Effects Of Ligustrazin On Epithelial-to-Mensenchymal Transition And Extracellular Matrix Accumulation In Human Renal Proximal Epithelial Cells Induced By High Glucose

Objective To investigate the effects of ligustrazin on epithelial-to-mesenchymal transition(EMT) and extracellular matrix accumulation(ECM) in human renal proximal tubular cells(HK-2) induced by high glucose, and to explore the possible mechanisms of ligustrazin for the inhibition of renal interstitial fibrosis.Methods HK-2 cells were treated with high glucose or a combination of high glucose and ligustrazin. Morphological changes were assessed by phase contrast microscopy. Transdiferentiation of tubular cells into myofibroblasts was assessed by immunofluorescence with monoclonal antibodies toα-SMA. The expression of mRNA levels ofα-SMA, E-cadherin, ColⅠα1, FN, PAI-1 and MMP2 were evaluated by RT - PCR respectively. The expression of protein levels ofα-SMA and E-cadherin were analysed by Western Blot. The secretion of ColⅠand FN proteins were quantitated by ELISA.Results The control cells displayed typical cobblestone morphology of epithelial cells. High glucose treatment of confluent HK-2 cells resulted in distinct morphological changes in a time and dose-dependent manner. High glucose treated cells showed evidence of gross elongation with filopodia formation but inhibited in the presence of 80μg/ml ligustrazin. Indirect immunofluorescence staining showed that expression ofα-SMA could be enhanced by high glucose but decreased after co-treatment of 80μg/ml ligustrazin. RT-PCR staining showed that high glucose up-regulated the expression of mRNA levels ofα-SMA, ColⅠα1,FN and PAI-1 but reduced E-cadherin and MMP2(p<0.01). Recombinant high glucose and ligustrazin restored the expression of mRNA levels of E-cadherin and MMP2 but decreasedα-SMA, ColⅠα1,FN and PAI-1 in a dose-dependent manner(p<0.05). Western Blot staining showed that high glucose up-regulated the expression of protein level ofα-SMA but reduced the level of E-cadherin(p<0.01). Recombinant high glucose and ligustrazin restored the expression of protein level of E-cadherin but decreasedα-SMA in a dose-dependent manner(p<0.05). ELISA staining showed that high glucose up-regulated the expression of protein levels of the secretions of ColⅠand FN(p<0.05). Recombinant high glucose and ligustrazin decreased the protein levels of the secretions in a dose-dependent manner(p<0.01).Conclusions High glucose might induce EMT and produce ECM components. Ligustrazin might block high glucose-induced EMT, prevent accumulation of ECM components and enhance degradation of ECM components induced by high glucose, prevent tubulointerstitial fibrosis in diabetic nephropathy ulteriorly.