The Effect Of HDAC Inhibitors And HDAC1 SiRNA On The Alveolar Typeâ…¡ Epithelial Cells EMT Induced By TGF-β₁

【Background and abjective】The concept of histone acetylation is to transfer acetyl of acetyl-COA to the histone lysine amino in the catalysis of the histone acetyl transferase(HATs), and acetyl can be removed from lysine in the catalysis of the histone deacetylases(HDACs). The histone acetylation and deacetylation are in the reversible and dynamic process under the regulation of these two enzymes. The therapeutic targets of histone deacetylase inhibitors(HDACis) are epigenetic. Because HDACis can regulate the gene expression, can lead to arrest cell cycle and induce cell apoptosis, and also other biological effects included. At last, HDACis could inhibit the proliferation of tumor cells and enhance the sensitivity of tumor cells to the chemotherapeutics. Recent studies have shown that HDAC is closely relsted the chronic dieases,such as chronic kidney diseases, myocardial hypertrophy and Idiopathic pulmonary fibrosis(IPF),which are characterized by fibrosis.Therefore, the application of HDACis in other diseases has always been explored, including the treatment of fibrosis. The experimental study in vivo and vitro has showed that some HDACis could inhibit the fibroblast anti-apoptosis and proliferation, and also inhibit the deposition of extracellular matrix in the progress of IPF, which indicate that HDACis have the potential effect of anti-fibrosis. At present, more and more researches at home an abroad have showed thatthe alveolar epithelial mesenchymal transition may play an important role in the pathogenesis of pulmonary fibrosis. So preventing the occurrence of EMT could reduce or even reverse the aggregation of myofibroblast. Therefore, the reversal of fibrosis at early stage may be expectly to improve the prognosis of the pulmonary fibrosis patients.The study found that HDAC inhibitors of histone deacetylase inhibitors( HDACIs),-Trichostatin a(TSA) can improve the cardiac, liver, kidney and skin fibrosis,and the application of HDACis is hardly any toxic to the normal liver and renal tubular cells.However, whether HDACis can inhibit the lung epithelial mesenchymal transition and its fibrosis, how the specific role of HDACis in pulmonary fibrosis is, and what the mechanism is, they are unclear, up to now. Our previous studies have shown that TGF-β1 could induce the EMT of Rar lung epithelial-6TN(RLE-6TN) cells in vitro, and HDAC1 is involved in the process of EMT of RLE-6TN cells induced by TGF-β1. In this study, with respectively addition of TSA and HDAC1 si RNA, accompany with exogenous TGF-β1, we observe the expression of HDAC1、HADC8、E-cad and α-SMA of RLE-6TN cells induced by TGF-β1, and definite the mechanism of inhibiting the expression of HDACA1、8 to protect the RLE-6TN cells and to preventing lung fibrosis.【Meterials and Methods】In cultured RLE-6TN cells, with respectively addition of HDAC1 inhibitor TSA and designation and synthesis HDAC1 si RNA, after addition of exogenous TGF-β1, we detect the expression of HDAC1 、 HADC8 、 the specific epithelial cell markers E-cad and the specific mesenchymal cell markers α-SMA respectively using Real-time RT-PCR and Western Blot materials, and detect the effect of TSA on the cell migration capability of RLE-6TN in vitro using Transwell chamber materials.【Results】Compared with the blank control group, with the TSA retreatment 6h and TGF-β1 treatment 6h, we detect the expression of α-SMA m RNA was down-regulated(P<0.05). The expression of E-cad m RNA was up-regulated at 6h and 12h(Though the difference was not statistically significant).HDAC8 m RNA was down-regulated at 24 h and 48h(Though the difference was not statistically significant). HDAC1 m RNA was down-regulated(P<0.01). Compared with the blank control group,with the TSA treatment,the expression of HDAC1、8 and α-SMA protein were down-regulated, andE-cad was up-regulated; Transwell assay found that compared with the blank control group, the number of cells(20× and 40×) through the microporous membrane with the treatment of TGF-β1 were significantly increased(P<0.01). Compared with the single TGF-β1 treatment group, the number of cells(20× and 40×) through the microporous membrane with the treatment of TSA and TGF-β1 were significantly decreased(P<0.01). Compared with the blank control group, after the RLE-6TN cells were treated with HDAC1 si RNA and TGF-β1, the expression of HDAC1 m RNA and HDAC8 m RNA was significantly decreased(P<0.01). Compared with the blank control group, after the RLE-6TN cells were treated with HDAC1 si RNA and TGF-β1, the expression of HDAC1 protein was significantly decreased(P<0.01). Transwell assay found that the result of with the treatment of HDAC1 si RNA was the same as with the treatment of TSA. The immune fluorescence detected that compared with the control group, the expression of E-cad protein with the treatment of TGF-β 1 was decreased; the expression of α-SMA protein with the treatment of TGF-β1 was increased, and be localized in the nucleus,and after adding the TSA, α-SMA protein was decreased. The expression of HDAC1 protein increased with the treatment of TGF-β1,and with the treatment of TSA, HDAC1 protein was decreased. The immune fluorescence detected the result of with the treatment of HDAC1 si RNA was the same as with the treatment of TSA. Compared with the blank control group and the TSA retreatment group, with the treatment of TGF-β 1,the intercellular space was larger, the cells discreted into a single,and the cells form was like a spindle. After with the treatment of TSA,the gap between cells was small, and the cells became confluent. As to the form changement,the result of with the treatment of HDAC1 si RNA was the same as with the treatment of TSA.【 Conclusion 】 In the cultured RLE-6TN cells in vitro, HDACis TSA and HDAC1 si RNA interference can inhibit the cultured cells to acquire the α-SMA phenotype and to lose the E-cad phenotype, thus they can partially reversed TGF-β1 induced alveolar epithelial EMT.