| BackgroundInflammatory response plays a key role in the pathological progression of ischemic stroke and has an important impact on its prognosis.Microglia are the first line of immune defense and play an important role in the inflammatory response.The state of microglia affects the inflammatory response of brain tissue,and its hyperactivation and apoptosis are associated with inflammation and damage in ischemic cerebrovascular disease.In addition,microglia are the main source of the NOD-like receptor family pyrin domain-containing 3(NLRP3)inflammasomes in the central nervous system.The NLRP3 inflammasome is an important mediator in regulating the inflammatory response after cerebral ischemia and may be a potential target.Salidroside(Sal)is a phenylpropanoid extracted from Rhodiola rosea,which has good anti-inflammatory effect.It is still unclear whether Sal can affect the activation of NLRP3 inflammasome and apoptosis in microglia after cerebral ischemia/reperfusion(I/R)injury to exert anti-inflammatory effects,and its possible control mechanism.ObjectivesBy constructing an oxygen-glucose deprivation/reoxygenation(OGD/R)model of microglia and middle cerebral artery occlusion/reperfusion(MCAO/R)model in rats.The purpose of this study was to investigate whether Sal can affect the activation of NLRP3 inflammasome and apoptosis in microglia after cerebral I/R injury to exert anti-inflammatory effect,and its possible regulatory mechanism.Methods1.Chapter 1(1)A microglia cell line(BV2)cell OGD/R model was constructed to simulate the pathophysiological process of brain I/R in vivo.(2)Cell Counting Kit-8(CCK-8)was used to detect BV2 cell viability,lactate dehydrogenase(LDH)release assay was used to detect cell damage,flow cytometry,terminal deoxynucleotidyl transferase dUTP nick-end labeling(TUNEL)methods were used to detect the rate of apoptosis.(3)Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of inflammatory factors in cell culture medium such as tumor necrosis factor-α(TNF-α),interleukin(IL)-6,IL-8.(4)Western blot was used to detect the expression levels of apoptosis-related proteins such as Bcl-2,Bax,and cleaved caspase-3;The expression levels of NLRP3 inflammasome related proteins such as NLRP3,apoptosis-associated speck-like protein containing a CARD(ASC),Caspase-1,IL-1β,and IL-18;The expression levels of key proteins in the toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)signaling pathway such as TLR4,myeloid differentiation primary response 88(MyD88),phosphorylated nuclear factor kappa B p65(p-NF-κB p65),NF-κB p65.(5)The expression levels of NLRP3,Caspase-1 and TLR4 were further detected by immunocytochemical staining.(6)In order to further verify the relationship between TLR4/NF-κB signaling pathway and NLRP3 inflammasome activation and apoptosis.The BV2 cells induced by OGD/R were incubated with TAK242,a specific inhibitor of TLR4,and then the TLR4/NF-κB signaling pathway,NLRP3 inflammasome and apoptosis were observed.(7)In addition,in order to further verify whether the effect of Sal on OGD/R-induced microglia has an effect on neuronal cells.The BV2 cell culture medium(CM)of different groups was collected and incubated with the normally cultured neuronal cell line(SH-SY5Y).The CCK-8 assay was used to detect the cell viability of SH-SY5Y cells,LDH release assay was used to detect cell damage.2.Chapter 2(1)7-8 weeks old SD male rats were selected to establish MCAO/R model,and intraperitoneal injection of Sal(50 mg/kg/d)was given for 7 days.(2)The cerebral infarct volume were detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining,modified Neurological Severity Score(mNSS)and weight change of rats were measured to detect the degree of neurological deficits in rats.(3)The open field test was used to test the motor ability of the rats,and the Morris water maze test was used to test the cognitive function of the rats.(4)The expression levels of TNF-α,IL-6 and IL-8 in rat serum were detected by ELISA.(5)Cell apoptosis in the ischemic penumbra of brain tissue was detected by TUNEL method.(6)Western blot was used to detect the expression levels of apoptosis-related proteins such as Bcl-2,Bax,and cleaved caspase-3;The expression levels of NLRP3 inflammasome related proteins,such as NLRP3,ASC,Caspase-1,IL-1β,and IL-18;The expression levels of key proteins in the TLR4/NF-κB signaling pathway such as TLR4,MyD88,p-NF-κB p65,NF-κB p65 in rat brain tissue.(7)Immunohistochemical staining was used to detect the expression levels of NLRP3 and TLR4 in rat brain tissue.(8)Immunofluorescence staining was used to detect the expression levels of IB A-1,NLRP3 and TLR4 in rat brain tissue.(9)The rats were injected intraperitoneally with TAK242,a specific inhibitor of TLR4,and then the TLR4/NF-κB signaling pathway,NLRP3 inflammasome and apoptosis were detected.3.Chapter 3(1)MicroRNA(miRNA)and mRNA sequencing was performed on the brain tissue of the sham group and the MCAO/R group in the early stage.According to the sequencing results,the kyoto encyclopedia of genes and genomes(KEGG)enrichment results of the two groups were analyzed,and it was found that the NOD-like receptor signaling pathway was a signaling pathway related to the NLRP3 inflammasome.Furthermore,NLRP3 is a differentially expressed factors involved in this signaling pathway.Further analysis revealed differentially expressed miRNAs(miR-370-3p,miR-133c,miR-1224)associated with NLRP3.