丹参素抗类风湿性关节炎成纤维样滑膜细胞增殖和迁移的机制:基于转录组学及实验验证

Mechanism of danshensu against the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes: a study based on transcriptomics and experimental validation

摘要:

目的 研究丹参素（SAA）对人类风湿性关节炎成纤维样滑膜细胞（MH7A）增殖和迁移的作用及其作 用机制。方法 通过 CCK8实验探究不同浓度的 SAA对肿瘤坏死因子 α（TNF-α）诱导的 MH7A细胞增殖的作用。 通过划痕愈合实验探究 SAA 对 TNF-α 诱导的 MH7A 细胞迁移的作用。利用转录组测序分析筛选 SAA 作用于 TNF-α诱导MH7A细胞的差异基因，构建差异基因的蛋白质互作（PPI）网络，对差异基因进行GO和KEGG分析，筛 选出核心基因，并进一步通过分子对接及动力学模拟，验证 SAA 对核心靶点的作用，利用 qPCR 和 Western blot在 MH7A 炎症细胞模型上进行核心靶点的生物学验证。结果 10 μg/L 的 TNF-α 能够诱导 MH7A 细胞增殖（P < 0.001），不同剂量的 SAA 干预 24、48、72 h后，10 ~ 160 µmol/L浓度组的 SAA 均能抑制 TNF-α诱导的 MH7A 细胞的 增殖（P < 0.05）。10 µmol/L的SAA干预24 h后，能抑制TNF-α诱导的MH7A细胞的迁移（P < 0.01）。转录组分析筛 选出100个SAA作用于TNF-α诱导的MH7A细胞的基因。GO分析结果显示SAA对TNF-α诱导的MH7A细胞的作用 主要与黏着斑、钙黏蛋白结合等相关。KEGG分析结果提示MAPK信号通路可能是SAA作用于TNF-α诱导的MH7A 细胞的主要信号通路。分子对接结果显示靶点超氧化物歧化酶2（SOD2）与SAA的对接分数为-7.290 kcal/mol，在 体内能够与 SAA自主稳定结合。分子生物学结果显示，与正常组相比，TNF-α诱导的 MH7A细胞 SOD2 mRNA表 达显著上升（P < 0.000 1）。与模型组相比，10 µmol/L SAA 的干预显著降低 SOD2 mRNA 的表达（P < 0.000 1）。与 正常组相比，TNF-α诱导的MH7A细胞SOD2、CCND1蛋白表达显著上升（P < 0.01），10 µmol/L SAA的干预显著降 低SOD2、CCND1蛋白的表达（P < 0.05）。结论 SAA能够抑制类风湿性关节炎成纤维样滑膜细胞的增殖和迁移， 其机制可能与MAPK信号通路、SOD2基因的调控有关。

Abstract:

Objective To investigate the effect and mechanism of Salvianic acid A (SAA) on the proliferation and migration of human rheumatoid arthritis fibroblast-like synoviocytes (MH7A cells). Methods The effect of different concentrations of SAA on the proliferation of tumor necrosis factor-α (TNF-α)-induced MH7A cells was examined using the CCK-8 assay. The effect of SAA on the migration of TNF-α-induced MH7A cells was investigated using a scratch wound healing assay. Transcriptome sequencing analysis was employed to screen differentially expressed genes in SAAtreated TNF-α -induced MH7A cells. A protein-protein interaction (PPI) network was constructed for these genes, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to identify core genes. Molecular docking and dynamics simulations were further conducted to validate the interaction between SAA and core targets. Finally, qPCR and Western blot were used for biological validation of the core targets in the MH7A inflammatory cell model. Results TNF-α (10 μg/L) significantly induced MH7A cell proliferation (P < 0.001). Treatment with SAA at concentrations ranging from 10 to 160 µmol/L for 24, 48, and 72 hours effectively inhibited the TNF- α -induced proliferation of MH7A cells (P < 0.05). Furthermore, SAA (10 µmol/L) treatment for 24 hours significantly suppressed the TNF- α -induced migration of MH7A cells (P < 0.01). Transcriptome analysis identified 100 differentially expressed genes in SAA-treated TNF-α-induced MH7A cells. GO analysis indicated that the effects of SAA on these cells were primarily associated with focal adhesion and cadherin binding. KEGG pathway analysis suggested that the MAPK signaling pathway might be a key pathway involved in SAA's action. Molecular docking results showed a binding score of -7.290 kcal/mol between the target Superoxide dismutase 2 (SOD2) and SAA, indicating a stable and autonomous binding in silico. Molecular biological results revealed that compared to the normal group, SOD2 mRNA expression was significantly increased in TNF-α-induced MH7A cells (P < 0.0001). Compared to the model group (TNF- α alone), intervention with 10 µmol/L SAA significantly reduced SOD2 mRNA expression (P < 0.0001). Furthermore, compared with the normal group, the protein expression levels of SOD2 and CCND1 were significantly upregulated in TNF- α -induced MH7A cells (P < 0.01). Treatment with 10 µmol/L SAA significantly downregulated the protein expression of SOD2 and CCND1 (P < 0.05). Conclusion SAA can inhibit the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes, and its mechanism may be related to the regulation of the MAPK signaling pathway and the SOD2 gene.