| Diabetic nephropathy (DN) is the most frequent and serious complication ofdiabetes mellitus, which is a leading cause of diabetes death. The principalmorphological characteristics of DN is progressing thickening of glomerularbasement membrane and extracellular matrix (ECM) accumulation in mesangialregion. Many studies have demonstrated that ECM accumulation plays animportant role in the onset of DN. The component and amount of ECM aredetermined by the dynamic process of synthesis and degradation. ECMaccumulation is the imbalance between synthesis and degradation, which isso-called ECM transforming disturbance. Increased synthesis and/or decreaseddegradation of ECM components may result in ECM accumulation. However, themechanism and regulating factors of ECM imbalance have not been illuminatedyet. In view of these considerations, it is important to find the penetratingmechanism and prevention of ECM accumulation.The pathogenesis of DN is very complicated, which is the results of manyfactors interaction. It has been shown that many cytokines participate thedevelopment of DN. At present, the main experimental therapeutic measures forthe renal fibrosis are neutralizing some cytokines, inhibiting fibroblastic activitiesand suppressing collagen formation, thereby reducing ECM deposition. TGF-β isone of the most important cytokines, which leads to tissue fibrosis, so blockade ofits expression or activities can alleviate the degree of the tissue fibrosis. In recentyears, a series of new anti-TGF-β strategies have be raised to interfere in TGF-βexpression. However, TGF-β has extensive target cells and complicated effects,which not only include inducting cell proliferation and promoting the tissuefibrosis, but also resisting inflammation and cell differentiation. Blockingcompletely its expression and activities may cause some side effects, such as lossof control of cell growth, immunization disproportion, and serious inflammation.Furthermore, TGF-β has no notable effects on maintaining fibroblast phenotypealthough it has significant promoting fibrogenesis. Therefore, targeted TGF-βtreatment just only blocked the onset and proceeding of fibrosis, the fibrosis wasnot reversed. Identification of more specific target for anti-fibrotic therapy is anurgent need. Connective tissue growth factor (CTGF), a new fibrosis-relatingcytokine, which is the downstream mediator of TGF-β, participates in many linkof the development of DN by adjusting fibroblast growth and ECM synthesis.Under physiological condition, CTGF, of which biological effect is singlecomparatively, expresses in low level and only mediates the fibrogenic effects ofTGF-β, its anti-inflammatory and anti-proliferation effects are not influenced.Thus, CTGF may become more specific and effective candidate for thetherapeutic target of DN. Intensive research of CTGF may provide a new and safeintervention for DN. The medicine inhibited CTGF production and activationmay reverse the renal fibrosis and provide a new and ideal target for anti-fibrosis.Trigonella foenum-graecum, the seeds of leguminous plant Trigonel1afoenum-graecum L, commonly called tonquin bean or vanilla, possess warmingkidney, eliminating cold, and relieving pain. It has mainly been applied to kidneycold caused by deficiency, hypogastrium psychroalgia, intestinal hernia,cold-damp beriberi and so on. It has been found in animal experiments thatTrigonella foenum-graecum has hypoglycemic effect. This fact has causedresearchers' attention. However, the active component of trigonellafoenum-graecum, cell and biochemistry are still unclear. Up to now, the effects oftrigonella foenum-graecum and its active component on function and structure indiabetic kidney, ECM accumulation, TGF-β, and CTGF expression have not beenreported.In the present study, STZ-induced diabetes model in rats was used in vivoand mesangial cells were cultured in vitro. The research methods employed inthis study include immunohistochemistry, cytobiology, RT-PCR, Western blot andELISA as so on. The following problems were investigated: (1) the