Role Of Histone Deacetylases (HDACs) In Progression And Reversal Of Liver Fibrosis In Mice

Fibrosis of liver refers to a overgenerous and invertible wound healing response caused by various chronic liver injury, is characterized by the excessive extracellular matrix(ECM) deposition. Studies have shown the excessive deposition of ECM mainly comes from activated hepatic stellate cells(HSCs). Although in normal liver, HSCs display a quiescent phenotype, upon liver insults from any etiological factors, such as alcohol, virus infection, auto-immune imbalances and so on, the resting state HSCs eventually trans-differentiate into myofibroblasts-like cells (MFBLC) with strong ability of proliferation, which is known as HSCs activation. Activation of HSCs is the critical step during liver fibrosis, and could be modulated by various cytokines and signaling pathways. Studies also found that activated HSCs can revert their phenotype to a quiescent status, eventually lead to reversal of liver fibrosis after removing risk factors.Accumulating evidence has demonstrated a potential role of histone deacetylases (HDACs) in liver fibrosis. However, the varieties features of HDACs in hepatic fibrosis are still remain largely unknown. Here, we systematically revealed the role of HDACs in liver fibrosis by establishing liver fibrosis models and liver fibrosis reversal models in vitro and in vivo, and may be useful in developing new therapy target for liver fibrosis. The main contents as follows: 1. Establishment of animal models of CC14-induced murine liver fibrosis models and murine liver fibrosis reversal models.C57BL/6J mice were randomly divided into three groups (twenty mice per group), including control group, model group (liver fibrosis group) and reversal group (spontaneously liver fibrosis reversal group).Liver fibrosis was generated by subcutaneous injection of a 10% diluted concentration of CC14 in olive oil (0.02 ml/g/mouse) twice a week for 4 weeks. Control mice were treated with olive oil(0.02 ml/g/mouse) at the same time intervals in the same method. The reversal mice were made by 8 weeks’ normal feeding after CC14 revocation.24h after the last injection, all mice were sacrificed and liver tissues were harvested for further hematoxylin eosin (H&E) and masson staining and immunohistochemical staining (IHC). And expressions ofCOL1α1 and α-SMA in liver tissues were measured by Real-time qPCR and Western blot analyses. All results proved in that CC14-induced liver fibrosis models and liver fibrosis reversal models have been well established.2. The mRNA levels of Class Ⅰ,Ⅱ and Ⅳ HDACs inCC14-induced murine liver fibrosis models and murine liver fibrosis reversal models1) The expression profile of Class Ⅰ, Ⅱ and Ⅳ HDACs inCC14-induced murine liver fibrosis models and murine liver fibrosis reversal models were verified by Real-time qPCR. Experimental results showed that the normal regulatory mechanisms of hepatic HDACs expressions have been disrupted during the progression of murine liver fibrosis, and the general mRNA expression pattern of HDACs in murine liver fibrosis tissues was elevated.2) Protein expression of histone H3 acetylation (Ac-H3) in CC14-induced murine liver fibrosis models and murine liver fibrosis reversal models were examined by Western blot analyses. Experimental results indicated the changes of Ac-H3 during the progression of murine liver fibrosis, and the protein expression of Ac-H3 in murine liver fibrosis tissues was decreased.3) Rat hepatic stellate cell line (HSC-T6) was used in the study, and administration with 10ng/ml TGF-β1 and 100nM TSA for 24h, harvesting all cells. Expressions of COL1α1 and a-SMA inTGF-β1-induced HSC-T6 cells and in bothTGF-β1and TSA treated cells were measured by Western Blot. Experimental results indicated that expressions of COLlaland a-SMA were up-regulated in TGF-β 1-induced activated HSC-T6 cells, but expressions of