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Salvianolic acid A (SA-A), a water-soluble compound extracted from traditional Chinese herb Radix Salvia miltiorrhiza, has anti-fibrotic effects on carbon tetrachloride (CCl4)-induced liver fibrosis. However, the underlying molecular mechanism remains unclear. Thus, this study aimed to elucidate the molecular mechanism underlying the anti-fibrotic effects of SA-A on CCl4-induced liver fibrosis in mice. All mice (except control group) were intraperitoneally administered CCl4 dissolved in peanut oil to induce liver fibrosis. Treatment groups were then gavaged with SA-A (20 or 40 mg/kg). The liver function index; liver fibrosis index; and superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) levels were determined. Furthermore, histopathological changes in liver tissues were observed via hematoxylin–eosin and Masson's trichrome staining. The expression of α-smooth muscle actin (α-SMA) and collagen I was detected using immunofluorescence, and the mRNA levels of inflammatory factors were determined using quantitative polymerase chain reaction. Finally, western blotting and immunofluorescence were used to determine the expression levels of proteins related to Nrf2/HO-1, NF-κB/IκBα, p38 MAPK, and JAK1/STAT3 signaling pathways. The results showed that SA-A could ameliorate CCl4-induced liver injury and liver fibrosis, improve morphology, and alleviate collagen deposition in the fibrotic liver. Moreover, SA-A could regulate the Nrf2/HO-1, NF-κB/IκBα, p38 MAPK, and JAK1/STAT3 signaling pathways; increase the levels of SOD and GSH-Px; and decrease MDA level in the fibrotic liver. Collectively, our study findings indicate that SA-A is effective in preventing liver fibrosis in mice by inhibiting inflammation and oxidative stress via regulating the Nrf2/HO-1, NF-κB/IκBα, p38 MAPK, and JAK1/STAT3 signaling pathways. [ABSTRACT FROM AUTHOR]

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFR β inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFR β inhibitors, salvianolic acid B and gomisin D, were screened out with K D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α -SMA and collagen Ӏ mRNA levels raised by TGF- β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo , Sal B could also attenuate CCl 4 -induced liver fibrosis by downregulating PDGFR β downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials. In-situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for drug screening and affinity determination. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • The target of Sal B against liver fibrosis was investigated in vitro and in vivo for the first time. • The binding ability of Sal B to PDGFRβ was identified. • The underlying mechanism of Sal B inhibiting HSC activation and improving liver fibrosis by targeting PDGFRβ was elucidated. • Sal B can promote the apoptosis of activated HSCs induced by PDGF-BB. • Sal B can inhibit the inflammatory response in CCl 4 -induced liver fibrosis mice. Liver fibrosis is a reversible pathological process and a wound healing response to liver injury. As an early stage of various liver diseases, liver fibrosis can develop into cirrhosis, liver failure, and even liver cancer if not controlled in time. Salvia miltiorrhiza is a medicinal plant with hepatoprotective effects. Salvianolic acid B (Sal B) is the representative component of S. miltiorrhiza. Many studies have reported the anti-liver fibrosis effects and mechanisms of Sal B. However, the direct anti-fibrotic targets of Sal B have not yet been reported. Platelet-derived growth factor receptor β (PDGFRβ) is one of the most classical targets in liver fibrosis, which is closely related to hepatic stellate cells (HSCs) activated. Previously, we established and applied a PDGFRβ affinity chromatography model, and found that Sal B binds well to PDGFRβ. Therefore, this study aimed to investigate the direct targets of Sal B against liver fibrosis. We confirmed the binding ability of Sal B to PDGFRβ by molecular docking and a surface plasmon resonance biosensor. Our findings indicated that Sal B targeted PDGFRβ to inhibit the activation, migration and proliferation of HSCs and suppressed the PDGF-BB-induced PDGFRβ signaling pathway. Annexin V-FITC/PI assay showed that Sal B reversed the PDGF-BB-induced decrease in HSC apoptosis rate. In the mouse liver fibrosis model, Sal B inhibited the PDGFRβ signaling pathway, HSC activation and reduced inflammatory response, ultimately improved CCl 4 -induced liver fibrosis. In summary, the direct anti-fibrotic targets of Sal B may be PDGFRβ, and this study clarified the anti-liver fibrosis effects and mechanism of Sal B. [ABSTRACT FROM AUTHOR]

Platelet-derived growth factor receptor β (PDGFRβ) plays an important role in hepatic fibrosis and is closely associated with hepatic stellate cells (HSCs) activation. Previously, by modeling PDGFRβ affinity chromatography, we found that gomisin D can target PDGFRβ. However, whether gomisin D has anti-fibrosis effects through targeting PDGFRβ remained unclear. In this study, the effect of gomisin D on hepatic fibrosis was evaluated in vivo and vitro. HSC cell lines and primary HSC were cultured and functionally we found that gomisin D promotes HSC apoptosis, inhibits HSCs activation and proliferation. A male BALB/c mouse liver fibrosis model was established to comfirm gomisin D (especially in 50 mg/kg) could improve liver fibrosis by inhibiting HSCs activation. In addition, gomisin D had a good binding ability with PDGFRβ (KD = 3.3e-5 M). Mechanically, gomisin D regulated PDGF-BB/PDGFRβ signaling pathway by targeting PDGFRβ, further more inhibited HSC activation, subsequently inhibited inflammatory factors, ultimately improved CCl 4 -induced liver fibrosis. Overall, gomisin D could inhibit HSC proliferation and activation, promote HSC apoptosis, and alleviate CCl 4 -induced hepatic fibrosis by targeting PDGFRβ and regulating PDGF-BB/PDGFRβ signaling pathway. This study provides a new drug for anti-liver firbosis therapy, and elucidates the deeper mechanism of gomisin D against HSCs activation by targeting PDGFRβ. [ABSTRACT FROM AUTHOR]