Carapax Trionycis extracts inhibit fibrogenesis of activated hepatic stellate cells via TGF-β1/Smad and NFκB signaling

Zuliang Hu  ¹ , Pengtao You  ² , Sha Xiong  ³ , Jianrong Gao  ¹ , Yinping Tang  ³ , Xiaochuan Ye  ³ , Yu Xia  ³ , Dongquan Zhang  ¹ , Yanwen Liu  ³

• ¹ Zhejiang Quhua Hospital, Quzhou, Zhejiang, 324004, PR China.
• ² Key Laboratory of Resources and Chemistry of Chinese Medicine, Hubei University of Chinese Medicine, Wuhan, Hubei, 430065, PR China. Electronic address: tptyou@hbtcm.edu.cn.
• ³ Key Laboratory of Resources and Chemistry of Chinese Medicine, Hubei University of Chinese Medicine, Wuhan, Hubei, 430065, PR China.

Abstract

Carapax Trionycis is used as a traditional Chinese medicine with a long history of clinical application in China, and it represents an essential medication used for liver fibrosis treatment. Previous studies demonstrated that Carapax Trionycis extracts protect liver against fibrosis in CCL₄-induced animal models. This study investigated the anti-fibrotic molecular mechanisms exerted by Carapax Trionycis extracts with molecular weight less than 6 KD (CT6) in rat hepatic stellate cell line HSC-T6 activated by TGF-β1. HSC-T6 cells induced by TGF-β1 were used to evaluate CT6 anti-fibrotic effect in vitro. CCK8 was used to evaluate cell viability and CT6 effect on HSC-T6 proliferation. ELISA was performed to detect the presence of inflammatory cytokines. Western blot and q-PCR were performed to explore the molecular mechanisms. Our data demonstrated that CT6 did not clearly affect cell viability but suppressed TGF-β1-induced HSC-T6 proliferation. Collagen I and α-smooth muscle actin (α-SMA) protein levels were decreased by CT6 in TGF-β1-induced HSC-T6, followed by the inhibition of TIMP1, TIMP2 and TGF-β1/Smad pathway. Furthermore, CT6 decreased Jun D and p-p65 protein levels, down-regulated Tgf-β1, Tnf-α, Il-1β, Il-6 mRNA and TNF-α, IL-1β and IL-6 expression in TGF-β1-treated HSC-T6. These results suggested that CT6 inhibited HSC-T6 activation induced by TGF-β1, indicating the potential therapeutic effect of these extracts against liver fibrosis.

Keywords: CT6; Carapax trionycis; Liver fibrosis; Mechanisms; Pathway.