The Study Of Protective Effect Of Xiexin Decoction And Its Ingredients On Experimental Diabetic Nephropathy

Diabetic nephropathy(DN) is one of the micro vascular complications of diabetes mellitus, which accounts for much of the mortality of diabetes, 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 about 1700 years 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 Janpan, but the effective substance and mechanism of it remains unclear.The present study was aimed to investigate the renal protective effect of Xiexin Decoction and effects of different components and their combination on the experimental diabetic nephropathy, which was expected to provide evidence for the clinical use of Xiexin decoction on DN.Part 1 The protective effect of Xiexin Decoction on the experimental diabetic nephropathyObject:To investigate the effects of constituents of Xiexin Decoction on the experimental diabetic nephropathy in rats and high glucose(HG)-induced mesangial cellsMethod:(1) The Male Sprague-Dawley rats were divided into two groups and fed with the normal pellet diet (NPD) or the high-fat diet (HFD) respectively for four weeks. The HFD-fed rats were then injected intraperitoneally with streptozotocin (STZ). The rats of which the fasting blood glucose≥16.7mmol/L and the urine glucose≥+++were then divided into three groups:the model group, the Xiexin decoction (XXD) group and the metformin group. The rats were then received oral normal saline, XXD and metformin respectively. The fasting blood glucose, the renal function, the kidney index, the albumin in urine and the ultra-structure of the kidneys of each rat were determined after twelve weeks. (2) Glomerular mesangial cells (HBZY-1) were cultured in vitro and divided into several groups:normal control group, high glucose group, and groups treated with Xinxie decoction (160ug/ml, 400ug/ml,lmg/ml), flavonoids of Radix Scutellariae(8ug/ml,20ug/ml,50ug/ml),and polysaccharides of Radix et Rhizoma Rhei (2ug/ml,10ug/ml,50ug/ml). Every group were treated for 24 hours before the MTT assay was taken to measure cell proliferation.Results:(1) Xiexin decoction (effective components) reduced the amount of the water intake, the food consumption by 17.68mmol/L, the creatinine clearance rate (CCr) by 3.92ml/min/kg, the micro albumin in urine by 0.54 mg/24h(p<0.01) and the fasting blood glucose by 7.12mmol/L (p<0.05 or p<0.01) compared with the model group. Xiexin decoction also decreased the incrassation of the glomerular basement membrane and the change of the foot process fusion.(2) Compared to HG-treated group,160ug/ml,400ug/ml,lmg/ml Xiexin decontion decreased cell viability by 14.84%,51.30%,95.54%respectively; 2ug/ml,5ug.ml,12.5ug/ml alkaloids of Rhizoma Coptidis decreased cell viability by 4%,29%,38%respectively; 8ug/ml,20ug/ml,50ug/ml flavonoids of Radix Scutellariae decreased cell viability by 21%,28%,70%; 2ug/ml,10ug/ml,50ug/ml polysaccharides of Radix et Rhizoma Rhei decreased cell viability by 44%,56%, 63%.Conclusion:Xiexin decoction (effective components) improved the diabetic nephropathy (DN) of the experimental diabetic rats. Xiexin decoction, flavonoids of Radix Scutellariae, flavonoids of Radix Scutellariae and polysaccharides of Radix et Rhizoma Rhei can decrease cell proliferation in high glucose (HG)-induced mesangial cells in a dose-dependent manner.Part 2 The protective effect of Xiexin Decoction on high glucose(HG)-induced mesangial cellsObject:To investigate the effects of constituents of Xiexin Decoction on the experimental diabetic nephropathy in rats and high glucose(HG)-induced mesangial cellsMethod:Glomerular mesangial cells (HBZY-1) were cultured in vitro and treated with high glocuce (30mM) in the absence or presence of various concentrations of berberine (lug/ml and 5ug/ml), baicalin (2ug/mland lOug/ml) or rhubarb extract (lug/ml and 5ug/ml) for 24 hours. Cell proliferation was measured by MTT assay. Collagen IV and MCP-1 were examined by Elisa assay respectively. Malonaldehyde (MDA) as well as SOD activities were measured by spectrophotometry using commercial kits.Results:(1) Compared to HG-treated group, lug/ml,5ug/ml berberine decreased relative cell viability by 12%,40%(p<0.05); 2ug/ml,10ug/ml baicalin decreased relative cell viability by 21%,48%(p<0.05).