platform for the activation of Caspase-1 by detecting various stimuluses.Upon the activation of NLRP3 inflammasome,large amounts of interleukin-1(IL-1)and interleukin-18(IL-18)will be released which further aggravates inflammation.Shixiao decoction(SXS)has been used to treat diseases caused by blood stasis for a long history and it was reported to improve microcirculation and alleviate pain.However,its effect on CIRI is still unknown.Hence,a better understanding of the mechanism of SXS on CIRI can provide solid evidence for clinical treatment.Objective:In this study,we tried to observe the protective effect of SXS on CIRI,its regulation of NLRP3 inflammasome and the suppression of inflammatory cytokines.Method:(1)Animals research:Male Sprague-Dawley rats were subjected to MCAO based on suture method.75 rats were divided into five groups randomly:Sham group(Sham),Model group(Model),SXS-L group(SXS-L),SXS-M group(SXS-M)and SXS-H group(SXS-H).Sham group:Rats of this group were administered with the same method of Model group but without inserting suture.SXS-L(0.63g/kg),SXS-M(1.26g/kg)and SXS-H(2.52g/kg)groups were given a gavage of SXS for 5 days before 2h ischemia.After 24h reperfusion,neurological function scores of each group were recorded.Then,all rats were sacrificed after anesthetized with 10%chloralhydrate,Their brains and serum were collected for following experiments.Firstly,we measured infarction area by TTC staining.Secondly,PCR was used to detect the expression of NLRP3,caspase-1,IL-?,IL-6,IL-18 and TNF-? in infarction cortex.Besides,we detected the expression of TNF-?,IL-?,IL-6 and IL-18 in infarction cortex and serum by ELISA.(2)Cells research:BV2 cells were divided into control group(CON),OGD/R(oxygen and glucose deprivation/reperfusion)group(Model)and treatment groups.Based on our previous research,cells of Model and treatment groups were administered with different concentrations of SXS extracts for 2h before 4h hypoxia,while cells of control group were cultured in normal medium without extracts and OGD/R operation.After 4h hypoxia,cells were reoxygenated for 24h.Firstly,we used CCK-8 kit to detect the viability of BV2 cells.Secondly,FCM(flow cytometry)was used to test the apoptosis of BV2 cells.Furthermore,we used ELISA to assay the expression of TNF-?,IL-18 and IL-1? in culture supernatant.NLRP3,caspase-1,IL-1?,IL-6,IL-18 and TNF-? mRNA were qualified by using PCR.Results:1.The neurological score of model group was significantly higher compared with sham group(P<0.001)while the scores of treatment groups were lower than model group(P<0.01?P<0.01).2.Significant cerebral infarction was seen in model group(49.8%,P<0.001)and infarction area of treatment groups were reduced to 19.6%,15.4%,4.1%respectively(P<0.001?P<0.001?P<0.001).3.The results of ELISA demonstrated that the level of inflammatory cytokines in serum and cortexes of model groups increased significantly compared with control group(P<0.001?P<0.01?P<0.01?P<0.01)and SXS obviously decreased the expression of inflammatory cytokines of treatment groups(P<0.001?P<0.05?P<0.05?P<0.05),which suggested anti-inflammatory effect of SXS.4.The expression of NLRP3?Caspase-1?IL-1 p?IL-6?IL-18?TNF-? mRNA in infarction cortex of model group increased significantly compared with sham group(P<0.001?P<0.01?P<0.001?P<0.001?P<0.001?P<0.001)and SXS reduced the expression of inflammatory factors of treatment groups compared with model group(P<0.001>P<0.01.P<0.001?P<0.001?P<0.001?P<0.001).The dates indicated inhibitory effect on NLRP3 inflammasome of SXS.5.The result of CCK-8 showed that no significant differences were found between treatment groups,which indicated SXS extracts has no obvious cytotoxicity to BV2 cells.6.The proportion of apoptosis in OGD/R group increased obviously compared with control group(P<0.01)and SXS extracts protected BV2 cells by decreasing apoptosis(P<0.05?P<0.05).7.Inflammatory factors in model group increased significantly compared with control group(P>0.05?P<0.05?P<0.001?P<0.001)and SXS extracts reduced the expression of inflammatory factors of treatment groups compared with OGD group(P<0.05?P<0.05?P<0.001?P<0.001).8.The level of NLRP3,caspase-1,IL-1?,IL-6,IL-18 and TNF-? mRNA of OGD/R group increased significantly compared with control group(P>0.05?P<0.05?P<0.001?P<0.05?P<0.05?P<0.01)and SXS extracts reduced the expression of inflammatory cytokines in treatment groups compared with model group(P>0.05?P<0.01?P<0.001?P<0.01?P<0.05?P<0.01)while no obvious differences were found between treatment groups.Conclusion:In conclusion,our results have demonstrated that SXS has protective effects against CIRI both in vitro and in vivo possibly through suppressing the activation of NLRP3 inflammasome and inflammatory cytokines mediated by NLRP3 inflammasome. |
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