二氢杨梅素(DHM)是一种生物活性的黄酮类化合物,从菊花中提取,有各种活动。在本研究中,通过网络药理学和实验验证,探讨二氢杨梅素(DHM)缓解肝硬化的分子机制。细胞模型由TGF-β1激活人肝星状细胞系(HSC;LX-2)诱导。α-SMA的蛋白质水平,胶原蛋白I,使用Westernblot检测LX-2细胞内的胶原蛋白III和通路相关蛋白。进行EdU染色以检测细胞增殖。免疫荧光染色检测α-SMA和I型胶原的表达水平。从PubChem数据库中筛选DHM的药物靶标。鉴定了肝硬化数据集GSE14323中的差异表达基因。使用qRT-PCR验证所鉴定的药物靶标在LX-2细胞中的表达。结果表明,TGF-β1处理显著增加LX-2细胞的活力,促进细胞增殖,和升高的α-SMA,胶原蛋白I,和胶原蛋白III含量。DHM治疗可以部分消除TGF-β1的影响,如抑制细胞活力和增殖和减少α-SMA所证明的,胶原蛋白I,和胶原蛋白III含量。经过网络药理学分析,9个差异表达的靶基因(MMP2,PDGFRB,PARP1,BCL2L2,ABCB1,TYR,CYP2E1,SQSTM1和IL6)在肝硬化中被鉴定。根据qRT-PCR验证,DHM能抑制MMP2、PDGFRB的表达,PARP1,CYP2E1,SQSTM1和IL6,并增强LX-2细胞内ABCB1的表达水平。此外,DHM抑制TGF-β1诱导的HSC中mTOR和MAPK信号通路。总之,DHM可以抑制HSC激活,这可以通过作用于MMP2、PDGFRB、PARP1、CYP2E1、SQSTM1、IL6和ABCB1基因及其下游信号通路,包括mTOR和MAPK信号通路。
Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present
study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-β1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-β1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-β1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-β1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.