目的:本研究旨在通过脂多糖(LPS)刺激PC12细胞,建立体外神经细胞损伤模型,并通过高通量序列技术和生物信息学分析研究黄芪甲苷对关键靶标的影响。
方法:将对数生长期的PC12细胞用终浓度为0.25、0.5、0.75、1和1.25mg/mL的LPS处理24小时。并计算细胞存活率。用LPS处理建立神经细胞炎症模型,达到50%的细胞存活率。PC12细胞分别用0.01、0.1、1、10或100μmol/L黄芪甲苷处理24h,选择不影响细胞存活率的黄芪甲苷浓度作为后续实验的处理组。用比色法检测NOS活性;ERCC2、XRCC4、XRCC2、TNF-α、IL-1β,RT-qPCR和Western印迹法检测TLR4、NOS和COX-2mRNA和蛋白的表达。使用第二代序列(倍数变化>2,P<0.05)筛选各组之间的差异表达基因(DEGs),并进行以下KEGG富集分析,采用RT-qPCR和Westernblotting检测不同组PC12细胞中与IL-17通路相关的DEGs的mRNA和蛋白表达。
结果:用0.01、0.1或1μmol/L黄芪甲苷处理24h后,PC12细胞的活力无明显变化(P>0.05)。然而,用0.5、0.75、1或1.25mg/mLLPS处理24小时后,活力稳步下降(P<0.01)。ERCC2、XRCC4、XRCC2、TNF-α、IL-1β,TLR4,NOS,1mg/mLLPS处理PC12细胞24h后,COX-2显著升高(P<0.01);当PC12细胞用0.01、0.1或1μmol/L黄芪甲苷预处理PC12细胞,然后用1mg/mLLPS处理24h时,这些变化得到逆转(P<0.05)。第二代测序显示1026个基因上调,而1287个基因下调。DEGs与自噬有关,TNF-α,白细胞介素-17,MAPK,P53,Toll样受体,和NOD样受体信号通路。此外,用1mg/mLLPS处理24小时的PC12细胞显示与IL-17途径相关的CCL2,CCL11,CCL7,MMP3和MMP10的mRNA和蛋白表达增加。RT-qPCR和Western印迹分析证实上面列出的DEG对应于序列测定结果。
结论:LPS可损伤PC12细胞,引起神经细胞炎症反应和DNA损伤。黄芪甲苷在体外发挥抗炎和DNA损伤保护作用,抑制IL-17信号通路发挥神经保护作用。
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 are associated with the IL-17 pathway. RT-qPCR and Western blotting analyses confirmed that the DEGs listed above corresponded to the sequence assay results.
CONCLUSIONS: 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.