Overexpression of estrogen receptor ß inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation.

BACKGROUND: Estrogen receptor ß (ERß) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERß. Here, we explore the effect of overexpressed ERß on human HSCs and the role of ERß in pharmacological action of calycosin. METHODS: LX-2 cells were transfected with lentivirus to overexpress ERß. In the presence or absence of overexpressed ERß, the effects of ERß and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-ß1-induced LX-2 cells and the role of ERß in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERß or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERß on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot. RESULTS: ERß overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERß or calycosin alone inhibited the TGF-ß1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERß and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERß overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed. CONCLUSIONS: ERß mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect.

Ershiwuwei Lvxue Pill alleviates rheumatoid arthritis by different pathways and produces changes in the gut microbiota.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease that often results in joint destruction. Ershiwuwei Lvxue Pill (ELP), a prescription of Tibetan medicine, has been used for centuries for the clinical treatment of RA in Tibet, China. In a previous study, we reported that ELP could ameliorate RA symptoms in CIA rats by inhibiting the inflammatory response and inducing apoptosis in synovial tissues. It is still needed further to clarify the mechanisms of action of ELP in mitigating RA. PURPOSE: In this study, we aim to elucidate the mechanism of action of ELP to improve RA joint damage and explore the changes in the intestinal flora and host metabolites. METHODS: Firstly, we analyzed the main absorbed constituents of ELP in the serum of rats by ultra-performance liquid chromatography quadrupole-time-flight mass spectrometry (UPLC-Q-TOF/MS). Then, we verified the alleviating effects of ELP on cartilage injury and bone erosion as well as the inflammatory response in CIA rats by microCT, H&E staining, safranin-O staining, and ELISA. Moreover, we investigated the main factors that mediate joint damage, including the production of matrix metalloproteinases (MMPs) and osteoclast activity in the ankle of rats by immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) staining. Further, we explored the molecular mechanisms of the MMPs production and osteoclast activity in CIA rats treated with ELP through various experiments such as ELISA, qRT-PCR, western blotting, and immunofluorescence assay. Besides, we investigated gut microbiota composition by 16S rDNA sequencing and serum metabolites through untargeted metabolomics. In addition, we analyzed the correlation between gut microbiota and metabolites by Spearman correlation analysis. RESULTS: In this study, we identified 20 compounds from rat serum samples, which could be the ELP components that improve RA. Moreover, we found that ELP could alleviate cartilage and bone injury by reducing MMP-1, MMP-3, and MMP-13 expression and osteoclast activity in CIA rats. Further studies demonstrated that ELP could reduce joint damage by inhibiting osteoprotegerin (OPG)/receptor activator for nuclear factor-κB ligand (RANKL) /nuclear factor-κB (NF-κB) and extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinases (JNK) signal pathways. The 16S rDNA sequencing analysis indicated that there was a significant difference in the gut microbiota composition between the normal and CIA rats, and these differences were changed after ELP administration. ELP could alter the gut microbiota by increasing the abundance of the genus Lactobacillus and decreasing the abundance of Dorea, [Eubacterium]_ventriosum_group, Anaerostipes, Collinsella, Coprococcus_1, Ruminiclostridium_5, Ruminococcus_1, Family_XIII_UCG-001, Butyricicoccus, Erysipelotrichaceae_UCG-003, Lachnoclostridium, Faecalibacterium, Lachnospiraceae_UCG-010, Roseburia, Rs-E47_termite_group_norank, Treponema_2 genera. Non-targeted metabolomics analysis showed that ELP reduced arachidonic acid levels. The serum arachidonic acid level was significantly correlated with the abundance of 41 genera, particularly Collinsella and Lactobacillus. CONCLUSION: Our study shows that ELP can improve RA joint damage by inhibiting MMPs production and osteoclast activity, and regulating intestinal flora and host metabolites, which provides a novel insight into the ELP in alleviating RA.