To Investigate The Effects And Mechanisms Of Ginsenoside Rd On Cell Pyroptosis Induced By Cerebral Ischemia-reperfusion Injury Based On MiR-139-5p/FoxO1/Keap1/Nrf2 Pathway

Objective: Ischemic stroke is a cerebrovascular disease characterized by decreased cerebral circulation blood flow.It has become one of the three major killers threatening human health due to its high incidence,high disability rate and high mortality.Recent studies have shown that cerebral ischemia/reperfusion injury may be related to cell pyrotosis in neurons.Ginsenoside Rd has been reported to attunate the neural damage induced by cerebral ischemia/reperfusion injury.However,the relevant mechanism of ginsenoside Rd on cell pyroptosis has not been fully explained.Therefore,in this study,cell pyroptosis was taken as the entry point to explore the mechanism of ginsenoside Rd on cerebral ischemia-reperfusion injury from both in vivo and in vitro,providing scientific basis for the development of ginsenoside Rd as an effective anti-cerebral ischemia neuroprotective agent.Methods: 1.In vivo experiments: Male C57BL/6 mice(6-8 weeks old,weighing 22-25 g,SPF grade)were randomly divided into 7 groups: Sham operation group,cerebral ischemia/reperfusion group(middle cerebral artery occlusion/reperfusion(MCAO/R)model),ginsenoside Rd low-dose group(10 mg/kg),ginsenoside Rd middle-dose group(20 mg/kg),ginsenoside Rd high-dose group(40 mg/kg),ginsenoside Rd(40mg/kg)+Nrf2 inhibitor(ML385)group,ginsenoside Rd(40 mg/kg)+mi R-139-5p inhibitor group,96 mice in each group.Except for the Sham group,MCAO/R model was established in other groups by silk suture embolization.In the ginsenoside Rd group,administration of Rd at the dose of 10 mg/kg,20 mg/kg or 40 mg/kg was carried out 30 min before MCAO/R via intraperitoneal injection.In the ginsenoside Rd+Nrf2 inhibitor group,ML385(30 mg/kg)pre-treatment was intraperitoneally administered 1 h before administration of Rd.In the ginsenoside Rd(40 mg/kg)+ mi R-139-5p antagomir group,mi R-139-5p antagomir applied 3 days before MCAO/R via intracerebroventricular injection.After 24 h of reperfusion,the neurological deficit behavior of mice was evaluated according to Longa scoring system.TTC staining was performed to measure the percentage of cerebral infarction by volume.Wet-dry weighting method was used to determine the water content of brain tissues.And the morphological and structural changes of neurons in each group were observed by Nissl staining.The brain tissues were sampled for Immunofluorescence staning to determine cell pyroptosis and the co-localization of NLRP3 and TXNIP,DCFH-FA fluorescence probe to detect the ROS level,RT-PCR to assess mi R-139-5p,Fox O1,Keap1,NLRP3,ASC,and Caspase-1 m RNA expression levels,Western blot to test Fox O1,Keap1,total Nrf2,nuclear Nrf2,HO-1,NQO1,Trx1,NLRP3,ASC,Caspase-1,and GSDMD protein expression levels,ELISA to measure levels of IL-18 and IL-1β in the homogenate of brain tissues.2.In vitro experiments: Primary cortical neurons were obtained from 18-day-old embryos of pregnant C57BL/6 mice under sterile conditions.After 1 week of normal culture,neurons were randomly divided into 10 groups: control group(control),model group(oxygen-glucose deprivation/re-oxygenation(OGD/R)model),ginsenoside Rd lowconcentration group(5 μM),ginsenoside Rd middle-concentration group(10 μM),ginsenoside Rd high-concentration group(20 μM),ginsenoside Rd(20 μM)+ Fox O1-overexpressing pc DNA3.1-plasmid transfection group,Fox O1-overexpressing pc DNA3.1-plasmid transfection group,Fox O1-sh RNA transfection group,mi R-139-5p mimic transfection group,mi R-139-5p inhibitor transfection group.Cell viability was detected by MTT method.Cell damage was determined by LDH release method.Cell pyroptosis was measured by flow cytometry.Intracellular ROS content was detected by DCFH-DA fluorescence probe method.Then the interaction between NLRP3 and TXNIP was detected by co-immunoprecipitation(Co-IP)assay.Furthermore,the m RNA expression levels of mi R-139-5p,Fox O1,Keap1,NLRP3,ASC,and Caspase-1 were evaluated by RT-PCR.The protein expression levels of Fox O1,Keap1,total Nrf2,nuclear Nrf2,HO-1,NQO1,Trx1,NLRP3,ASC,Caspase-1,and GSDMD was detected by Western blot.Lastly,the level of IL-18 and IL-1β in cell supernatants was assessed by ELISA.The negative regulation of Fox O1 by mi R-139-5p was verified by dual luciferase reporter genes.The binding level of Fox O1 to Keap1 gene promoter region was detected by chromatin