[Angelicae Sinensis Radix extract attenuates synovial fibrosis via H3K18la/ METTL3/m~6A axis-dependent modification...

This study explores whether Angelicae Sinensis Radix extract alleviates fibrosis of human fibroblast-like synoviocytes(FLSs) by regulating the histone H3 lysine 18 lactylation(H3K18la)/methyltransferase-like protein 3(METTL3)/N~6-methyladenosine(m~6A) axis-mediated m~6A methylation of Netrin-1. FLSs were grouped as follows: control, transforming growth factor(TGF)-β1, TGF-β1+Angelicae Sinensis Radix extract drug-containing serum(AS), and TGF-β1+AS+sodium lactate(Nala). Other groups except the control group were stimulated with 10 ng·mL~(-1) TGF-β1 for 24 h for the modeling of knee osteoarthritis(KOA)-like fibrosis. The TGF-β1+AS group was treated with 10% AS-containing serum for 24 h, while the TGF-β1+AS+Nala group received a combination of AS and 5 mmol·L~(-1) Nala(a METTL3 inhibitor) for 24 h. Following the interventions, hematoxylin-eosin(HE) staining and Masson's trichrome staining were performed to evaluate the effects of AS on cellular morphology and collagen deposition. Western blot was performed to analyze the levels of pan-Kla, H3K18la, METTL3, inflammatory cytokines [interleukin-6(IL-6), tumor necrosis factor-alpha(TNF-α), and interleukin-1 beta(IL-1β)], and fibrosis-related proteins [collagen Ⅰ, alpha-smooth muscle actin(α-SMA), tissue inhibitor of matrix metalloproteinase-1(TIMP-1), and vimentin]. Immunofluorescence staining was employed to measure the levels of H3K18la and α-SMA, and qPCR was employed to measure the mRNA level of METTL3. ELISA quantified the levels of IL-6, TNF-α, and IL-1β in supernatants. Online tools were used to predict the m~6A methylation sites on Netrin-1. Lentiviral transfection was used to knock down METTL3(sh-METTL3) and blank plasmid(sh-NC) or overexpress METTL3(oe-METTL3), Netrin-1(oe-Netrin-1), and blank plasmid(oe-NC). RNA immunoprecipitation sequencing(RIP) was conducted to study the mRNA interaction between METTL3 and Netrin-1. MeRIP-qPCR was employed to assess the m~6A level of Netrin-1, while qPCR and Western blot were employed to evaluate the mRNA and protein levels, respectively, of METTL3 and Netrin-1. Compared with the control group, the TGF-β1 group exhibited significantly elevated levels of pan-Kla, H3K18la, METTL3, IL-6, TNF-α, IL-1β, collagen Ⅰ, α-SMA, TIMP-1, and vimentin. AS reduced the levels of these factors, while the TGF-β1+AS+Nala group showed increased levels compared with the TGF-β1+AS group. Netrin-1 contained multiple m~6A methylation sites. Compared with the IgG control group, sh-METTL3 lowered the levels of Netrin-1 and m~6A-modified Netrin-1, whereas oe-METTL3 increased the levels of Netrin-1 and m~6A-modified Netrin-1. In addition, sh-METTL3 downregulated the mRNA/protein levels of METTL3 and Netrin-1. AS regulates the H3K18la/METTL3/m~6A axis to inhibit the m~6A methylation of Netrin-1, reduce inflammatory cytokine secretion, and down-regulate fibrosis-related gene expression, thereby alleviating synovial fibrosis in KOA.