基于WGCNA探讨葛根素对呕吐毒素诱导C6细胞损伤的保护作用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0130

摘要/Abstract

摘要：

【目的】利用呕吐毒素（deoxynivalenol, DON）构建C6细胞损伤模型，分析葛根素（puerarin, PUE）对DON诱导C6细胞损伤的保护作用机制。【方法】通过RNA测序（RNA-seq）和加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA），挖掘与PUE缓解DON诱导C6细胞损伤最相关的关键模块和关键核心靶点基因，探究PUE缓解DON诱导的神经毒性作用的分子机制。【结果】PUE可以显著抑制DON诱导的C6细胞活力下降，对损伤的C6细胞形态有所恢复，可有效缓解DON诱导的C6细胞损伤。RNA-seq和WGCNA结果表明，Blue模块是与PUE缓解DON诱导C6细胞损伤表型最相关的关键模块，从中筛选出S100a11、Pdia3、Hsp90b1等基因是PUE发挥对DON诱导C6细胞损伤保护作用的关键核心基因。【结论】PUE可能是通过靶向关键核心基因参与内质网中的蛋白质加工途径从而缓解DON诱导的C6细胞损伤。

关键词: 呕吐毒素, 葛根素, 加权基因共表达网络分析, C6细胞

Abstract:

【Objective】The experiment used deoxynivalenol（DON）to construct a C6 cell injury model and analyzed the protective mechanism of puerarin（PUE）in alleviating DON-induced C6 cell injury. 【Method】By using RNA sequencing（RNA-seq）and weighted gene co expression network analysis（WGCNA）, we aimed to identify the key modules and core target genes, which was most closely related to the alleviation of PUE on DON-induced C6 cell injury. Furthermore, we explored the molecular mechanism of PUE in alleviating DON- induced neurotoxicity. 【Result】The PUE significantly inhibited the decrease in the cell viability induced by DON, restored the morphology of damaged C6 cells, and effectively alleviated DON-induced cytotoxicity. The RNA seq and WGCNA results indicated that the Blue module was the most relevant key module to the alleviation of PUE on DON-induced C6 cell injury, and S100a11, Pdia3 and Hsp90b1 genes were selected as the key core genes. 【Conclusion】These results suggest that PUE may alleviate DON-induced C6 cell injury by targeting key core genes and regulating in endoplasmic reticulum signaling pathway.

Key words: deoxynivalenol, puerarin, weighted gene co-expression network analysis（WGCNA）, C6 cell

赵海平, 刘林, 王昕璐, 岳鹏飞, 孔维军, 王蒙. 基于WGCNA探讨葛根素对呕吐毒素诱导C6细胞损伤的保护作用[J]. 生物技术通报, 2024, 40(9): 301-310.

ZHAO Hai-ping, LIU Lin, WANG Xin-lu, YUE Peng-fei, KONG Wei-jun, WANG Meng. Exploring the Protective Effect of Puerarin on Deoxynivalenol-induced C6 Cell Injury Based on WGCNA[J]. Biotechnology Bulletin, 2024, 40(9): 301-310.

图/表 6

图1 PUE对C6细胞活力的影响 A：PUE对正常C6细胞活力的影响；B：PUE对DON致C6细胞活力下降的影响。###：与Control组相比，P<0.001；***：与DON组相比，P<0.001

Fig. 1 Effect of PUE on C6 cell viability A：Effect of PUE on normal C6 cell viability. B: Effect of PUE on DON induced decrease in C6 cell viability. ###: Compared with the Control group, P<0.001.***: Compared with the DON group, P<0.001

图2 明场下不同处理组的细胞形态

Fig. 2 Cell morphologies in different treatment groups in the brightfield

图3 差异表达基因火山图 A：DON组较Control组差异表达基因火山图；B：PUE组较DON组差异表达基因火山图

Fig. 3 Volcano map of differentially expressed genes A: Volcano map of differentially expressed genes between the DON group and the control group; B: Volcano map of differentially expressed genes between the PUE group and the DON group

图4 加权基因共表达网络分析 A：标度独立性图；B：平均连通性图；C：基因聚类模块。上部为分层聚类树，下部为不同模块；D：基因共表达模块与不同表型之间的相关性热图。图中横坐标为性状，纵坐标为各个模块，每个格子中的数字表示模块与性状的相关性，值越接近1，表示该模块与性状正相关性越强；越接近-1，表示模块与性状负相关性越强，括号里的值代表显著性，值越小说明显著性越强

Fig. 4 WGCNA analysis A: Scale independence plot. B: Mean connectivity plot. C: Gene clustering module. The upper part is a hierarchical clustering tree, the lower part is a different module. D: Heat map of correlation between gene co-expression modules and different therapeutic phenotype. The abscissa is the trait, and the ordinate is each module, the number in each cell indicates the correlation between the module and the trait, and the closer the value is to 1, the stronger the positive correlation between the module and the trait. The closer to -1, the stronger the negative correlation between the module and the trait. The values in parentheses indicate significance, and the lower the value, the stronger the significance

图5 Blue模块中基因的富集分析 A：GO富集分析；B：KEGG富集分析

Fig. 5 Enrichment analysis of genes in the Blue module A: GO enrichment analysis. B: KEGG enrichment analysis

图6 关键核心基因的筛选 A：Blue模块中MCC排名前10基因的共表达网络。颜色的深浅表示基因连接度的高低，颜色越深表明连接度越高；B：韦恩图。红色圆圈为PUE组较DON组之间的差异表达基因，橘色圆圈为根据MCC算法筛选出来排名前10的核心基因，蓝色圆圈为Blue模块中的基因；C：关键核心基因在不同组中的相对表达量

Fig. 6 Screening of key core genes A: Co-expression network of top 10 MCC genes in the Blue module. The shade of color indicates the degree of gene connectivity, and the darker the color, the higher the degree of connectivity. B: Venn diagram. The red circles show the differentially expressed genes between the PUE group and the DON group, the orange circles are the top 10 core genes screened according to the MCC algorithm, the blue circles are the genes in the Blue module. C: Relative expression of key core genes in different groups

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