| ObjectiveDiabetic nephropathy(DN) is one of the major microvascular complications of diabetes mellitus patients, which mainly involved in renal corpuscle as well as increasedrate of glomerular filtration and increased protein filtration, which lead to a large amount of proteinuria, which has been also the main cause of end-stage renal disease(End stage renal disease, ESRD). As the number of diabetes is growing, how to prevent the occurrence of diabetic nephropathy and its progression more and more be taken seriously. Multiple previous studies have been observed that TWP has stronger anti-inflammatory and immunomodulatory effects, which could significantly treat various kinds of autoimmune diseases. What is more, its character of playing role in diabetic progression is variable and changeable, which not only played role in T cell, dendritic immune cells and so on but also suppressedsecretion of inflammatory factors, adhesive factors and chemotactic factors so that it could achieve the effect of therapy of a variety of autoimmune diseases. Amounts of researches have demonstrated TWP could prolong the progression of glomerular sclerosis and down-regulate secretion of proteinuria via anti-inflammation, inhibition of immune However, the regulating mechanism of TWP on effecting of renal fibrosis and deposition of extracellular matrix are temporarily unclear. Many previous studies showed that the TGF-?1/ Smad signal pathway played a vital role in heart fibrosis, lung fibrosis and renal fibrosis, but the mechanism of TGF-?1/ NF-?B signal pathway on renal fibrosis is not obvious. Therefore, we adopt ELISA technology, q-PCR technology, Western bloting technology and immunohistochemistry, our study aim at the effect of TWP on expression of the associated closely renal fibrosis pathway--TGF-?1/NF-?B signal pathway and its associated protein in the streptozotocin(STZ)-induced diabetic rat kidney tissue.MethodsSelecting randomly 10 from these 70 clean grade male SD rats as prevention normal control(NC, n=10), which were kept up with normal feedstuff. Other 60 rats were kept up with high-sugar and high-fat diet and streptozocin(STZ, 30mg/kg) was injected through tail intravenous injection, in order to induce T2 DM animal model after 8 weeks. Then these successful model of T2 DM rats were randomly divided into diabetes model(DM) group. After being DM mode, rats in treatment group began to gavage, these TWP treatment groups were divided into the low dose of TWP(L-TWP, 1mg/kg·d)?the medium dose of TWP(M-TWP, 3mg/kg·d)and the high dose of TWP(H-TWP, TWP 6mg/kg·d), there are fifteen rats in every group, while NC and DM groups were gavaged by the same amount of distilled water(2ml), all rats were executed after eight weeks of intragastric administration. Keep a record of the body weight(BW),blood glucose(GLU) and 24 hours urine microalbumin(UMA); keep a record of the BW and kidney weight(KW) when rats were executed; leave the blood specimen to detect GLU,the hepatic-renal function,the blood lipid and blood routine examination; take use of ELISA technology to detect levels of TGF-?1, NF-?Bp65, Collagen IV(COL-IV), Fibronection(FN), Laminin(LN); leave renal tissue samples to observe the change of glomerular filtration membrane ultrastructure in diabetic rats nephropathy; utilize respectively q-PCR and Western blotting to detect levels of m RNA and protein of TGF-?1, NF-?B p65, COL-IV, FN and LN in renal tissues of diabetic rats.Results1.The results of animal experiments1.1. Comparing with the NC group, levels of TG?TC?FINS?HOMA-IR and HOMA-? is higher in the high-sugar and high-fat group(P<0.05). After injecting STZ, the body weight is reducing, along with a series of symptoms, such as polydipsia? polyuria and polyphagia and so on, which showed the model of diabetic rats had been established successfully.1.2. General indicators:(1)BW?GLU: Comparing with the NC group, the GLU in DM group increased significantly(P<0.05), while the BW decreased obviously(P<0.05);(2)Kidney weight/Body weight(KW/BW): Comparing with the NC group, the index of KW/BW in DM group increased clearly(P<0.05), while the level of KW/BW down-regulated in these groups after lavage of TWP(P<0.05), especially in H-TWP group;(3)Blood lipid: Comparing with the NC group, the level of TG and TC in the DM group up-regulated obviously(P<0.05), while no significant effects were observed afterintragastric administration of TWP(P>0.05);(4) The hepatic-renal function and blood routine examination: the levels of hepatic-renal function and blood routine were not obvious differences(P>0.05) in every intervention group.1.3. 24 h UMA:(1)Comparing with the NC group, the level of UMA up-regulated outstandingly in the DM group(P<0.05); Comparing with the DM group, there were down-regulating the level of UMA clearly in TWP treatment groups in a dose-dependent manner(P<0.05).1.4. Glomerular pathological morphological changes in every group of rats: There was not clear abnormity in the NC group. The results of HE staining, Masson staining and PAS staining shows that pathological ultrastructurein the DM group.These above pathological ultrastructurewere alleviated in different doses of TWP groups.1.5. Adopting ELISA technology to detect TGF-?1, NF-?B p65, COL-IV, FN, LN in serum of rats:(1)Comparing with the NC group, levels of TGF-?1, NF-?B p65, COL-IV, FN, LN were obviously higher in DM group(P<0.05); comparing with the DM group, levels of TGF-?1, NF-?B p65, COL-IV, FN, LN down-regulated in different doses of TWP(P<0.05).1.6. Taking advantages of q-PCR technology to detect expression of TGF-?1, NF-?B p65, COL-IV, FN, LN m RNA in the rat kidney tissue:(1) Comparing with the NC group, the expression of TGF-?1, NF-?B p65, COL-IV, FN, LN m RNA up-regulated obviously in DM group(P<0.05);(2) Comparing with the DM group, the expression of TGF-?1, NF-?B p65, COL-IV, FN, LN down-regulated clearly in a dose-dependent manner(P<0.05).1.7. Adopting technology of Western blotting to detect the expression of TGF-?1, NF-?B p65, COL-IV, FN, LN protein:(1) Comparing with the NC group, the expression of TGF-?1, NF-?B p65, COL-IV, FN, LN protein up-regulated obviously(P<0.05);(2) Comparing with the DM group, TGF-?1, NF-?B p65, COL-IV, FN, LN protein were down-regulated clearly by different doses of TWP in a dose-dependent manner(P<0.05).1.9. Adopting immunohistochemical method to detect the protein expression of TGF-?1, NF-?B p65, COL-IV, FN, LN in kidney of rats:(1) Comparing with the NC group, there were marked cellular infiltration of TGF-?1, NF-?B p65, COL-IV, FN, LN in glomerular in diabetic groups(P<0.05); after lavage of different doses of TWP individually, the expression of TGF-?1, NF-?B p65, COL-IV, FN, LN decreased significantly in kidney of rats(P<0.05), especially in M-TWP group and H-TWP group.Conclusions:1.Comparing to the DM group, there is better of KW/BW, 24 h UMA and pathological ultrastructure in different doses of TWP group, especially in the median and high doess of TWP group.2.Tripterygium glycosides plays a role in anti-inflammatory and immunosuppression, which can inhibit hyperplasia of mesangial cell, so as to improve electric charge of glomerular basement membrane and alleviate fibrosis of glomerular.3.TGF-?1 and the active NF-?B signaling pathwaymediated the process of synthesis of ECM, which can commonly accelerate the synthesis of type IV collagen, fibronection and laminin, mediated commonly the occurrence of diabetic nephropathy development process.4. TWP could inhibit the TGF-?1/ NF-?Bsignaling pathway, so as to alleviateglomerular structure of DM, which is a dose-dependent manner. |
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