(2)The differentially expressed miRNAs were verified by quantitative polymerase chain reaction(qPCR).(3)The expression levels of miRNAs were detected by qPCR after Sal treatment.(4)The qPCR detection showed that Sal could increase the expression level of miR-370-3p in the brain tissue of MCAO/R rats,but had no effect on the expression of miR-133c and miR-1224.In order to further verify whether the anti-inflammatory effect of Sal on rat cerebral I/R injury is related to the expression of miR-370-3p.The lentivirus of miR-370-3p inhibitor was injected stereotaxically into the lateral ventricle of Sal-treated MCAO/R rats to interfere with the function of miR-370-3p.(5)The expression level of IBA-1 in rat brain tissue was detected by immunofluorescence staining.(6)Western blot was used to detect the expression levels of NLRP3 inflammasome related proteins such as NLRP3,ASC,Caspase-1,IL-1β,and IL-18;The expression levels of key proteins in the TLR4/NF-κB signaling pathway such as TLR4,MyD88,p-NF-κB p65,NF-κB p65 in rat brain tissue.Results1.Chapter 1(1)Sal can increase the viability of BV2 cells induced by OGD/R,inhibit the release of LDH,reduce the apoptosis rate of BV2 cells,and reduce the expression levels of the pro-apoptotic proteins Bax and cleaved caspase-3,and increase the expression levels of the anti-apoptotic protein Bcl-2.(2)Sal can reduce the expression levels of inflammatory factors TNF-α,IL-6 and IL-8 in the cell culture supernatant of BV2 cells induced by OGD/R.(3)In BV2 cells induced by OGD/R,and the expression levels of NLRP3 inflammasome related protein such as NLRP3,ASC,Caspase-1,IL-1β,and IL-18 were up-regulated,and the expression levels of key protein of TLR4/NF-κB signaling pathway such as TLR4,MyD88,p-NF-κB p65/NF-κB p65 were up-regulated,and these changes were reversed by Sal treatment.(4)Intervention with TLR4-specific inhibitor TAK242 can inhibit the TLR4/NF-κB signaling pathway,and at the same time inhibit the activation of NLRP3 inflammasome and apoptosis in OGD/R-induced BV2 cells.(5)SH-SY5Y cells showed decreased cell viability and increased LDH release after co-incubation with OGD/R BV2-CM.However,SH-SY5Y cells incubated with OGD/R+Sal BV2-CM showed increased cell viability and decreased LDH release.2.Chapter 2(1)Sal can reduce cerebral infarction volume,mNSS and weight loss in MCAO/R rats.(2)Sal can improve motor function and cognitive function in MCAO/R rats.(3)Sal can reduce the expression of pro-apoptotic proteins Bax and cleaved caspase-3 and increase the expression of anti-apoptotic proteins Bcl-2 in the brain tissue of MCAO/R rats.Sal can inhibit the apoptosis rate in the brain tissue of MCAO/R rats.(4)Sal can inhibit the levels of inflammatory factors TNF-α,IL-6 and IL-8 in the serum of MCAO/R rats.(5)Sal can inhibit the expression levels of NLRP3 inflammasome related proteins such as NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the brain tissue of MCAO/R rats.(6)Sal can inhibit the expression levels of key proteins of TLR4/NF-κB signaling pathway such as TLR4,MyD88,p-NF-κB p65/NF-κB p65 in the brain tissue of MCAO/R rats.(7)Sal can inhibit the expression levels of IBA-1,NLRP3 and TLR4 in the brain tissue of MCAO/R rats.(8)Inhibition of TLR4 can inhibit the TLR4/NF-κB signaling pathway in the brain tissue of MCAO/R rats,and at the same time inhibit the activation of NLRP3 inflammasome and apoptosis.3.Chapter 3(1)miRNA sequencing showed that compared with the sham group,the expressions of miR-133c and miR-1224 were up-regulated,and the expression of miR-370-3p was down-regulated in the brain tissue of rats in MCAO/R group;After qPCR verification,it was found that compared with the sham group,the expression of miR-133c was down-regulated,and the expression of miR-1224 was up-regulated,and the expression of miR-370-3p was down-regulated in the MCAO/R group.(2)Sal can increase the expression level of miR-370-3p in the brain tissue of MCAO/R rats.However,Sal had no effect on the expression levels of miR-133c and miR-1224.(3)Sal can inhibit the expression levels of IBA-1 in the brain tissue of MCAO/R rats by increasing the expression of miR-370-3p.(4)Sal can inhibit the expression levels of NLRP3 inflammasome related proteins such as NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the brain tissue of MCAO/R rats,by increasing the expression of miR-370-3p.(5)Sal can inhibit the expression levels of key proteins of TLR4/NF-κB signaling pathway such as TLR4,MyD88,p-NF-κB p65/NF-κB p65 in the brain tissue of MCAO/R rats,by increasing the expression of miR-370-3p.Conclusions1.Sal can inhibit the activation of NLRP3 inflammasomes and apoptosis in microglia induced by OGD/R by inhibiting the TLR4/NF-κB signaling pathway;In addition,the protective effect of Sal on OGD/R-induced microglia may help reduce neuronal damage.2.Sal can inhibit the activation of NLRP3 inflammasome and cell apoptosis in microglia in rats cerebral I/R injury by inhibiting the TLR4/NF-κB signaling pathway.3.Sal can inhibit the activation of NLRP3 inflammasome and the activation of TLR4/NF-κB signaling pathway in rats cerebral I/R injury by increasing the expression level of miR-370-3p. |
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