Change Of Cytokines Expression In Real Of Stz-Induced Diabetic Rats And Intervention Effect Of Salvia Miltiorrhiza Bunge Combined Losartan

BACKGROUND AND OBJECTIVE: Diabetic Nephropathy (DN) is one of the most common chronic complications of diabetes mellitus and accounts for increased mortality and morbidity in diabetics. DN is now the leading course of end stage renal disease (ESRD) in the western world. Thus the expenditure on dialysis and kidney transplantation increases year by year, which brings heavy economical burdens on patients and the society. But the origin and pathogenesis of DN is extremely complex and has not been clarified completely yet. Genetic factors, abnormal hemodynamic disturbances and glycometabolism disorder are believed to be the main causes of DN now. The pathological hallmarks of early DN are glomerular hypertrophy, basement membrane thickening and mesangial expansion while the features of the later stage of DN is glomerular sclerosis and interstitial fibrosis. Underlying all these pathological changes is the progressive excess accumulation of extracelluar matrix (ECM), a hallmark of DN. And in the metabolic process of ECM, cytokines play r. key role. Among all those cytokines promoting glomerular sclerosis and interstitial fibrosis, transforming growth factor-p (TGF-β) appears to play a pivotal roll. However, it will bring some unwanted effects if we block up TGF-β1 function completely due to it's anti-hypertrophy and anti-inflammatory effects besides profibrotic activity- Recently, attf tition has focuced on connective tissue growth factor (CTGF). which mimics the biological activity of TGF-p in profibrotic tissue formation. Thus, acting as a downstream mediator of the profibrotic activity of TGF-β.However, the regulation of ECM deposition is dynamic, and involves not only synthetic, but also degrading processes. Two major ECM protease systems,plasminogen activator (PA)/PA inhibitors (PAI) system and matrix metalloproteinases (MMP)/ti,ssue inhibitors of matrix metalloproteinases (TIMP) system, are interralated and invoS.ved in matrix degradation. Among these factors PAI-1 play the most important roll in the pathogenesis of DN.In brif, overexpression of TGF-01, CTGF and PAI-1 will lead to increased synthase and decreased degradation of ECM in diabetic kidney, which may play a pivotal role in the irritation and development of DN. However, there was little experiments to prove this idea.There was a large amount of published literatures which demonstrated the fact mat, besides the strict control of blood glucose (BG) and blood pressure (BP), the application of angiotensin receptor blockers (ARBs) can both postpone the onset and delay the progression of DN effectively. It seems that we should take angiotensin receptor antagonists as the first choice for primary prevention of DN. Some scholars suggested that we should applied ARBs as early as possible for all diabetic patients even though they were not hypertensive. However, how early should we begin to block the renin-angiotensin system with ARBs? There is no established evidence or proof for it by now. At the same time, Salvia Miltiorrhiza has been proved to have satisfactory therapeutic effect on DN although the mechanism of its action is still unkown. Nevertheless, microalbuminuria has long been recognized as the first clinical hallmark of DN. But little has done to prove whether we should begin our treatment for DN before or after the appearance of microalbuminuria.Therefor, we focused our study on cytokines, the main mediators of DN, such as TGF-J31, CTGF and PAI-1, in order to elucidate the exact role of cytokines and ECM accumulation in the pathogenesis of DN. And we also want to make clear the effect of Salvia Miltiorrhiza and Losartan, an angiotensin receptor antagonist, applying before or after microalbuminuria, on cytokines expression in STZ-induced diabetic rats. This will help both to reveal the mechanism of DN medical intervention and to chose the proper therapeutic strategy and appropriate time to begin treatment in clinical practice.RESEARCH DESIGN AND METHODS: 80 uninephrectomized male Wistar rats are randomatically divided into 8 groups: normal control (NC), diabetic nephropathy control (DN), Salvia Miltiorrhiza early treatment group (DS1), Losartan early treatment group (DL1) and Salvia Miltiorrhiza combined Losartan earlytreatment group (DLS1), Salvia Miltiorrhiza late treatment group (DS2), Losartan late treatment group (DL2) and Salvia Miltiorrhiza combined Losartan late treatment group (DLS2). Rats were made diabetic by single intraperitoneal injection of streptozotocin (STZ) diluted in citrate buffer at a dose of 60mg/kg body weight. Rats of NC group just received the same amount of buffer instead of STZ. Measure the random blood glucose 72 hours after the injection of STZ, the one whose blood glucose was above 16.7mmol/L was enlisted.The three early treatment groups were treated immediately after the diabetic mould was made, while the three late treatment group began their treatment 8 weeks later. Rats of DS1 and DS2 group were given Salvia Miltiorrhiza (15g-kg*!