Effects Of S-adenosyl-methionine On Proliferation And Collagen Expression Of Acetaldehyde-induced Rat Hepatic Stellate Cells

Abstract:Objective:Hepatic fibrosis(HF) is a middle stage from varied chronic liver injuries to cirrhosis; its occurrence and development are regulated by many factors. The central process is the activation and proliferation of hepatic stellate cells(HSCs) stimulated by factors, resulting in production and deposition of a large deal of extracellular matrix(ECM) in the liver tissue, and then developed to cirrhosis. Therefore, inhibition of HSCs activation and proliferation, inducing apoptosis is a key issue to prevent liver fibrosis occurrence and development. S-adenosyl-methionine(SAM) is a natural compound, presenting in various tissue and body fluids. It is formed from the methionine and adenosine triphosphate, catalyzed by methionine adenosyltransferase. It involves varied biochemical reactions in vivo. SAM is an essential intermediate for methylation, it provide methyl group to receptor directly. In addition, SAM can be transformed into a strong reducing substance—glutathione(GSH) by sulphur-transferring in vivo, which can resist hurt to cell by oxygen free radicals and inflammatory substances. Currently, exogenous SAM is used to treat cholestasis in clinical. Its mechanism relates to SAM providing methyl group and transferring sulphur. There is strong correlation between hypomethylation and liver fibrosis induced by alcoholic liver disease. In theory, SAM may affect the process of hepatic fibrosis. This study aims to investigate the effects of SAM on the proliferation, morphological changes of rat HSCs and collagenⅠ,Ⅲexpression of rat HSCs stimulated by acetaldehyde (AA). It provides theoretical basis of SAM application in the clinical to protect against liver fibrosis. Methods:Hepatic stellate cells(HSC-T6) were cultured in vitro and were divided into seven groups:control group, acetaldehyde treated group,0.15 mmol/L SAM group,0.5 mmol/L SAM group,1.5 mmol/L SAM group,5.0 mmol/L SAM group,15.0 mmol/L SAM group. Acetaldehyde was added to the cells cultured with different concentrations of SAM for 1h, and they were cultured for 24h. Then morphological changes of cells were observed by optical microscopy and the cell proliferation was detected by MTT. According to the test results, two dosages were selected for the next experiments. Cells were divided into four groups:the control group, acetaldehyde treated group,1.5 mmol/L SAM group and 5.0 mmol/L SAM group. Acetaldehyde was added to the cells cultured with different concentrations of SAM for 1h, and they were cultured for 24h. The expressions of type I and III collagen were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western-blot. Results:1. Under inverted microscope, the HSC-T6 cells in control group grew well, the cells bodies were spindle or stellate. Compared with the control group, the acetaldehyde treated group appeared more cells, arrayed more intensive, showed a smaller gap between cells, the pseudopod of cells in peripheral appeared more and longer and the cells bodies were stellate or irregular shape. Compared with acetaldehyde treated group, all SAM treated groups, the number of cells reduced, the gap between cells increased, the pseudopod of cells in peripheral reduced, the cells bodies were short spindle or flat. Furthermore, the higher of the concentration of SAM, the more obvious of the change.2. MTT assay showed that the absorbance in control group, acetaldehyde group,0.15 mmol/L SAM group,0.5 mmol/L SAM group,1.5 mmol/L SAM group,5.0 mmol/L SAM group,15.0 mmol/L SAM group were (x±s):0.463±0.038,0.698±0.014,0.445±0.026, 0.415±0.029,0.391±0.012,0.342±0.024,0.223±0.025, respectively. Compared with the control group, the absorbance in acetaldehyde treated group were elevated, there was significant difference between them (P＜0.01). Compared with acetaldehyde treated group, the absorbance in different concentrations of SAM treated groups was significantly decrease, there was significant difference between acetaldehyde treated group and all different concentrations of SAM treated groups (P＜0.01).3. RT-PCR results showed that the mRNA relative expression of collagen-I in the control group, acetaldehyde treated group,1.5 mmol/L SAM group and 5.0 mmol/L SAM group were (x±s):1.416±0.020,1.972±0.038, 1.681±0.031,1.461±0.032, respectively; the mRNA relative expression of collagen-III were (x±s):0.996±0.008,1.875±0.028,1.537±0.056, 1.053±0.062, respectively. Compared with the control group, the mRNA relative expression of collagen-I and collagen-III in acetaldehyde treated group was significantly increased, there was significant difference between them (P<0.01). Compared with acetaldehyde control group, the mRNA relative expression of collagen-I and collagen-III in 1.5 mmol/L SAM,5.0 mmol/L SAM treated group were significantly decreased, there was significant difference between acetaldehyde treated group and all different concentrations of SAM treated groups (P＜0.01).4. Western-blot results showed that the protein relative expression of collagen-I in the control group, acetaldehyde treated group,1.5 mmol/L SAM group and 5.0 mmol/L SAM group were as (x±s):1.109±0.070,1.298±0.101, 0.970±0.058,0.829±0.030, respectively; the protein relative expression of collagen-III were (x±s):1.017±0.013,1.204±0.024,1.089±0.015, 1.048±0.019, respectively. Compared with the control group, the protein relative expression of collagen-I in acetaldehyde treated group was significantly increased, there was significant difference between them (P＜0.05); the protein relative expression of collagen-III in acetaldehyde treated group was significantly increased, there was significant difference between them too (P＜0.01). Compared with acetaldehyde control group, the protein relative expression of collagen-I and collagen-Ⅲin 1.5 mmol/L SAM,5.0 mmol/L SAM treated group were significantly decreased, there was significant difference between acetaldehyde treated group and all different concentrations of SAM treated groups (P＜0.01).Conclusion:SAM can inhibit the proliferation and collagen-I, collagen-Ⅲ, synthesis of HSCs stimulated by acetaldehyde in a concentration dependent manner in vitro. This study demonstrated that SAM has the anti-hepatic fibrosis effect; it is can be used to prevent and cure hepatic fibrosis induced by alcoholic.