Thymosin β4 suppresses CCl(4) -induced murine hepatic fibrosis by down-regulating transforming growth factor β receptor-II.
The present study aimed to clarify the effects of thymosin β4 (Tβ4) on CCl4 -induced hepatic fibrosis in mice and to further explore the underlying mechanisms.
Expression of Tβ4 in fibrotic liver tissues was assessed by a quantitative real time-reverse transcriptase polymerase chain reaction and immunohistochemistry. The effects of intraperitoneal adeno-associated virus-Tβ4 (AAV-Tβ4) on CCl4 -induced hepatic fibrosis were observed by the evaluation of collagen deposition, hepatic stellate cell (HSC) activation and pro-fibrotic cytokine expression. In vitro tests with HSCs and hepatocytes were performed to confirm the effects of Tβ4.
The expression of Tβ4 was down-regulated in fibrotic mouse livers but was rapidly up-regulated by CCl4 -induced acute injury. AAV-Tβ4 pre-treatment significantly attenuated liver injury, collagen deposition, HSC activation and pro-fibrotic cytokine over-expression, such as transforming growth factor β1 (TGF-β1), platelet-derived growth factor B (PDGF-B), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) in CCl4 -intoxicated mouse livers. In vitro experiments showed that Tβ4 suppressed HSC proliferation, blunted TGF-β1-induced HSC activation and reduced TGF-β1-induced TGF-β1, PDGF-B, CTGF and PAI-1 expression in both HSCs and hepatocytes. Ectopic Tβ4 ameliorated the over-expression of TGF-β receptor-II (TGF-βRII) in the fibrotic mouse livers. Exogenous Tβ4 down-regulated TGF-βRII expression, whereas neutralizing endogenous extracellular Tβ4 with a specific antibody up-regulated TGF-βRII expression in cultured HSCs and hepatocytes.
Tβ4 possesses anti-fibrotic activity in the liver, which is attributable, at least partly, to down-regulating TGF-βRII and thereby blunting TGF-β1-mediated fibrogenetic signaling in both HSCs and hepatocytes.
Li H
，Li Q
，Zhang X
，Zheng X
，Zhang Q
，Hao Z
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Experimental study on H(2)O(2) activation of HSC-T6 and hepatic fibrosis in cholestatic mice by "Yajieshaba".
Yajieshaba (YJSB), approved by the Yunnan Provincial Food and Drug Administration in 2008, are known for their anti-inflammatory, antiviral, and pro-apoptotic properties, effectively treating Hepatic fibrosis (HF). However, its mechanism of action remains unclear.
The objective of this investigation is to explore how YJSB influences the TGF-β1/Smad signaling pathway as a strategy for reducing HF.
The establishment of a HF model in mice involved ligation of the common bile duct, followed by administration of YJSB. Body and liver weights were measured, and the liver index calculated. Serum levels of ALT, AST, ALP, TBA, and TBIL were assessed using colorimetric methods. Additionally, liver homogenates were analyzed for PIIINP, Col-IV, LN, HA, and Hyp, as well as TGF-β1 activity, using ELISA. Histological analyses of liver sections, stained with H&E, Ag, and Masson's trichrome, were performed to examine inflammation and the accumulation of collagen and reticular fibers. These studies aimed to elucidate the pharmacodynamic effects of YJSB on HF in mice with bile duct obstruction. The target pathways of YJSB were preliminarily identified through immunofluorescence detection of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, P-Smad3C, and Smad4 proteins. In vitro experiments included the induction of hepatic stellate cell (HSC-T6) activation by H2O2. A cell injury model was established for HSC-T6, and the CCK-8 assay was used to determine the optimal YJSB concentration and treatment duration. After pirfenidone (PFD) administration, which inhibits the TGF-β1/Smad pathway, the effects of YJSB on HSC-T6 cell proliferation were observed. ELISA assays quantified Col-III, α-SMA, and Col-I in cell lysates to assess YJSB's impact on collagen synthesis in HSC-T6 cells. Western blot analysis was performed to assess the protein levels within the TGF-β1/Smad signaling cascade.
In the HF mouse model, administration of YJSB notably augmented the body weight and reduced the liver index. Concurrently, there was an elevation in serum concentrations of ALP, AST, ALT, TBA, and TBIL. Similarly, in the liver homogenates of HF mice, increases were observed in the levels of HA, PIIINP, Col-IV, LN, Hyp, and TGF-β1. Histological assessments using H&E, Ag, and Masson stains indicated a substantial diminution in liver tissue damage. Through immunofluorescence analysis, it was discerned that YJSB modulated the expression of TGF-β1, P-Smad2L, P-Smad2C, and P-Smad3L downwards, while elevating P-Smad3C and Smad4 protein expressions. Additional investigations revealed a significant reduction in α-SMA, Col-I, and Col-III levels in cell culture fluids, suggesting a decrease in collagen synthesis and a protective role against cellular damage. Western blot analyses demonstrated that the TGF-β1/Smad pathway inhibitor, PFD, acted in synergy with YJSB, enhancing its regulatory effects on this pathway, decreasing levels of TGF-β1, P-Smad2L, P-Smad2C, P-Smad3L, and promoting the expression of P-Smad3C.
YJSB demonstrates a pharmacodynamic effect against HF, enhancing liver functionality and effectively mitigating the damage associated with bile duct obstruction. The proposed action mechanism of YJSB involves modulation of the TGF-β1/Smad signaling pathway. Research indicates that YJSB might play a role in suppressing the movement, programmed cell death, and activation of HSC-T6, potentially decelerating the advancement of hepatic fibrosis.
Bai Y
，Liu F
，Luo S
，Wan Y
，Zhang L
，Wu X
，Chen Q
，Xie Y
，Guo P
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