The Roles Of TRPV4 In Rat HSC

Hepatic fibrosis(HF) occurs as a reversible wound-healing response to cellular injury factors, such as alcohol, drugs, vira infection, or autoimmune imbalances and so on, and characterized by the accumulation of extracellular matrix(ECM) accompanied with liver injury. Uncontrolled HF can eventually lead to liver cirrhosis or even hepatocellular carcinoma(HCC) and become a serious threat to human health. So it is great significant to reverse and diagnose HF by clarifying the molecular mechanism of HF.Hepatic stellate cells(HSCs) were proved to be intimately associated with the progression and development of hepatic fibrosis. Activated HSCs increase the migration and deposition of ECM components, which accelerates the activated HSCs differentiate into myofibroblast-like cells(MFBLC), and produces collagens, ultimately cause HF. As one of the primary effector cells in liver, to promote the activated HSCs to apoptosis become a effective method to prevent HF. Study after study have demonstrated that autophagy played a important role in HF. Furthermore, studies revealed that there is a close relationship between autophagy and apoptosis. It is indicated that the activation of human HSCs led to the increased of calcium(Ca2+) influx. It has great important to promote the activated HSCs apoptosis by inhibiting Ca2+ influx. Furthermore, the change of Ca2+ concentration has a close realationship with cells autophagy. Our preliminary data indicated that the non-selective Ca2+ channel transient receptor potential vanilloid 4(TRPV4) act as an important regulator in HF. Current study of TRPV4 focuses on its rolesi in HSC, thus providing new targets for the cure of liver fibrosis.The main contents are divided into three sections, as follows:1. TRPV4 expression in rat hepatic stellate cells and the effect of TRPV4 on rat hepatic stellate cells apoptosis protein.The study was performed in rat HSC-T6 cell line. The q RT-PCR and Western blot analysis revealed that TRPV4 m RNA and protein level were substantially induced in response to 10ng/ml TGF-b1 for 24 h compared to un-treated group, indicating that TRPV4 may play a significant role in HSC-T6. Then we inhibit the expression of TRPV4 by transfection of si RNA using Lipofectamine TM2000. Flow cytometry result indicated that inhibiting the expression of TRPV4 could significant caused apoptosis of HSC-T6 compared to the TGF-β1-treated group. The q RT-PCR and Western blot results demonstrated that a significant up-regulated expression of caspase3 m RNA, pro-caspase3 protein and Bax levels compared to the TGF-β1-treated group. Furthermore, over-expression of TRPV4 by using 4α-PDD result in a significant down-regulated caspase3 m RNA, pro-caspase3 protein, Bax m RNA and protein levels compared to the TGF-β1-treated group, indicating TRPV4 could inhibit apoptosis protein expression and may be play a role in apoptosis.2. The level of autophagy in rat hepatic stellate cells and the effect of TRPV4 on rat hepatic stellate cells autophagyThe q RT-PCR and Western blot results indicated that LC3 m RNA and protein level were substantially up-regulated P62 m RNA and protein level were substantially down-regulated in response to 10ng/ml TGF-b1 compared to un-treated group. Then we inhibit the expression of TRPV4 by transfection of si RNA using Lipofectamine TM2000. The q RT-PCR and Western blot results revealed that a significant down-regulated expression of LC3 m RNA, protein and up-regulated expression of P62 m RNA, protein levels compared to the TGF-β1-treated group. The acridine orange staining results indicated a significant decrease of autophagosome compared to the TGF-β1-treated group. Furthermore, over-expression of TRPV4 by using 4α-PDD result in a significant up-regulated LC3 m RNA, protein and down-regulated P62 m RNA, protein levelscompared to the TGF-β1-treated group. Consistently, the acridine orange staining results showed that the autophagosome was up-regulated compared to the TGF-β1-treated group. To further study the mechanism of TRPV4 on autophagy. It has indicated that AKT pathway was an important contributor pathway of autophagy. The western blot study found that inhibition of TRPV4 expression result in a significant of p-AKT expression compared to the TGF-β1-treated group. These results indicated that TRPV4 up-regulated autophagy through activating AKT pathway.3. The effect of autophagy on apoptosis in rat hepatic stellate cellsWe use chloroquine(CQ) to inhibit the autophagy level. The qRT-PCR and western blot results demonstrated that a significant down-regulated expression of caspase3 m RNA, pro-caspase3 protein and Bax levels compared to the TGF-β1-treated group, indicating that autophagy could inhibit apoptosis in rat hepatic stellate cells. Finally, inhibiting autophagy by CQ, we found there is no distinct changes of apoptosis when over-expression of TRPV4 or not, suggesting that TRPV4 activated autophagy may be through inhibiting apoptosis protein expressison.