Exploring the Protective Effect of Puerarin on Deoxynivalenol-induced C6 Cell Injury Based on WGCNA

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

Abstract

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

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.

Figures/Tables 6

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

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

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

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

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

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

References 33

[1]	郭红艳, 杨家庆, 刘园, 等. 呕吐毒素的食品污染、吸收代谢及肠道毒性研究进展[J]. 食品科学, 2022, 43(19): 382-390.
	Guo HY, Yang JQ, Liu Y, et al. Progress in research on food pollution by and absorption, metabolism and intestinal toxicity of vomitoxin[J]. Food Sci, 2022, 43(19): 382-390.
[2]	Ganesan AR, Mohan KN, Karthick Rajan D, et al. Distribution, toxicity, interactive effects, and detection of ochratoxin and deoxynivalenol in food: a review[J]. Food Chem, 2022, 378: 131978.
[3]	Gruber-Dorninger C, Jenkins T, Schatzmayr G. Global mycotoxin occurrence in feed: a ten-year survey[J]. Toxins, 2019, 11(7): 375.
[4]	Dong JN, Zhao ZK, Wang ZQ, et al. Impact of deoxynivalenol on rumen function, production, and health of dairy cows: insights from metabolomics and microbiota analysis[J]. J Hazard Mater, 2024, 465: 133376.
[5]	Zhang JJ, You L, Wu WD, et al. The neurotoxicity of trichothecenes T-2 toxin and deoxynivalenol(DON): current status and future perspectives[J]. Food Chem Toxicol, 2020, 145: 111676.
[6]	刘林, 王昕璐, 赵海平, 等. 天然酚类化合物对呕吐毒素诱导毒性损伤保护作用的研究进展[J]. 食品安全质量检测学报, 2023, 14(13): 211-220.
	Liu L, Wang XL, Zhao HP, et al. Research progress of protection of natural phenolic compounds against deoxynivalenol-induced toxicity[J]. J Food Saf Qual, 2023, 14(13): 211-220.
[7]	Li CX, Liu Y. Puerarin reduces cell damage from cerebral ischemia-reperfusion by inhibiting ferroptosis[J]. Biochem Biophys Res Commun, 2024, 693: 149324.
[8]	Hou BY, Ma P, Yang XY, et al. In silico prediction and experimental validation to reveal the protective mechanism of Puerarin against excessive extracellular matrix accumulation through inhibiting ferroptosis in diabetic nephropathy[J]. J Ethnopharmacol, 2024, 319(Pt 2): 117281.
[9]	He LD, Wu XY, Zhang X, et al. Puerarin protects against H₂O₂-induced apoptosis of HTR-8/SVneo cells by regulating the miR-20a-5p/VEGFA/Akt axis[J]. Placenta, 2022, 126: 202-208.
[10]	Lin SP, Zhu LD, Shi HJ, et al. Puerarin prevents sepsis-associated encephalopathy by regulating the AKT1 pathway in microglia[J]. Phytomedicine, 2023, 121: 155119.
[11]	刘畅, 刘思章, 于靖辉, 等. 基于权重基因共表达网络分析挖掘人参皂苷Rh₁生物合成相关基因[J]. 中草药, 2024, 55(8): 2723-2733.
	Liu C, Liu SZ, Yu JH, et al. Mining of genes related to biosynthesis of ginsenoside Rh₁ based on WGCNA in Panax ginseng[J]. Chin Tradit Herb Drugs, 2024, 55(8): 2723-2733.
[12]	谢雯, 任昊, 魏涛, 等. 基于RNA-seq技术分析microRNA 29a对八眉猪仔猪小肠上皮细胞基因表达的影响[J]. 农业生物技术学报, 2022, 30(8): 1547-1558.
	Xie W, Ren H, Wei T, et al. Analysis of the effect of micro RNA 29a on gene expression in small intestinal epithelial cells of bamei piglets(Sus scrofa)based on RNA-seq technology[J]. J Agric Biotechnol, 2022, 30(8): 1547-1558.
[13]	李玉清, 王洪连, 胡琼丹, 等. 基于转录组测序和WGCNA分析挖掘黄芪三七合剂治疗IRI急性肾损伤的关键基因[J]. 中药药理与临床, 2024, 40(2): 40-47.
	Li YQ, Wang HL, Hu QD, et al. Key genes of astragali radix and notoginseng radix et rhizoma compounds(an)in treating iri-induced aki through rna-seq and wgcna analysis[J]. Pharmacol Clin Chin Mater Med, 2024, 40(2): 40-47.
[14]	原佳妮, 赵延辉, 侍玉梅, 等. 利用WGCNA挖掘种公鸡睾丸和附睾中影响精子活力的核心基因[J]. 江苏农业学报, 2023, 39(3): 762-769.
	Yuan JN, Zhao YH, Shi YM, et al. Mining of hub genes affecting sperm motility in testes and epididymides of breeder cocks by WGCNA method[J]. Jiangsu J Agric Sci, 2023, 39(3): 762-769.
[15]	孔德平, 亓会斌, 冯兆阳, 等. 星形胶质细胞和小胶质细胞对中枢神经系统的作用[J]. 中国药理学与毒理学杂志, 2021, 35(9): 678-679.
	Kong DP, Qi HB, Feng ZY, et al. Effects of astrocytes and microglia on central nervous system[J]. Chin J Pharmacol Toxicol, 2021, 35(9): 678-679.
