Xing Lu, Hu Xu-Dong, Tao Yan-Yan, Peng Yuan, Liu Cheng-Hai
Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine Shanghai 201203, China.
Department of Biology, School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine Shanghai 201203, China.
Zhongguo Zhong Yao Za Zhi. 2022 Jun;47(11):3029-3037. doi: 10.19540/j.cnki.cjcmm.20211214.401.
We have demonstrated that Fuzheng Huayu Recipe(FZHY) plays an anti-liver fibrosis role by regulating the polarization of intrahepatic macrophages, while the key targets in macrophages and the effective components of FZHY remain unclear. In this study, we obtained the potential anti-liver fibrosis target set of FZHY through network pharmacological analysis, and the differentially expressed gene set of FZHY for the prevention and treatment of mouse liver fibrosis through RNA-Seq of the liver tissue. The potential core targets of FZHY against liver fibrosis were obtained by degree value analysis of the common target proteins between the above two sets. Then, through the retrieval of PubMed database, we identified the potential key targets in macrophages. After that, the effective components in FZHY corresponding to key targets were obtained by reverse pharmacological analysis. Finally, we verified the regulatory effects of these effective components on the expression of key target genes by using the lipopolysaccharide-induced M1 macrophages derived from THP-1 cells. The RNA-Seq data combined with network pharmacological analysis showed that FZHY might alleviate liver fibrosis by regulating the expression of CCL2, TIMP1, and MMP2 genes in macrophages. The results of in vivo experiments showed that FZHY significantly inhibited the expression of CCL2 and TIMP1 genes and promoted the expression of MMP2 genes in liver tissues of liver fibrosis mice. The results of in vitro experiments demonstrated that FZHY and its four effective components(luteolin, ursolic acid, quercetin, and danshensu) significantly inhibited the expression of CCL2 and TIMP1 genes in M1 macrophages derived from THP-1 cells. In addition, the expression of MMP2 gene was up-regulated by luteolin, ursolic acid, and quercetin, not affected by FZHY, and down-regulated by danshensu. FZHY could inhibit the expression of CCL2 and TIMP1 genes in M1 macrophages by the four effective components to achieve the anti-inflammatory and anti-liver fibrosis effects.
我们已经证明，扶正化瘀方（FZHY）通过调节肝内巨噬细胞的极化发挥抗肝纤维化作用，而巨噬细胞中的关键靶点以及FZHY的有效成分仍不清楚。在本研究中，我们通过网络药理学分析获得了FZHY潜在的抗肝纤维化靶点集，并通过对肝组织进行RNA测序获得了FZHY防治小鼠肝纤维化的差异表达基因集。通过对上述两组共同靶蛋白的度值分析，获得了FZHY抗肝纤维化的潜在核心靶点。然后，通过检索PubMed数据库，我们确定了巨噬细胞中的潜在关键靶点。之后，通过反向药理学分析获得了FZHY中与关键靶点对应的有效成分。最后，我们使用脂多糖诱导的源自THP-1细胞的M1巨噬细胞，验证了这些有效成分对关键靶基因表达的调节作用。RNA测序数据与网络药理学分析表明，FZHY可能通过调节巨噬细胞中CCL2、TIMP1和MMP2基因的表达来减轻肝纤维化。体内实验结果表明，FZHY显著抑制肝纤维化小鼠肝组织中CCL2和TIMP1基因的表达，并促进MMP2基因的表达。体外实验结果表明，FZHY及其四种有效成分（木犀草素、熊果酸、槲皮素和丹参素）显著抑制源自THP-1细胞的M1巨噬细胞中CCL2和TIMP1基因的表达。此外，木犀草素、熊果酸和槲皮素上调MMP2基因的表达，FZHY对其无影响，丹参素下调MMP2基因的表达。FZHY可通过这四种有效成分抑制M1巨噬细胞中CCL2和TIMP1基因的表达，从而实现抗炎和抗肝纤维化作用。
