Optimised formula of Fufang Biejia Ruangan tablets alleviates renal fibrosis by suppressing matrix-stiffness-induced fibroblast activation via inhibition of integrin αVβ1 binding.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Biejia Ruangan Tablets (FFBJ), a traditional Chinese medicine, has long been used in the management of fibrosis, demonstrating notable efficacy and safety. However, the bioactive components of FFBJ and their mechanisms of action remain poorly understood.

AIM OF THE STUDY: To identify the core bioactive components of FFBJ and elucidate their mechanisms of action against renal fibrosis.

MATERIALS AND

METHODS

The constituents of FFBJ were characterised using high-performance liquid chromatography-High-resolution mass spectrometry (HPLC-HRMS). A multi-tiered strategy integrating pharmacokinetic evaluation, ADMET predictions, molecular docking, and efficacy assessments (MTT/Sirius-Red assays) identified five core bioactive compounds. The synergistic ratios of these compounds were optimised using the Box-Behnken design. In vitro, TGF-β1-stimulated NRK49F cells were treated with FFBJ-COF, a compound composed optimised formula from FFBJ, at low (half-medium dose), medium (30 μM taxifolin, 7.732 μM turtle-shell heptapeptide [OC1], 13.623 μM quercetin, 13 μM curcumenol, and 3.3 μM hederagenin), or high (double-medium dose) concentrations. In vivo, mice with unilateral ureteral obstruction (UUO) were treated with FFBJ-COF via gavage at low (half-medium dose), medium (64.85 mg/kg/d taxifolin, 30.30 mg/kg/d turtle-shell heptapeptide [OC1], 29.25 mg/kg/d quercetin, 21.83 mg/kg/d curcumenol, and 10.07 mg/kg/d hederagenin), or high (1.5-fold medium dose) levels. Polyacrylamide hydrogels (1-20 kPa) were employed to mimic fibrotic-matrix stiffness. Co-immunoprecipitation and western blotting were used to investigate the regulation of integrin αVβ1 binding and the downstream FAK/RhoA/MRTF-A signalling.

RESULTS

HPLC-HRMS analysis revealed 76 components. FFBJ-COF, comprising curcumenol, hederagenin, taxifolin, quercetin, and OC1, was formulated based on efficacy screening and ratio optimization. FFBJ-COF treatment attenuated renal injury and fibrosis by reducing fibroblast activation and extracellular matrix (ECM) deposition in both in vivo and in vitro model. Mechanistic studies showed that ECM stiffness activated fibroblasts, which was inhibited by FFBJ-COF. Co-immunoprecipitation demonstrated that matrix stiffness enhanced the binding of integrin αVβ1, whereas FFBJ-COF suppressed this interaction and inhibited the downstream FAK/RhoA/MRTF-A signalling pathway.

CONCLUSION

FFBJ-COF was identified as the core bioactive fraction of FFBJ. FFBJ-COF exerts anti-fibrotic effects by inhibiting integrin αVβ1 binding, thereby suppressing the FAK/RhoA/MRTF-A signalling and disrupting the positive feedback loop between ECM stiffness and fibrosis. These findings highlight the potential of FFBJ as a novel therapeutic agent for renal fibrosis.