immunocoprecipitation(Ch IP)assay.Results:In vivo experiments: 1.Ginsenoside Rd with 10 mg/kg,20 mg/kg and 40mg/kg improved neurological function of mice with OGD/R,reduced cerebral infarction volume and cerebral edema ratio,and alleviated the pathological damage of neurons in the cortical tissue,with 40 mg/kg ginsenoside Rd had the best effect.2.Ginsenoside Rd with10 mg/kg,20 mg/kg and 40 mg/kg decreased the percentage of pyroptotic cells,downregulated the protein expression levels of pyroptosis-related proteins NLRP3,ASC,Caspase-1 p20,GSDMD-N,reduced the levels of IL-18 and IL-1β,suppressed the intracellular ROS level,and alleviated the interaction between NLRP3 and TXNIP,with40 mg/kg ginsenoside Rd had the best effect.3.Ginsenoside Rd with 10 mg/kg,20 mg/kg and 40 mg/kg down-regulated the m RNA and protein expression levels of Keap1,but upregulated the nuclear translocation level of Nrf2 and the protein expression levels of HO-1,NQO1 and Trx1,with 40 mg/kg ginsenoside Rd had the best effect.4.After intraperitoneal injection of Nrf2 inhibitor ML385,the inhibitory effects of 40 mg/kg ginsenoside Rd on pyroptosis-related protein expression level,IL-18 and IL-1β secretion level,intracellular ROS level,and the co-localization of TXNIP and NLRP3 were reversed by ML385.5.Ginsenoside Rd up-regulated the expression level of mi R-139-5p in a dosedependent manner.However,after intracerebroventricular injection of mi R-139-5p antagomir in MCAO/R mice,The inhibitory effects of 40mg/kg ginsenoside Rd on pyroptosis-related protein expression level,IL-18 and IL-1β secretion level,intracellular ROS level,and the co-localization of TXNIP and NLRP3 were reversed by mi R-139-5p antagomir.In vitro experiments: 1.Ginsenoside Rd with 5 μM,10 μM and 20 μM significantly improved cell viability of OGD/R-treated neurons,and suppressed LDH release,with 20μM ginsenoside Rd had the best effect.2.Ginsenoside Rd with 5 μM,10 μM and 20 μM decreased the proportion of pyroptotic cells,down-regulated the expression levels of pyroptosis-related proteins NLRP3,ASC,Caspase-1 p20,GSDMD-N,reduced the secretion levels of IL-18 and IL-1β,suppressed intracellular ROS level,and weakened the interaction between TXNIP and NLRP3,with 20 μM ginsenoside Rd had the best effect.3.Ginsenoside Rd with 5 μM,10 μM and 20 μM down-regulated the m RNA and protein expression levels of Keap1 in OGD/R-treated neurons,but up-regulated the nuclear translocation level of Nrf2 and the protein expression levels of HO-1,NQO1,and Trx1,with 20 μM ginsenoside Rd had the best effect.4.Ginsenoside Rd up-regulated the expression level of mi R-139-5p in a dose-dependent manner.Mi R-139-5p mimic significantly increased cell viability of neurons treated with OGD/R and attenuated OGD/R-induced cell injury.Furthermore,mi R-139-5p mimic suppressed the proportion of pyroptotic cells,down-regulated the expression levels of pyroptosis-related proteins NLRP3,ASC,caspase-1 and GSDMD-N,reduced IL-18 and IL-1β secretion,restained intracellular ROS overproduction,and reduced the interaction between TXNIP and NLRP3.Meanwhile,mi R-139-5p mimic negatively regulated Fox O1 expression.However,the effect of mi R-139-5p inhibitor was exactly opposite to that of mi R-139-5p mimic.5.The m RNA and protein levels of Keap1 were upregulated by Fox O1 overexpression.Furthermore,Overexpression of Fox O1 markedly decreased the transcriptional activity of Nrf2 and the expression levels of downstream target genes of Nrf2,including HO-1,NQO1,and Trx1.The result of Ch IP assay was verified that Fox O1 bound directly to the Keap1 promoter region.However,the above effects were opppsite in Fox O1-sh RNA transfected neurons group.5.Overexpression of Fox O1 reversed the 20 μM ginsenoside Rd-induced downregulation of Keap1 and upregulation of nuclear Nrf2,HO-1,NQO1,and Trx1.In addition,overexpression of Fox O1 abolished the 20 μM ginsenoside Rdmediated downregulation of TXNIP,NLRP3,ASC,Caspase-1 p20,and GSDMD-N,reduced IL-18 and IL-1β release,suppressed ROS production,decreased the pyroptotic cell ratio and weakened the co-localization of TXNIP with NLRP3.Conclusions: Ginsenoside Rd has a protective effect on neurons after cerebral ischemia/reperfusion injury,and this effect may be related to the up-regulation of mi R-139-5p expression level,the reduction of Fox O1 and Keap1 expression levels,and the activation of Nrf2 antioxidant signaling pathway,and the inhibition of cell pyroptosis induced by ROS-TXNIP-NLRP3 inflammosome axis.