| Diabetic nephropathy(DN) is one of the micro vascular complications of diabetes mellitus,which accounts for much of the mortality of diabetes.Diabetic nephropathy is characterized by accumulation of extra cellular matrix and thickening of glomerular-tubular basement membrane and leads to proteinuria and renal failure eventually.Xiexin Decoction (XXD) is a traditional Chinese medicinal formula containing Radix et Rhizoma Rhei (Rheum palmatum L.), Rhizoma Coptidis (Coptis chinesis Franch) and Radix Scutellaria (Scutellaria baicalensis Georgi). It has been used for about1700years and is still widely used to treat inflammation-related diseases such as upper respiratory tract infection and gastritis. Xiexin Decoction has been used to treat DN in modern times in China and Japan, but the effective substance and mechanism of it remains unclear. In vivo study confirmed that Xiexin Decoction can decrease the level of blood glucose. But the relationship between the effect of anti-DN and decrease blood glucose is still unknown.In this study, we determined the effect of Xiexin Decoction in high-glucose induced HK-2EMT model,to find the substance and mechanism of the effect of anti-DN.Part1the protect effect of Xiexin Decoction on the oxidative stress induced by high glucose in HK-2Aims:To investigate the effects of constituents of Xiexin Decoction (Rhein, Berberine and Baicalin) on high glucose-induced oxidative stress in HK-2.Methods:HK-2were cultured by high glucose and treated with different concentrations of Rhein, Berberine and Baicalin for24h. MDA activities were measured by thiobarbituric acid method and SOD activities were measured by Xanthine oxidase method.Results:1. after high glucose-cultured for24h, the MDA activities was higher than control group by25.21%(p<0.01), and no change in mannitol (osmotic control) group vs. control group. After drug treatment, the MDA activities was lower than high glucose group in Rhein (2μg/ml、5μg/ml) by6.16%、18.90%(p<0.05or p<0.01), Berberine (2μg/ml、5μg/ml、10μg/ml) by12.24%、20.95%,9.53%(p<0.05or p<0.01) and Baicalin (2μg/ml、5μg/ml、10μg/ml) by21.45%,15.04%.18.41%(p<0.05or p<0.01).2. After high glucose-cultured for24h, the SOD activities was less than control group by27.96%(p<0.01), and no change in mannitol (osmotic control) group vs. control group. After drug treatment, the SOD activities was higher than high glucose group in Rhein (5μg/ml、10μg/ml) by21.41%,27.37%(p<0.05or p<0.01), Berberine (2μg/ml、5μg/ml、10μg/ml) by23.15%,28.12%,30.02%(p<0.05or p<0.01) and Baicalin (2μg/ml、5μg/ml、10μg/ml) by40.59%,27.35%,38.52%(p<0.05or p<0.01).Conclusions:The constituents of Xiexin Decoction (Rhein, Berberine and Baicalin) all can increase the activities of SOD and decrease the activities of MDA, indicating that the protect effect of Xiexin Decoction on the oxidative stress induced by high glucose in kidney.Part2The anti-high glucose-induced trans-differentiated of Xiexin Decoction Aims:To investigate the effect of constituents of Xiexin Decoction (Rhein, Berberine and Baicalin) on high glucose-induced trans-differentiated in HK-2.Methods:1. HK-2were cultured by high glucose and treated with different concentrations of Rhein, Berberine and Baicalin. The expression of a-SMA was detected by Western Blot.2. The microscope was used to observed the change in form of HK-2induced by high glucose (30mmol/L) and Xiexin Decoction (5μg/ml).Results:1. a-SMA was not expressed in control group and mannitol (osmotic control) group. After high glucose treated for48h, the expression of a-SMA was20.03%higher than normal group (p<0.01). Rhein (2μg/ml、5μg/ml、10μg/ml) can remarkably decrease the level of α-SMA (11.67%,13.33%and12.50%, p<0.05or p<0.01) and Berberine (5μg/ml、10μg/ml) and Baicalin (5μg/ml、10μg/ml) can dose-dependent suppression the expression of a-SMA(10.05%,17.65%and9.09%,11.88%, p<0.05or p<0.01).2. HK-2cells exhibited the typical cobblestone when grown in culture. After high glucose treated for48h, they became elongated, disassociated from neighboring cells, and lose their cobblestone monolayer pattern. The mannitol (osmotic control) group was not typically changed. The number of the elongated cells decresed in XXD groups.Conclusions:The constituents of Xiexin Decoction (Rhein, Berberine and Baic alin) all can decrease the level of a-SMA, indicating that the protect effect of Xiexin Decoction in kidney not dependent on the control of blood sugar or b1ood pressure.Part3the mechanism of the effect of anti-high glucose-induced cell migration of Xiexin DecoctionAims:To investigate the effect and mechanism of constituents of Xiexin Decoction (Rhein, Berberine and Baicalin) on high glucose-induced cell migration in HK-2.Methods:1. HK-2were cultured by high glucose and treated with different concentrations of Rhein, Berberine and Baicalin. The rate of cell-migration was measured by the Wound-healing assay and the Transwell experiment.2. HK-2were cultured by high glucose and treated with different concentrations of Rhein, Berberine and Baicalin. The expression of FAK was detected by Western Blot.Results:1. In the Transwell experiment, after high glucose-cultured for48h, the rate of cell-migration was higher than control group (66.47±7.29vs10.47±1.33cells/filed, p<0.01),and no change in mannitol (osmotic control) group vs. control group (10.60±1.56vs10.47±1.33cells/filed). The Rhein (5μg/ml), Berberine (5μg/ml) and Baicalin (5μg/ml) can suppression the cell-migration (45.53±9.72,17.73±3.48,19.93±4.39vs66.47±7.29cells/filed, p<0.05or p<0.01).2. After high glucose-cultured for7d, the expression of FAK was higher than control group by28.73%(p<0,01), and no change in mannitol (osmotic control) group vs. control group. The Rhein cannot decrease the high expression of FAK induced by high glucose, but Berberine (2μg/ml、5μg/ml、10μg/ml) can suppression the expression of FAK (22.18%,26.59%, p<0.05or p<0.01vs. high glucose group), and Baicalin (5μg/ml、5μg/ml) also can significantly suppression the expression of FAK (25.18%,22.93%, p<0.01vs. high glucose group).Conclusions:The constituents of Xiexin Decoction (Rhein, Berberine and Baicalin) all can inhibition the cell-migration induced by high glucose in HK-2, indicating the protect effect of Xiexin Decoction on decrease the cell-migration induced by high glucose in kidney. The expression of FAK increased in the process of cell trans-differentiation in HK-2, the Berberine and Baicalin can decrease the expression of FAK to play a part in cell trans-differentiation induced by high glucose in kidney. |
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