effects ofpolysaccharide A2(A), an active component in trigonella foenum-graecum, onfunction and structure of diabetic kidney;(2) the effects of A2 on TGF-β1 andCTGF expression in glomeruli of diabetic rats;(3) the effects of A2 on ECMmajor component as well as the relationship between CTGF and renal ECM.The main results were as follows:1. A2 decreased obviously the contents of blood glucose (P<0.01), DMF(P<0.01) and HbAlc (P<0.05)of diabetic rats. Moreover, A2 ameliorated the renalhypertrophy and decreased kidney weight and kidney/body weight ratio indiabetic rats.2. A2 treatment decreased significantly the contents of blood TG (P<0.01),TC (P<0.01) and LDLC (P<0.01), and increased HDLC levels (P <0.01).3. Administration of A2 reduced urine protein excretion (P<0.01),Scr(P<0.05) and BUN (P<0.01) levels in diabetic rats, which curative effects getclose to Lotensin, a positive control drug. Compared with diabetes group, A2group had a smaller glomerular volume (Ρ<0.01). Histological changes in kidneyof diabetic rats included glomerular mesangial cells hyperplasia and PAS positivesubstance excessively depositing. All the changes were alleviated in A2 group.Electron microscopel examination showed segmental thickness of glomerularbasement membrane, hyperplasia of mesangial cells and fused widely podocytein diabetic rats. The ultrastructural changes were lightened in A2-treated group.4. As compared with control group, activities of CAT, GSH-Px and SOD inrenal cortex of 12-week diabetic rats were reduced and MDA content wassignificantly increased. Compared with diabetes group, activities of CAT,GSH-Px and SOD were increased and MDA content was markedly decreased inrenal cortex of rats treated with A2 (Ρ<0.01).5. Immunohistochemical study revealed that there was a decreased TGF-β1,CTGF, Col IV and FN immunostaining in A2-treated rats compared with diabeticrats.6. TGF-β1 and CTGF expression (including mRNA and protein) wasdecreased in A2-treated rats as compared with those of diabetic rats (Ρ<0.01).7. Compared with those of high glucose group, the contents of FN (Р<0.05),Col IV (Ρ<0.01), TGF-β1 (Ρ<0.01) and CTGF (Р<0.05) were significantlydecreased in 15%serum contained A2 group. The results were same among 20%,10% and 5% serum contained A2 group, but their effect was no more effectivethan 15% serum contained A2 group. Except 20%serum contained A2 group, theinhibiting effects of other serum contained A2 groups (15%, 10% and 5%) on thecontents of FN, Col IV, TGF-β1 and CTGF were concentration-dependent.8. The contents of FN, Col IV, TGF-β1 and CTGF(Ρ<0.01)in mesangial cellmedia with high level of glucose were reduced by A2 treatment.9. Expression of TGF-β1 and CTGF mRNA was down-regulated inA2-treated mesangial cells (Ρ<0.01).The main conclusions are as follow:1. A2 improves the general status of diabetic rats, and regulates the bloodglucose and blood lipid metabolism.2. A2 treatment ameliorates the changes of renal function and structure indiabetic rats.3. Administration of A2 increases the antioxidase activities and inhibitsoxidative damage in diabetic kidney.4. A2 decreased TGF-β1 and CTGF expression (including mRNA and protein)and inhibited ECM accumulation in kidney of diabetic rats are firstly discovered.5. The increased contents of FN, Col IV, TGF-β1 and CTGF (Ρ<0.01) inmesangial cell media with high level of glucose were decreased by A2.6. A2 treatment down-regulates TGF-β1 and CTGF expression in mesangialcells treated with high glucose .7. A2 ameliorates the development of DN through decreasing CTGFexpression.The main creative results:1. Trigonella foenum-graecum polysaccharide A2 down-regulated TGF-β1and CTGF expression in the renal cortex of diabetic rat.2. The abnormalities of the renal function and structure in diabetic rats wereameliorated by Trigonella foenum-graecum polysaccharide A2.3. A2 adjusted the blood glucose and lipid disturbance as well as protectedthe renal function and structure in diabetic rats. A2 had a complex andmulti-target therapeutic function.
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