The Effect Of Salidroside On Expression Of Nrf2, γ-GCS And PPAR-γ In Kidney Of Diabetic Rats

Object: Currently, diabetic nephropathy disease (Diabetic Nephropathy,DN) has become one of the main end-stage renal disease, the study of themechanism of the development of diabetic nephropathy is particularlyimportant,but has not been fully elucidated, the mechanism may be related togenetics, glucose metabolism, hemodynamic, and inflammatory cytokinesand other factors. To investigate the underlying mechanism may provide a newtheoretical basis for the prevention and treatment of diabeticnephropathy.Studies have demonstrated that oxidative stress has closeconnection with the pathogenesis of diabetic nephropathy. High glucose-induced oxidative stress can contribute to excessive reactive oxygen species,then inflammatory mediators and cytokines production induce synthesis ofextracellular matrix, to accelerate glomerulosclerosis. The formation oflong-term high blood glucose can decrease capacity of carrying oxygen by redblood cells,the production of hydrogen peroxide, oxygen free radicals intissue cells increased and glutathione reduced, leading to cell injury, affect thestructure and function of the kidneys. Nrf2/ARE signaling pathway isimportant for the body’s endogenous protective pathway, this pathway isconsidered to be a key pathway in vivo numerous antioxidants, with thebalance of oxidizing environment-reduced state, anti-inflammatory and otherbiological effects. Nrf2/ARE signaling pathway’s activation makesdownstream gene transcription phase Ⅱdetoxifying and antioxidant enzymegene increasing, increases the body ’s resistance to oxidative stress.Peroxisome proliferator-activated receptors (PPARs) are one of nucleartranscription factor superfamily with three subtypes, which PPAR-γ andkidney is most closely related. Activation and inhibition of PPAR-γ in thebody may play a protective role. PPAR-γ agonists mainly includes two types, i.e., the natural ligand and synthetic ligands, such as15d-PGJ2former, thelatter including thiazolidinediones, such as TZDs. Currently，we have knowna few antagonists, such as BADGE, GW9662.Studies have shown thatNrf2/ARE signaling pathway and PPAR-γ have some intrinsic connection. Asan herbal extract, salidroside has a wide range of pharmacological effects,such as antioxidantion, anti-inflammatory, anti-tumor. In animals’ studies,whether salidroside can reduce oxidative stress and proteinuria in diabetickidney by effecting Nrf2/ARE signaling pathway and PPAR-γ factor, thereports in the literature is very rare. In this experiment by modeling theexperimental diabetic rats given salidroside, we can explore the relationshipbetween Nrf2/ARE signaling pathway with PPAR-γ factors and the role ofSalidroside in the development of diabetic nephropathy, so as to provide newideas to treat diabetes nephropathy.Materials and Methods: We choose SPF level30healthy male SD rats,weighing200±20g, were randomly divided into two groups,10normal group(NC group), the others were made into diabetes mellitus. After purchasingrats we feed them adaptively, before intraperitoneal injection of streptozotocin(STZ),all rats require fasting12hours, then the others SD rats were givenSTZ65mg/kg.After72hours, when the blood glucose≥16.7mmol/L, Urineglucose≥3+，we consider the model of diabetes mellitus success. Then theywere were randomly divided into two groups, DM group and SALgroup. TheNC group were given the same amount of citric acid-sodium citrate bufferIntraperitoneal injection. After successding models, Salidroside was given toSAL group30mg/kg·d orally. The rats of the cage to feed, to a normal diet,free food water, temperature, humidity, light.The whole experiment wereconducted12weeks. Respectively, for6weeks and12weeks, rats wererandomly selected five,24-hour urine specimens to detect protein, called afterthe body weight by intraperitoneal injection of10%chloral hydrate anesthesia,we take the heart blood to detect blood glucose, creatinine and blood ureanitrogen. After remove the envelope,we weigh the kidneys.The right kidneywas used to detect the indicators of oxidative stress like CAT, GSH and the expression of Nrf2, γ-GCS, PPAR-γ by RT-PCR. The left kidney was slicedinto thin pieces, then they were stained with hematoxylin and eosin (HE) andPAS staining in the light microscope kidney tissue pathology,Immunohistochemistry was used to detect the expression of Nrf2, γ-GCS, andPPAR-γ in kidney, the result of expression was analysised by applying theimage analysis system. Experimental data was descripted with mean±standard deviation (X±S), The comparition of each group data use method ofone-way ANOVA, two groups’ data were compared using two independentsamples t test, if the data does not meet the normality and homogeneity, weuse non-parametric tests. We use SPSS19.0statistical analysis software toanalysis the experimental data, and considering P <0.05statisticalsignificantly.Results: we statistics the weight, kidney index (kidney weight/bodyweight),24-hour urine protein and blood biochemistry in each rats’ group,P>0.05statistically significant. Which, along with the progression of diabeticrats emergence polydipsia, polyphagia, polyuria, weight loss, listlessness,unresponsive and other symptoms gradually,24-hour urine protein increased.Urinary protein excretion in rats SAL group compared with DM groupdecreased,and its blood glucose, blood urea nitrogen, serum creatininedecreased compared DN group, P <0.05considered statistically significant.From Light microscope in kidney tissue of each group,HE and PAS stainingshows that with the continued presence of high blood sugar, NC group ratkidney had no significant disease, the cells and mesangial matrix of the othertwo groups in the6weeks had mild hyperplasia, Lesions gradually increasedover time, in12weeks mesangial matrix is hyperplasia more obvious.Comparing with DM group significantly, SAL group had milder renaldiseases. Application of image analysis system on immunohistochemicalstaining, the results show: Nrf2, γ-GCS mainly expressed in intrinsicglomerular cells, tubular epithelial cells, the expression intensity: SAL group>DM group> NC group (P <0.05); PPAR-γ is mainly expressed in the epithelialcells of renal tubular, duct,glomerular had rare expression, the factor expression intensity: NC group>DM group>SAL group (P <0.05). Oxidativestress indicators, kidney CAT and GSH significantly reduced at6and12weeks after successful modeling, SAL group was significantly higher thanDM group (P <0.05). RT-PCR results showed a consistent trend with theexpression of Nrf2、γ-GCS、PPAR-γ immunohistochemical results.Conclusion: After successful modeling diabetic rats, oxidative stressenhanced. Nrf2/ARE signaling pathways was activated, and expression ofγ-GCS enhanced, expression of PPAR-γ reduced；SAL can enhance activationof Nrf2/ARE pathway, inducing expression of γ-GCS and inhibit expressionof PPAR-γ，to lower blood sugar, reduce proteinuria and the renal injury ofoxidative stress.