[16]	潘东宁, 傅攀峰, 王红, 等. 核糖核酸酶抑制因子对H₂O₂损伤的大鼠神经胶质瘤细胞系C6的影响[J]. 中国生物化学与分子生物学报, 2002, 18(6): 115-119.
	Pan DN, Fu PF, Wang H, et al. Effects of ribonuclease inhibitor on the rat glial cell line C6 injured by H₂O₂[J]. Chin J Biochem Mol Biol, 2002, 18(6): 115-119.
[17]	Kalagatur NK, Abd Allah EF, Poda S, et al. Quercetin mitigates the deoxynivalenol mycotoxin induced apoptosis in SH-SY5Y cells by modulating the oxidative stress mediators[J]. Saudi J Biol Sci, 2021, 28(1): 465-477. doi: 10.1016/j.sjbs.2020.10.030 pmid: 33424329
[18]	Wang XC, Chu XY, Cao L, et al. The role and regulatory mechanism of autophagy in hippocampal nerve cells of piglet damaged by deoxynivalenol[J]. Toxicol In Vitro, 2020, 66: 104837.
[19]	Hou SL, Ma JJ, Cheng YQ, et al. DON induced DNA damage triggers absence of p53-mediated G2 arrest and apoptosis in IPEC-1 cells[J]. Toxicology, 2024, 501: 153707.
[20]	Wang N, Zhang YM, Wu L, et al. Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J]. Neuropharmacology, 2014, 79: 282-289. doi: 10.1016/j.neuropharm.2013.12.004 pmid: 24333675
[21]	雷雨广, 徐继鹏, 陈超, 等. 干扰或过表达PDIA3对过氧化氢诱导的星形胶质细胞损伤的影响[J]. 中国老年学杂志, 2020, 40(3): 585-589.
	Lei YG, Xu JP, Chen C, et al. Effects of interference or overexpression of PDIA3 on hydrogen peroxide-induced astrocyte damage[J]. Chin J Gerontol, 2020, 40(3): 585-589.
[22]	储天琪, 刘峰, 陈红林, 等. 小黄鱼肝脏中hsp90b1和hspb1对温度胁迫的响应[J]. 农业生物技术学报, 2022, 30(3): 528-538.
	Chu TQ, Liu F, Chen HL, et al. Response of hsp90b1 and hspb1 to temperature stress in the liver of Larimichthys polyactis[J]. J Agric Biotechnol, 2022, 30(3): 528-538.
[23]	李杰峰. 缺氧微环境下DON诱发RAW_264.7细胞衰老的分子机制[D]. 荆州: 长江大学, 2023.
	Li JF. Molecular mechanism of DON-induced RAW_264.7 cell senescence under hypoxic microenvironment[D]. Jingzhou: Yangtze University, 2023.
[24]	Wan D, Wang X, Wu QH, et al. Integrated transcriptional and proteomic analysis of growth hormone suppression mediated by trichothecene T-2 toxin in rat GH3 cells[J]. Toxicol Sci, 2015, 147(2): 326-338. doi: 10.1093/toxsci/kfv131 pmid: 26141394
[25]	Li WQ, Han FB, Tang KF, et al. Inhibiting NF-κB-S100A11 signaling and targeting S100A11 for anticancer effects of demethylzeylasteral in human colon cancer[J]. Biomed Pharmacother, 2023, 168: 115725.
[26]	Zhang LQ, Zhu TT, Miao HL, et al. The calcium binding protein S100A11 and its roles in diseases[J]. Front Cell Dev Biol, 2021, 9: 693262.
[27]	成秀梅, 杨雅, 魏楚蓉, 等. 白藜芦醇通过Ca²⁺/Caspase-3途径减轻内质网应激诱导的神经细胞凋亡[J]. 南京医科大学学报: 自然科学版, 2023, 43(5): 678-683.
	Cheng XM, Yang Y, Wei CR, et al. Resveratrol attenuated endoplasmic reticulum stress-induced apoptosis through Ca²⁺/Caspase-3 pathway[J]. J Nanjing Med Univ Nat Sci, 2023, 43(5): 678-683.
[28]	Oakes SA, Papa FR. The role of endoplasmic reticulum stress in human pathology[J]. Annu Rev Pathol, 2015, 10: 173-194. doi: 10.1146/annurev-pathol-012513-104649 pmid: 25387057
[29]	Zhang J, Zhao QB, Xue ZH, et al. Deoxynivalenol induces endoplasmic reticulum stress-associated apoptosis via the IRE1/JNK/CHOP pathway in porcine alveolar macrophage 3D4/21 cells[J]. Food Chem Toxicol, 2023, 180: 114033.
[30]	杨敏. 脱氧雪腐镰刀菌烯醇、2, 5-己二酮对猪卵巢颗粒细胞的凋亡影响及转录组分析[D]. 合肥: 安徽农业大学, 2019.
	Yang M. Effect of deoxynivalenol and 2, 5-hexanedione on apoptosis of porcine ovarian granulosa cells and transcriptome analysis[D]. Hefei: Anhui Agricultural University, 2019.
[31]	Wang K, Zhu X, Zhang K, et al. Puerarin inhibits amyloid β-induced NLRP3 inflammasome activation in retinal pigment epithelial cells via suppressing ROS-dependent oxidative and endoplasmic reticulum stresses[J]. Exp Cell Res, 2017, 357(2): 335-340. doi: S0014-4827(17)30332-4 pmid: 28583762
[32]	Liu X, Huang R, Wan JY. Puerarin: a potential natural neuroprotective agent for neurological disorders[J]. Biomed Pharmacother, 2023, 162: 114581.
[33]	杨航, 杨晶欣, 郑兰兰, 等. 核糖体蛋白25(RPS25)通过调节c-Myc促进肾透明细胞癌的发生发展[J]. 基础医学与临床, 2019, 39(8): 1168-1174.
	Yang H, Yang JX, Zheng LL, et al. Ribosomal protein 25(RPS25)promotes the development of renal transparent cell carcinoma through regulating c-Myc[J]. Basic Clin Med, 2019, 39(8): 1168-1174.