OBJECTIVE To explore the effect of total flavonoids of Rhododendra simsii(TFR)on improving cerebral ischemia/reperfusion injury(CIRI)and its relationship with STIM/Orai-regulated operational Ca^(2+)influx(SOCE)pathway.METHODS Oxygen-glucose deprivation/reoxygenation(OGD/R)PC12 cells were used to simulate CIRI in vitro,and the intracellular Ca^(2+)concentration and apoptosis rate of PC12 cells were detected by laser confocal microscope and flow cytometry,respectively.The regulation of STIM/Orai on SOCE was analyzed by STIM/Orai gene silencing and STIM/O rai gene overexpression.The CIRI model was established by MCAO in SD rats.The activities of inflammatory cytokines IL^(-1),IL-6 and TNF-αin serum were detected by ELISA.The pathological changes of ischemic brain tissue and the infarction of rat brain tissue were detected by HE staining and TTC staining.The protein and mRNA expression levels of STIM1,STIM2,Orai1,caspase-3 and PKB in brain tissue were detected by Western blotting and RT-qPCR,respectively.RESULTS The results of in vitro experiment showed that the fluorescence intensity of Ca^(2+)and apoptosis rate in PC12 cells treated with TFR were significantly lower than those in OGD/R group,and this trend was enhanced by SOCE antagonist 2-APB.STIM1/STIM2/Orai1 gene silencing significantly reduced apoptosis and Ca^(2+)overload in OGD/R model,while TFR combined with overexpression of STIM1/STIM2/Orai1 aggravated apoptosis and Ca2+overload.In the in vivo experiment,TFR significantly reduced the brain histopathological damage,infarction of brain tissue,the contents of IL^(-1),IL-6 and TNF-αin the serum in MCAO rats and down-regulated the expression of STIM1,STIM2,Orai1 and caspase-3 protein and mRNA in the brain tissue,and up-regulated the expression of PKB.The above effects were enhanced by the addition of 2-APB.CONCLUSION The above results indicate that TFR may reduce the contents of inflammatory factors and apoptosis,decrease Ca2+overload and ameliorate brain injury by inhibiting SOCE pathway mediated by STIM and
LU Jia-junJIANG Chen-chenHE Yu-xiangSHI LeiYIN Xiu-yunCHEN ZhuoCAO DiHAN Jun
目的观察胱硫醚γ裂解酶(CSE)siRNA转染人脐静脉内皮EAhy926细胞后CSE的表达及对H2S的影响。方法体外培养EAhy926细胞,经siRNA转染以沉默CSE基因表达,分为5组进行培养:正常对照组(A组):只加入转染试剂;阴性对照组(B组):转染Neg-siRNA;转染CSEsiRNA1组(C组);转染CSEsiRNA2组(D组);转染CSEsiRNA3组(E组)。采用Lipofectamine 2000介导转染EAhy926细胞,在荧光显微镜下观察细胞以检测转染效率;Western blot法检测CSE蛋白表达;收集细胞检测H2S含量。结果观察转染效率为60%左右;A组CSE蛋白表达为(153.41±23.43),B组24 h CSE蛋白表达为(150.22±17.56),而D组24 h CSE蛋白表达降至(59.43±21.81),差异有统计学意义(P<0.01);并且A组H2S含量为(0.96±0.05),B组24 h H2S含量为(0.95±0.06),D组24 h H2S含量降至(0.54±0.08),差异有统计学意义(P<0.01)。结论 CSE-siRNA能够有效地抑制EAhy926中CSE的表达,并且CSE-siRNA2沉默作用最强。
Objective:Securidaca inappendiculata is a medicinal plant frequently used in the treatment of inflammatory diseases in south China.In this study,we aimed to explore its bioactive constituent which contributes to the anti-inflammatory activity.Methods:Polyphenol-enriched and polyphenol-deprived fractions(PRF and PDF,respectively)were separated from the ethanolic extract by HPD300 macroporous resin-based method,and their antiinflammatory activities were investigated on a lipopolysaccharide(LPS)-induced acute lung injury(ALI)model in rats.The possible mechanism of action in alleviating acute inflammation was studied using RAW264.7 cells.Results:Both Folin-Ciocalteu and^(1)H nuclear magnetic resonance(NMR)analyses showed that polyphenolic content in PRF was approximately 10 times higher than that of PDF,and this observation reflected in their antioxidative capacities.PRF but not PDF significantly decreased the level of malondialdehyde,suppressed the expression of nicotinamide phosphoribosyltransferase(NAMPT)protein,and improved the severity of ALI in rats.PRF at 10μg/mL effectively downregulated the expression of proteins NAMPT,HMGB1,TLR4,and p-p65,and scavenged the intracellular reactive oxygen species(ROS)in LPS-primed RAW264.7 cells.N-acetyl-L-cysteine exhibited similar inhibitory effects on ROS production and NAMPT-mediated TLR4/NF-κB activation in vitro,whereas nicotinamide mononucleotide antagonized all the changes induced by PRF during cotreatments.Conclusion:As an antioxidant,PRF exhibited potent anti-inflammatory activity under both in vivo and in vitro conditions by downregulating NAMPT and TLR4/NF-κB.Accordingly,polyphenols were identified as important bioactive constituents in S.inappendiculata targeting oxidative stress-sensitive proinflammatory pathways.
