查看更多>>摘要：Objective:This study aimed to establish a neural cell injury model in vitro by stimulating PC12 cells with lipopolysaccharide(LPS)and to examine the effects of astragaloside Ⅳ on key targets using high-throughput sequence technology and bioinformatics analyses.Methods:PC12 cells in the logarithmic growth phase were treated with LPS at final concentrations of 0.25,0.5,0.75,1,and 1.25 mg/mL for 24 h.Cell morphology was evaluated,and cell survival rates were calculated.A neurocyte inflammatory model was established with LPS treatment,which reached a 50％cell survival rate.PC12 cells were treated with 0.01,0.1,1,10,or 100 μmol/L astragaloside Ⅳ for 24 h.The concentration of astragaloside Ⅳ that did not affect the cell survival rate was selected as the treatment group for subsequent experiments.NOS activity was detected by colorimetry;the expression levels of ERCC2,XRCC4,XRCC2,TNF-α,IL-1β,TLR4,NOS and COX-2 mRNA and protein were detected by RT-qPCR and Western blotting.The differentially expressed genes(DEGs)between the groups were screened using a second-generation sequence(fold change＞2,P＜0.05)with the following KEGG enrichment analysis,RT-qPCR and Western blotting were used to detect the mRNA and protein expression of DEGs related to the IL-17 pathway in different groups of PC12 cells.Results:The viability of PC12 cells was not altered by treatment with 0.01,0.1,or 1 μmol/L astragaloside Ⅳ for 24 h(P＞0.05).However,after treatment with 0.5,0.75,1,or 1.25 mg/mL LPS for 24 h,the viability steadily decreased(P＜0.01).The mRNA and protein expression levels of ERCC2,XRCC4,XRCC2,TNF-α,IL-1β,TLR4,NOS,and COX-2 were significantly increased after PC12 cells were treated with 1 mg/mL LPS for 24 h(P＜0.01);however,these changes were reversed when PC12 cells were pretreated with 0.01,0.1,or 1 μmol/L astragaloside Ⅳ in PC12 cells and then treated with 1 mg/mL LPS for 24 h(P＜0.05).Second-generation sequencing revealed that 1026 genes were upregulated,while 1287 genes were downregulated.The DEGs were associated with autophagy,TNF-α,interleukin-17,MAPK,P53,Toll-like receptor,and NOD-like receptor signaling pathways.Furthermore,PC12 cells treated with a 1 mg/mL LPS for 24 h exhibited increased mRNA and protein expression of CCL2,CCL11,CCL7,MMP3,and MMP10,which arc associated with the IL-17 pathway.RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results.Conclusion:LPS can damage PC12 cells and cause inflammatory reactions in nerve cells and DNA damage.astragaloside Ⅳ plays an anti-inflammatory and DNA damage protective role and inhibits the IL-17 signaling pathway to exert a neuroprotective effect in vitro.
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