ABSTRACT
BACKGROUND: The cell growth, proliferation and differentiation caused by p38 mitogen-activated protein kinase (p38MAPK) might act as the common pathway in the onset and development of diabetic vascular complication.OBJECTIVE: To investigate the effect of atorvastatin on p38MAPK signal pathway and the influence of atorvastatin on cell proliferation and expression of transforming growth factor-β1 (TGF-β1) at transcriptional level in human glomerular mesangial cells (HGMCs) cultured with oxidative modification of low-density lipoprotein (ox-LDL).DESIGN: A randomized, parallelized, controlled and open trial.SETTING: Endocrinology Department, First Hospital Affiliated to China Medical University; Endocrinology Department,Respiratory Department, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA.MATERIALS: The experiment had been done in the laboratories for Pharmaceutical Department of China Medical University and Respiratory Department of Shenyang Military Area Command of Chinese PLA from May 2004 to May 2005. The sample was cut from renal cortex from the healthy segment of nephroectomy from a tumor patient (Provided by Xiang Jun, Urology Department, the General Hospital of Shenyang Military Area Command of Chinese PLA; Informed consent was obtained). OX-LDL was purchased from biochemistry institute of Peking Union Medical College (Batch No.20040711 ). ox-LDL was 5.3±1.0 nmol in 100 μg protein. Atorvastatin was purchased from Pfizer Pharmaceutical Co. Ltd (No. 45837088); p38MAPK monocloncal antibody was purchased from Santa Cruz.METHODS: ① 6.0-8.0 cm3 blocks of cortex were cut from renal cortex from the healthy segment of nephroectomy from a tumor patient, glomerular mesangial cells were isolated. When grew into 80% confluent monolayer, the cells were digested and performed passage. After the first two passages, the cells were pure based on morphology and characterized by DAB staining for Vimentin antigen and actin antigen was positive, whereas cytokeratin antigen was negative. Oil red "O" staining confirmed that ox-LDL was intaken by HGMCs. The 4-8th passages of cells were used to study. ②HGMCs were seeded into 96-well plates with 5×103 cells per well and grown in 200 μL culture medium. The study was divided into 5 groups (6 wells each group): 1.2, 6,12 mg/L atorvastatin group, ox-LDL group and blank control group. The cells were pre-incubated with atorvastatin for 30 minutes, then exposed to 80 mg/L ox-LDL. The cells in blank control group were untouched. After 24 hours, MTT was added. The absorbance of each sample at the wavelength of 492 nm was measured with immunosorbant assay system. The inhibitory rate of cell proliferation was calculated. ③1×106 to 3×106 cells were seeded into six 200 mL flasks. The trial was divided into 5 groups randomly:control group, 10, 40, 80 mg/L ox-LDL groups and atorvastatin group (12 mL/g). The cells in each group were pre-incubated for 30 minutes, then exposed to 80 mg/L ox-LDL for 24-routine culture. The expressions of TGF-β1mRNA of harvested cells were detected with semi-quantitative reverse transcription-polymerase chain reaction and p38MAPK signal pathway activation was detected by Western blot.MAIN OUTCOME MEASURES: ①Identification results of HGMCs. ② Proliferation of HGMCs. ③ TGF-β1 expression of HGMCs. ④p38MAPK signal pathway activation of HGMCs.RESULTS: ①When the cells were sub-cultured to the second generation, cell volume was big. Most of the cells were spindle-shaped, irregular stellate or branch-like, filled with microfilaments which paralleled axis. Cells overlapped in the intensive area. After DAB staining, cytoplastic actin and vimentin were positive and keratin was negative. Oil red "O"staining confirmed that ox-LDL was intaken by HGMCs with red granules in the cytoplasma, while control group did not.It was proved that the cells cultured for passage were HGMCs. ② As compared with control group, the inhibitory rate of cell proliferation in ox-LDL group was significantly decreased, but that in atorvastatin 1.2, 6 and 12 mg/L groups was significantly increased (0, -17.4%, 6.4%, 22.5%, 61.5%, respectively, P ＜ 0.05 or 0.01) on concentration-dependent manner. ③ As compared with control group, ox-LDL (10, 40, 80 mg/L) increased the expression of TGF-β1 and activation of p38MAPK in concentration-dependent manner, the effect of 80 mg/L ox-LDL group was the most significantly (P ＜ 0.01). Atorvastatin decreased the increment of TGF-β1 expression and the activation of p38MAPK pathway induced by ox-LDL significantly. There was significant difference when compared with 80 mg/L ox-LDL group (P ＜ 0.01).CONCLUSION: Atorvastatin can antagonize the activation of p38MAPK pathway, decrease the secretion of TGF-β1 and inhibit mesangial cell proliferation induced by ox-LDL, suggesting that it may exert beneficial effect in the prevention and treatment of diabetic nephropathy with dyslipidemia.