Nrf2 mediates the protective effects of homocysteine by increasing the levels of GSH content in HepG2 cells.

Homocysteine (Hcy) and glutathione (GSH) are crucial reduction­oxidation mediators. The underlying mechanisms governing the effects of Hcy on GSH generation in the progression of alcoholic liver disease has so far received little attention. The present study hypothesized that the antioxidant transcriptional factor nuclear factor (erythroid­derived 2)­like 2 (Nrf2) may participate in Hcy­mediated regulation of GSH production in HepG2 human liver cancer cells. MTT assay was used to study the cytotoxicity of homocysteine, western blot analysis and immunofluorescence staining were used to determine the effect of Hcy on Nrf2 expression. Our data demonstrated that HepG2 cells exposed to exogenous levels of Hcy (0­100 µM) exhibited elevated GSH levels in a concentration­dependent manner. Furthermore, 4­hydroxynonenal (4­HNE)­induced cell injury was attenuated by Hcy; however, this protective effect was blocked by the GSH­production inhibitor buthionine sulfoximine. Hcy treatment was able to induce Nrf2 protein expression in HepG2 cells. Treatment with the Nrf2 activator tert­butylhydroquinone (0­100 µM) increased GSH expression in a concentration­dependent manner; however, Nrf2­siRNA abolished the Hcy­induced increase in GSH expression and cellular protection in 4­HNE­stressed HepG2 cells. In conclusion, the antioxidant transcriptional factor Nrf2 was demonstrated to mediate the Hcy­induced increase in GSH expression levels and cellular protection in HepG2 cells.

Inhibitory effect of baicalin on collagen-induced arthritis in rats through the nuclear factor-κB pathway.

This study focused on the potential therapeutic effect of baicalin on collagen-induced arthritis (CIA) in rats and the underlying mechanisms. The CIA rats were injected with baicalin (50, 100, or 200 mg/kg) once daily for 30 days. The rats were monitored for clinical severity of arthritis, and joint tissues were used for radiographic assessment and histologic examination. We quantified tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in experimental animals and used Western blots to assess levels of protein abundance, phosphorylation, and acetylation of nuclear factor (NF)-κB p65 and sirtuin 1 (sirt1) protein expression in joint tissues. Human fibroblast-like synoviocytes from rheumatoid arthritis (HFLS-RA) were adopted in further mechanistic investigations. Baicalin intraperitoneal injection for 30 days dose-dependently blocked clinical manifestations of CIA, such as functional impairment and swollen red paws. Meanwhile, it alleviated collagen-induced joint inflammation injury and inhibited the secretion of TNF-α and IL-1ß in both rat synovium and HFLS-RA. Further mechanistic investigations revealed that baicalin suppresses NF-κB p65 protein expression and phosphorylation in synovial tissue and human-derived synoviocytes. Moreover, the acetylation of NF-κB p65 was downregulated by baicalin, which negatively correlates with the baicalin-induced upregulation of sirt1 expression in the same conditions. The data indicate that CIA in rats can be alleviated by baicalin treatment via relieving joint inflammation, which is related to the suppression of synovial NF-κB p65 protein expression and the elevation of its deacetylation by sirt1.