-d'', ig) for 8 weeks; DL1 and DL2 group were given Losartan (20mg-kg'l-d"1, ig) for 8 weeks; DLS1 and DLS2 group were given Salvia Miltiorrhiza (lSg-kg^-d"1, ig) combined Losartan (20mg-kg"1-d'1, ig) for 8 weeks. During the experiment, every rat has its weight and blood glucose measured each 4 weeks. At the end of the 8th week and the 16th week, collect the 24h urine and urinary albumin excretion rate (UAER) was detected. At the end of the 16th week, all rats were sacrificed. Before execution, collect the 24h urine and detect UAER, urine creatinine (uCr). Then kill the rats, gather the blood from the abdominal aorta immediately and measure its serum creatinine (sCr) for future calculation of the creatinine clearance rate (Ccr), meanwhile total cholesterol (TC) and triglycerides (TG) were also detected. After that, the kidneys were removed, peel the envelope away, weighed, and calculate the ratio of kidney weight and body weight (KW/BW). Put part of the kidney tissue into 10% formalin to make paraffin slices for HE and PAS stain, mean glomerular volume (MGV) and fractional mesangial area (FMA) were calculated by medical image analysis system. The other part of the kidney tissue wee put into liquid nitrogen for conservation and the mRNA expression of TGF-pl, CTGF, PAI-1 and FN were detected by quantitative real time RT-PCR.RESULTS:?Body weight decreased significantly (P<0.05) at all the 4th, 8th, 12th and 16th week in diabetic rats compared with NC, meanwhile blood glucose increased. Body weight and blood glucose have no significant differences among all 7 diabetic groups (P>0.05).?Compared with NC group, UAER increased 12.68 folds at the end of the 8th week and 21.92 folds at the end of the 16th week in group DN. Compared with group DN, UAER in all six treated diabetic groups were decreased (P<0.05), but still higher than NC group (P<0.05). UAER in DLS1 and DLS2 were even lower significantly than that rats of group DSl, DLl, DS2 and DL2 (P<0.05). And there is no significant differences among DSl, DLl, DS2 and DL2 (P>0.05). At the same time, Scr in rats of group DN elevated significantly (136%, P<0.05) at the end of the 16th week. While in rats of all treated diabetic groups, Scr was almost equal to that in group NC (P>0.05). However, Ccr in rats of group DN began to decline already (14.8%, P< 0.05), which in rats of all treated diabetic groups was still higher than that in group NC (P<0.0:>).?TG and TC elevated in diabetic rats (P<0.05). Treating with losartan alone failed to change them. But treating with Salvia Miltiorrhiza, either alone or combined with losartan, reduced the level of TG and TC significantly (P<0.05).?KW/BW, MGV and FMA in DN elevated significantly, which were 129%, 98% and 183% higher than that in NC seperately. Compare with DN group, KW/BW, MGV and FMA in all six treatment diabetic groups were decreased significantly (P< 0.05), but still higher than that in group NC (P<0.05). KW/BW, MGV and FMA in DLS1 and DLS2 were even lower than that in DSl, DLl, DS2 and DL2 (P<0.05). While KW/BW, MGV and FMA in rats of group DSl, DLl, DS2 and DL2 were similar (P> 0.05).?Compared with NC group, the mRNA expression of TGF-pl, CTGF, PAI-1 and FN in diabetic groups increased 22.92, 13.42, 28.61 and 5.96 folds separately. Compared with DN group, the mRNA expression of TGF-|31, CTGF, PAI-1 and FN in all six treated diabetic groups decreased (P<0.05). There was no significant differences either between group DSl and DS2, or between group DLl and DL2(P> 0.05). Moreover, the mRNA expression decreased to the most largest extent in group DLS1 and DLS2 (P<0.05). and the decreasing extent is similar between the two groups (P>0.05). In addition, the mRNA expression level of TGF-pl, CTGF and FN in group DLl and DL2 is lower than that in group DSl and DS2(P<0.05), but themRNA expression level of PAI-1 is higher than group DS1 and DS2(P<0.05),?FMA is negatively correlated with Ccr in rats of group DN at the 16th week (r=-0.829, P<0.05). And the mRNA expression level of TGF-pi, CTGF, PAI-1 and FN is positively correlated with UAER, KW/BW, MGV and FMA.CONCLUSIONS:? The mRNA expression level of TGF-pl, CTGF, PAI-1 and FN is higher in kidneys of diabetic rats.?Over-expression of TGF={31, CTGF, PAI-1 and FN in diabetic rats were responsible for renal hypertrophy, excess ECM deposition and increased UAER.(3)The ECM of mesangium expands appear to be the main cause of declining renal function in DN. As the mesangial matrix expands, it impinges glomerular capillaries, reducing the surface available for filtration and narrowing or occluding the lumen.?Both Salvia Miltiorrhiza and Losartan could down-regulate the mRNA expression of TGF-pl, CTGF, PAI-1 and FN, and therefore reduce ECM accumulation, inhibit kidney hypertrophy, lessen urine protein and prevent the onset and progress of DN partly.(DSalvia Miltiorrhiza and Losartan have the similar therapeutic effect on DN. However, Losartan has a larger depressing eifcct on renal mRNA expression of TGF-pi and CTGF, while Salvia Miltiorrhiza has a larger depressing effect on renal mRNA expression of PAI-1.?The combination of Salvia Miltiorrhiza with Losartan has larger depressing effect on TGF-pl, CTGF, PAI-1 and FN expression than each used separately, and therefore has better renoprotetive effect on DN, which may be a new therapeutic avenues to prevent or reverse established DN.(7)The therapeutic effec is similar no matter we began our treatment before or after microalbuminuria, which suggested that microalbuminuria be the appropriate time to begin clinical treatment of DN.