The Effect Of Oxymatrine Against High Glucose-induced Fibrosis In Rat Renal Mesangial Cells

Diabetic nephropathy is one of diabetic microvascular complications, it seriously affect the living quality of patients, and even endanger the patients’ life. To explore the pathogenesis of diabetic nephropathy and to delay the diabetic nephropathy is imperative. The pathological characteristics of diabetic nephropathy are mainly contain those symptoms of glomerular hypertrophy, extracellular matrix(ECM)’s increase and abnormal deposition, the worst influence is a continuous progress of renal tubule interstitial fibrosis even the end-stage glomerular sclerosis. In addition, the renal interstitial fibrosis is a key link of disease progression, which is characterized by an accumulation of large amount of ECM. Research shows the transforming growth factorβ1(TGF-β1) is the strongest cytokine which can cause the renal fibrosis, TGF-β1/Smads signal transduction pathways mediates the fibrosis effect of TGF-β1.Connective tissue growth factor(CTGF) is a downstream factor of TGF-β1,which plays an important role in causing the renal fibrosis, by adjusting the growth of fibroblasts and the secretion of ECM.Bone morphorgenetic protein-7(BMP-7) is a subgroup of TGF-β superfamily, which plays a prominent part in renal protection. Studies show that BMP-7 can protect the renal through various ways, and it can inhibit TGF-β1/Samds signal transduction pathways and, in so doing, to prove BMP-7 has a strong anti-fibrosis effect, and this effect is independent of the renal protection. Oxymatrine(OMT), with the molecular formula of C15H24N2 O, which is a Chinese medicine monomer and which is mainly extracted from those traditional Chinese medicines, like sophora, sophora alopecuroides and broad beans’ root. Oxymatrine, it possesses a wide range of pharmacological effects, such as antiviral, anti-inflammatory, immune regulation and anti-fibrosis. Our previous studies have proved that in the model of rat diabetic nephropathy, Oxymatrine can cause the expression of Smad1 increased in glomerular, the glomerular mesangial matrix concentrated, and CTGF increased. Previous studies found that Oxymatrine playing an important role in improving renal fibrosis, but it still not clear that Oxymatrine through what to exert its function of anti-fibrosis, therefore, we put forward a hypothesis that, whether Oxymatrine reverse the renal fibrosis by effecting the accumulation of ECM?This study intends to set up a model of high glucose-induced fibrosis in rat mesangial cells, use the Oxymatrine to interpose the model, thus to determine the content of collagen IV and expression level of BMP-7, TGF-β1,CTGF,Smad2,Smad3 and Smad7 in the model, then to discuss the relationship between BMP-7,TFG-β1/Smads signal transduction pathways and renal fibrosis at the cellular level, with this support, to reveal the improvement effect of Oxymatrine in the molecular mechanism of diabetic renal interstitial fibrosis,thus to provide some theoretical basis for clinical medication.Objective: To study the effect of Oxymatrine in high glucose inducing the rat renal fibrosis, then to discuss the mechanism of Oxymatrine in improving the renal fibrosis.Methods: The rat renal mesangial cell line(RMCs) maintained in Dulbecco’s modified Eagle’s medium(DMEM 5.5m M) supplemented with 15% fetal bovine serum and anti-biotics. At approximately 60% confluence, RMCs were divided into control group(Con) and high-glucose group(HG) randomly. The RMCs in Con group were cultured in low glucose(5.5 m M) with free serum DMEM, at the same time, the RMCs in HG group were cultured in high-glucose(30 m M) with free serum DMEM. After building the model of high glucose induced the fibrosis of rat mesangial cells successfully, they were subdivided randomly into five groups: high-glucose group(HG); low, middle and high dose of oxymatrine group(0.04, 0.08, 0.16 mg/m)(O-L,O-M,O-H) and candesartan group(0.005mg/ml)(Can) for 24 h, 48 h and 72 h separately. We used RMCs which cultured in low glucose(5.5m M) with free serum DMEM as a control group(Con), then observe the morphologic changes of cells. We used MTT colorimetry to detect the cell survival rate of groups, and ELISA kit to detect the content of IV collagen in the supernatant among the group cells, thus to make sure that the model has built successfully. After oxymatrine and candesartan intervening 48 h, we used electron microscope to observe the morphological changes of mesangial cells by intervened. In the meantime, we used RT-PCR to detect the gene expression of the major signaling pathway molecules including BMP-7, CTGF, TGF-β1, Smad2, Smad3 and Smad7, then we used Western-blot analysis to determine the protein expression levels of BMP-7, CTGF, TGF-β1, phosphorylated Smad3(p-Smad3) and Smad3.Results:1 After high glucose stimulating the RMCs, there are changes of cells’morphology and relevant indicators.