COL1α1and a-SMA were down-regulated after administration activated HSC-T6 cells with TSA.3. Expression analysis of HDAC2 in murine liver tissues and human liver tissuesAbove results indicated that among all tested HDACs, the expression of HDAC2 was up-regulated in the progression, but down-regulated in the reversal stage, of liver fibrosis. Although HDAC2 has been well characterized in hepatocellular carcinoma (HCC) and increasing evidence hints the potential role of HDAC2 in the progression of renal fibrosis and pulmonary fibrosis, the role of HDAC2 in liver fibrosis still beyond our understanding. Moreover, expression of HDAC2 in murine liver tissues was measured by IHC and Western Blot analysis. Experimental results indicated that the protein expression of HDAC2 in murine liver fibrosis tissues was significantly elevated, and protein expression of HDAC2 in murine liver fibrosis reversal tissues was obviously decreased. In addition, the up-regulated expression of HDAC2 was also confirmed in human liver fibrosis tissues by Western Blot analysis. So, the potential functional role of HDAC2 in liver fibrosis will be further studied.4. Expression of HDAC2 in HSC-T6 cells stimulated by TGF-β1 and MDI.HSC-T6 cells were used for experimental study, and administration with 0,5,10 and 15 ng/ml TGF-β1 for 24h, harvesting cells. Expressions of COL1α1, α-SMA and HDAC2 in HSC-T6 cells stimulated with different concentrations of TGF-β1 were analysed by Western Blot. Similarly, administration HSC-T6 cells withlOng/ml TGF-β1 for 0,12,24 and 48 h, harvesting cells. Expressions ofCOL1α1,α-SMA and HDAC2 in HSC-T6 cells stimulated at different time points of TGF-β1 were analysed by Western Blot. After administration HSC-T6 cells withlOng/ml TGF-β1 for 24h, incubating HSC-T6 cells with MDI for 48h, harvesting cells. Expressions of COL1α1,α-SMA and HDAC2 in HSC-T6 cells with different treatments were analysed by Western Blot. Experimental results indicated that expression of HDAC2 was elevated in dose- and time-dependently by administration HSC-T6 cells with TGF-β1, accompanied by the induction of COL1α1and α-SMA expressions. However, the expression of HDAC2 was obviously down-regulated in reverted HSC-T6 cells.5. Effect of HDAC2 inhibition on the activation of HSC-T6 cells stimulated by TGF-β1 and some of mechanisms study.1) HSC-T6 cells were used for experimental study. According to the nucleotide sequence of HDAC2, small interfering RNA of HDAC2(HDAC2-siRNA)was designed and synthesized, then using LipofectamineTM 2000transfected into HSC-T6 cells. Administration HSC-T6 cells withlOng/ml TGF-β1 for 24h. Cell cycle distribution was analyzed by flow cytometry. Flow cytometry results showed that compared to scrambled-siRNA transfection group, inhibition of HDAC2 could cause a delay in G0/G1 cells cycle progression. Prompting that HDAC2 silencing could inhibit the proliferation of HSC-T6 stimulated by TGF-β1.2) HSC-T6 cells were used for experimental study samely. TransfectedHDAC2-siRNA into HSC-T6 cells by LipofectamineTM 2000. Administration HSC-T6 cells with10ng/ml TGF-β1 for 24h, harvesting cells. Expressions of COL1α1、α-SMA and HDAC2 in HSC-T6 cells were analysed by Western Blot. Experimental results indicated that compared to scrambled-siRNA transfection group, expressions ofCol1α1 and α-SMA were significantly repressed in HDAC2-siRNAtransfeted cells. Prompting that HDAC2 silencing could obviously inhibit the activation of HSC-T6 cells stimulated byTGF-β1.3) HSC-T6 cells were used for experimental study. Transfected HDAC2-siRNA into HSC-T6 cells by LipofectamineTM 2000, harvesting cells. The expression of Smad7 was measured by Western Blot analysis. Experimental results indicated that HDAC2silencing could restore the lower-expression ofSmad7 in HSC-T6 cells caused by TGF-β1 exposure. Prompting that HDAC2 may regulate the activation of HSC-T6 by suppression expression of Smad7.