Intervention Study on the Regulation of circAno6/miR-296-3p/TLR4 Signaling Axis by Shugan Jianpi Formula to Alleviate...

Liver fibrosis (LF) is a fibrotic and inflammatory condition resulting from chronic liver damage, and the activation of hepatic stellate cells (HSCs) is the central event. Our prior research has revealed that the circAno6/miR-296-3p/Toll-like receptor (TLR4) pathway is a key signaling axis in the pathogenesis of LF. Shugan Jianpi Formula (SGJPF) is a traditional Chinese medicinal treatment widely used to treat LF. We hypothesized that SGJPF exerts anti-LF effects by modulating the circAno6/miR-296-3p/TLR4 signaling axis, attenuating inflammatory responses, and inhibiting HSC activation, thereby ultimately mitigating LF. In this study, an SGJPF extraction solution was first prepared and administered to rats via gavage at a specified dosage. Blood samples were subsequently collected from the abdominal aorta to prepare SGJPF-containing serum. Optimal SGJPF intervention conditions were determined using the Cell Counting Kit-8 assay. Flow cytometry was used to assess JS-1 cell cycle progression. The levels of interleukin-1 beta (IL-1β) and IL-18 were measured by enzyme-linked immunosorbent assay. The expression levels of collagen type I (collagen I), α-smooth muscle actin (α-SMA), circAno6, TLR4, miR-296-3p, and NOD-like receptor protein 3 (NLRP3) were evaluated using Western blotting, immunofluorescence, and real-time quantitative polymerase chain reaction. The results indicated that the optimal intervention condition involved serum containing 20% SGJPF, which was administered for a duration of 48 h. SGJPF-containing serum has been shown to reduce the viability of JS-1 cells and decrease the cell count in the G2 + S phase, thereby affecting HSC function. Furthermore, SGJPF-containing serum effectively suppressed the expression levels of NLRP3, IL-1β, IL-18, collagen I, α-SMA, circAno6, and TLR4 while upregulating miR-296-3p expression. These findings suggest that SGJPF can modulate the activity of the circAno6/miR-296-3p/TLR4 signaling axis, attenuate inflammatory responses, and inhibit HSC activation, thereby mitigating LF.