(1) The morphology of RMCs: Compared with the control group, the high glucose groups with 24 h, 48 h, 72 h were changed in morphology, but among three groups, there no obvious difference.(2) The content of collagen IV: Compared with the control group, the content of collagen IV in supernatant of high glucose medium was obvious increased(P<0.05). It showed that the model of high glucose-induced fibrotic of RMCs was successfully built; The content of collagen IV in supernatant of rat mesangial cells medium was increased after cultured by high glucose for 24 h, 48 h, 72 h separately, but there was no statistically difference(P>0.05). It showed that high glucose induced the fibrosis of mesangial cells was increased with the cultured time extended, but the function was limited.(3) BMP-7, TGF-β1/Smads signaling pathway: The m RNA and protein expression level of BMP-7, Smad2, Smad7 was significantly decreased in HG group compared with Con group after high glucose intervening for 48h(P<0.05). The results above indicated that the high glucose could promote the fibrosis of mesangial cells. Compared with the Con group, the ratio of Smad2/Smad3 decreased in high glucose groups(P<0.05), and a remarkable ratio increase of p-Smad3/Smad3(P<0.05), at this aspect, the differences were all statistically significant(P<0.05).2 Drugs intervening for 48 h, the changes of cells’ morphology, content of collagen IV, the relevant indicators of BMP-7 and TGF-β1/Smads signaling pathway(1)The morphology of RMCs: the cells’ morphology is recovered by drugs’ intervening, but it still different from the normal Con group.(2) The content of collagen IV: Compared with HG group, the content of collagen IV in supernatant of high glucose medium with the intervention of oxymatrine and candesartan was significantly decreased(P<0.05). but it was still exceeded the Con group(P<0.05); The content of collagen IV was decreased along with the dosage of Oxymatrine increased, it showed that the oxymatrine has the function of promoting the renal fibrosis, and the function was depended on the concentration(P<0.05).And at this pact, the function of candesartan was better than oxymatrine.(3) The m RNA level of BMP-7, TGF-β1/Smads signaling pathwayCompared with the HG group, the m RNA level of BMP-7, Smad2 and Smad7 was significantly increased in the oxymatrine and candesartan group(P<0.05), but it was still lower than the Con group(P<0.05); the m RNA level of TGF-β1, Smad3, CTGF was decreased clearly, and it still exceeded the Con group(P<0.05). The ratio of Smad2/Smad3 in oxymatrine and candesartan groups exceeded the HG group(P<0.05). It showed that oxymatrine can promote the m RNA expression of BMP-7 and meanwhile, it can inhibitfor TGF-β1/Smads signaling pathway.(4) The protein expression level of BMP-7, TGF-β1, CTGF and p-Smad3/Smad3Compared with the HG group, the total protein expression level of BMP-7 in oxymatrine and candesartan group was increased(P<0.05), there was no significant difference in protein expression level between Con and O-H group(P>0.05), however the O-M and O-H group was still lower than the Con group(P<0.05). The protein expression level of CTGF, TGF-β1 in drug intervention group was under the HG group(P<0.05), but exceeded the Con group(P<0.05). Compared with the HG group, the protein expression level of p-Smad3/Smad3 in oxymatrine and candesartan group was decreased(P<0.05), but it was still exceeded the Con group(P<0.05). It showed that the degree of oxymatrine intervened mesangial cells fibrosis got improved.Conclusions:1 Oxymatrine can surely improve the morphology changing of high glucose stimulate rat mesangial cells.2 Oxymatrine can reduce collagen IV, and improve rats’ renal fibrosis.3 Oxymatrine through up-regulating the expression of BMP-7, Smad2, Smad7, and inhibit TGF-β1/Smads signaling pathway, thus to reverse the rats renal fibrosis, then to delay diabetic nephropathy.