(2) Compared to HG-treated group, lug/ml,5ug/ml berberine reduced collagen IV by 2.54ug/ml,5.17ug/ml(p<0.05 or 0.01); 2ug/ml,10ug/ml baicalin reduced collagen IV by 3.06ug/ml,6.37ug/ml(p<0.05 or 0.01); lug/ml,5ug/ml rhubarb extract reduced collagen IV by 2.22ug/ml,4.96ug/ml (p<0.05 or 0.01).(3) Compared to HG-treated group, lug/ml,5ug/ml,10ug/ml berberine reduced MCP-1 level by 113.18ng/ml,164.95ng/ml,197.36ng/ml; 2ug/ml,1 Oug/ml baicalin reduced MCP-1 level by 94.93ng/ml,153.15ng/ml; lug/ml,5ug/ml,10ug/m reduced MCP-1 level by 81.80ng/ml,130.46ng/ml,160.98ng/ml respectively(p<0.05 or 0.01).(4) Compared to HG-treated group, lug/ml,5ug/ml,10ug/ml berberine elevated SOD activities by 1.93U/ml,5.71U/ml,7.09U/ml(p<0.05 or 0.01); 2ug/ml,lOug/ml berberine elevated SOD activities by 3.47U/ml,7.92U/ml(p<0.05 or 0.01); lug/ml,5ug/ml, lOug/ml rhubarb extract elevated SOD activities by 2.12U/ml,5.57U/ml,8.42U/ml.(5) Compared to HG-treated group, lug/ml,5ug/ml,1 Oug/ml berberine decreased MDA level by 0.51nmol/ml,2.02nmol/ml,7.09nmol/ml; 2ug/ml,1 Oug/ml baicalin decreased MDA level by 1.44nmol/ml,4.08nmol/ml,4.26nmol/ml; rhubarb extract decreased MDA level by 0.433nmol/ml,2.02nmol/ml,3.68nmol/ml. Conclusion:Berberine, baicalin and rhubarb extract of Xiexin Decoction could ameliorate renal function by inhibiting proliferation, fibrosis and inflammation which maybe through reduction of oxidative stress.Part 3 The protective effects of combination of components of Xiexin Decoction on high glucose(HG)-induced mesangial cellsObject:To investigate the protective effects of combinations of components of Xiexin Decoction on the experimental diabetic nephropathy in high glucose (HG)-induced mesangial cells.Method:Glomerular mesangial cells (HBZY-1) were cultured in vitro and treated with high glocuce (30mM) in the absence or presence of berberine (lug/ml), baicalin (2ug/ml), rhubarb extract (lug/ml) or their combination for 24 hours using an L8(27)Orthogonal experiment design. Cell proliferation was measured by MTT assay. Collagen IV and MCP-1 were examined by Elisa assay respectively. MDA as well as SOD activities were measured by spectrophotometry using commercial kits.Results:(1) Compared to HG-treated group, berberine-baicalin, berberine-rhubarb extract, baicalin-thubarb extract group and the three-component group decreased relative cell viability by 50.26%,35.22%and 44.65%respectively (p<0.01).(2) Compared to HG-treated group, lug/ml,5ug/ml berberine reduced collagen IV by 2.54ug/ml,5.17ug/ml(p<0.05 or 0.01); 2ug/ml,10ug/ml baicalin reduced collagen IV by 3.06ug/ml,6.37ug/ml(p<0.05 or 0.01); lug/ml,5ug/ml rhubarb extract reduced collagen IV by 2.22ug/ml,4.96ug/ml (p<0.05 or 0.01).(3) Compared to HG-treated group, lug/ml,5ug/ml,10ug/ml berberine reduced MCP-1 level by 113.18ng/ml,164.95ng/ml,197.36ng/ml; 2ug/ml,10ug/ml baicalin reduced MCP-1 level by 94.93ng/ml,153.15ng/ml; lug/ml,5ug/ml,10ug/m reduced MCP-1 level by 81.80ng/ml,130.46ng/ml,160.98ng/ml respectively(p<0.05 or 0.01).(4) Compared to HG-treated group, lug/ml,5ug/ml,10ug/ml berberine elevated SOD activities by 1.93U/ml,5.71U/ml,7.09U/ml(p<0.05 or 0.01); 2ug/ml,10ug/ml berberine elevated SOD activities by 3.47U/ml,7.92U/ml(p<0.05 or 0.01); lug/ml,5ug/ml, l0ug/ml rhubarb extract elevated SOD activities by 2.12U/ml,5.57U/ml, 8.42U/ml(p<0.05or0.01).(5) Compared to HG-treated group, lug/ml,5ug/ml, lOug/ml berberine decreased MDA level by 0.51nmol/ml,2.02nmol/ml,7.09nmol/ml; 2ug/ml, lOug/ml baicalin decreased MDA level by 1.44nmol/ml(p>0.05),4.08nmol/ml,4.26nmol/ml(p<0.05); rhubarb extract decreased MDA level by 0.433nmol/ml(p>0.05),2.02nmol/ml, 3.68nmol/ml(p<0.05,0.01).Conclusion:Any two components of the three had additive effects in inhibiting high-glucose induced mesangial cell proliferation and cllagen IV secretion, and synergic effect in decreasing MCP-1. Combination of the three ingredients of Xiexin Decoction had the best effects.