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  • Title: [Effect of norcantharidin on the expression of FN, Col IV and TGF-β1 mRNA and protein in HK-2 cells induced by high glucose].
    Author: Chen Q, Li Y, Luo J, Yang Y, Li J, Sun L, Xiao L, Xu X, Peng Y, Liu F.
    Journal: Zhong Nan Da Xue Xue Bao Yi Xue Ban; 2012 Mar; 37(3):278-84. PubMed ID: 22561496.
    Abstract:
    OBJECTIVE: To observe the effect of norcantharidin (NCTD) on the expression of mRNA and protein of fibronectin (FN), collagen IV(Col IV) and transforming growth factor-β1(TGF-β1) in human kidney proximal tubular epithelial (HK)-2 cells induced by high glucose. METHODS: HK-2 cells were incubated with serum-free DMEM for 24 h to synchronize cell growth, and then the cells were divided into 4 groups: Group C (5.5 mmol/L D-glucose), Group M (5.5 mmol/L D-glucose + 24.5 mmol/L-mannitol), Group HG (30 mmol/L D-glucose), and Group HG + NCTD (30 mmol/L D-glucose + 0.5-40 mg/L NCTD). Cytotoxicity of HK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assay. The effect of NCTD on the proliferation of HK-2 cells in high glucose was determined by MTT. The cells were collected to extract total RNA and protein at 6, 24 and 48 h after the incubation. The expression of FN, Col IV and TGF-β1 mRNA was examined by RT-PCR, and FN, Col IV and TGF-β1 protein was analyzed by Western blot. RESULTS: Trypan blue dye exclusive assay showed NCTD concentrations over 5 mg/L were rather toxic in HK-2 cells. The proliferation of HK-2 cells in high glucose was interrupted by interfered with 5 mg/L NCTD as measured by MTT (P<0.05). NCTD at 5 mg/L had a stronger inhibitory effect than NCTD at 2.5 mg/L. Real-time PCR and Western blot showed that the mRNA and protein expression of FN, collagen IV and TGF-β1 increased in HK-2 cells treated with high glucose (P<0.05), while that in cells treated by NCTD was dramatically inhibited (P<0.05). No change in these parameters was detected in the 30 mmol/L D-mannitol control group (P>0.05). CONCLUSION: NCTD can downregulate FN, collagen IV and TGF-β1 mRNA and protein expression in HK-2 cells stimulated by 30 mmol/L D-glucose.
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