Effect Of Shenkangwan On The MiR-192 Signaling Pathway In Diabetic Nephropathy

Diabetic nephropathy (DN) is one of the most familiar and refractory vascular complications of diabetes mellitus (DM) in clinic. And it is a leading cause resulting in death among the end-stage renal disease and DM patients. About 40% type 1 and 5～20% type 2 DM patients might run to DN. DN whose pathogenesis has not been fully understood, doesn’t been well treated in the days. So how to treat and prevent DN effectively is becoming a common concerned problem among the international medical researchers.DM might damage the patients’ kidney from varies of ways and including all structure of the kidney, which could cause the proliferation and hypertrophy in glomerular cells, thickening of the glomerular basement membrance, ceaseless accumulation of extracellular matrix (ECM) and lead to glomerulosclerosis in the end. Transforming growth factor beta 1 (TGF-β1)is a multiple functional cytokine and has a assured effect in the pathogenesis of DN. These include inducing the proliferation and hypertrophy of the cells in the kidney, increasing the synthesizing and secretion of the ECM, decreasing the decomposition of the ECM via increasing the active effect of inhibitors of metalloproteinases and so on.. The activation of TGF-β1 can be stimulated and mediated by almost all confirmed media and the molecular pathway in Kidney damage of DM. Therefore, It has important clinical significance to find a new method for treatment of DN by antagonizing TGF-β1, and to find its more specific and efficient downstream factors or links.MicroRNAs (miRs) are short noncoding RNAs that recently have been shown to play important roles in mammalian gene expression. Recent evidence suggests that miR-192 involves in TGF-β1 mediated collagen regulation. Thus, TGF-β1 induced down-regulation of SIP1 via miR-192 and can enhance collagenⅠexpression via derepression at E-box elements. The observations provide the first functional role for a miR expressed in the kidney. Small noncoding RNAs and miRs such as miR-192 and their inhibitors may be targets for diseases such as DN and other diabetic complications.The majority of DN patients, once enter the clinical proteinuria, irreversible kidney damage has occurred, the disease will show a progressive development, so early detection and treatment is of great clinical value in DN. Now the treatment of the DN in western medicine mainly aims at its symptom which include controlling the intensive blood glucose, reducing the albuminuria, control the hypertension, and so on. But so far neither one medicine can prevent the progression of the DN. So it is important to prevent and treat DN by exerting the superiority and characteristic of traditional chinese medicine (TCM) in treating DN and explore actively effective treatment from TCM.DN belongs to the domain of the "Shenxiao", "Dropsy", "Shenlao", "Guange", and so on in TCM. "Splenic and renal weaken" is the basal pathogenesis of DN and the "Phlegm and damp", the "Grime and toxin", the "Gore" are the stimulative factors. So the "fundamentality weaken and sign mighty" is the TCM characteristic pathogenesis of DN. Early DN (Ⅲstages) always appears the symptoms of "Yin weaken" or "both Qi and Yin weaken". Clinic DN (Ⅳstages)always comes forth the symptoms of "splenic and renal weaken", "liquid and humidity logjam" or "gore stagnateing vas". While end DN (Ⅴstages) always emergences the symptoms of "downfall of Yin and Yang in spleen and kidney" and "grime and toxin blocking". So the therapeutic principle of DN inⅢ-Ⅴstages includes "increasing the Qi and toughen the spleen", "nourishing the kidney and maintain Yin" and "impeling blood stream and dredging vas".The compound preparation Shenkangwan was mainly constituted of astragalus mongholicus, rhubarb, leech, chrokee rose fruit, and so on. It has the effect of increasing the Qi and toughen the spleen, nourishing the kidney and constringency, diuresis and detumescence, impeling blood stream and dredging vas. So it accords with the TCM characteristic of pathogenesis of DN fitly and can treat the DN from the fundamentality to the sign. As a hospital preparation, Shenkangwan has been used to treating DN patients in clinic for many years and has a assured curative effect. The study from clinic observation and animal experiment indicated that Shenkangwan could ameliorate the general symptom, protect the kidney and stay the development of the DN. But its molecular mechanism in treating DN is not clear and need farther research.So we carried out the experiment to investigate the expression of TGF-β1、Ets-1、miR-192 in the kidneys of the DN rats and the expression of TGF-β1、Ets-1、SIP1、Collagenl、miR-192 in the cultured mesangial cells (MC) induced by high glucose on the basis of experiment results before, to explore the molecular and genic mechanism of Shenkangwan in treating DN from TGF-β1 induced collagen expression via miR-192 signaling pathway and provide the laboratorial evidence for its clinic application. The details were as follows:Chart I Laboratory study on the protective effect of Shenkangwan on the kidney of the DN RatsObjective:To observe the effect of Shenkangwan in treating rats of DN model and protecting their kidneys and provide the laboratorial foundation for further studying its therapeutic mechanism.Methods:Firstly, we established the DM rat models by intraperitoneal injection of streptozotocin(STZ) according to the dose of 55 mg·kg-1 body weight. In the following two weeks, we detected the blood glucose, the 24 hour urine protein and the urine volume. The rats, which value of blood glucose exceeding 16.6 mmol·L-1 and 24 hour urine protein exceeding 30 mg/kg/24h, urine volume 1.5 times as baseline were taken for the successful DM rat models and calculated the rate of them. Then they were randomly divided into 3 groups:model control group, micardis group, and Shenkangwan group. Other eight normal rats were used as normal control group. All rats were treated with corresponding drugs for 8 weeks. During and after the treatment, the general state, blood and uric glucose levels of the rats in every group were observed. After treated in the last time, all rats were put into the metabolizing cage to be measured the volume of their 24 hours urine and drinking water. Then we hocused all the rats with 10% chloral hydrate, collected the blood via ventral artery, gathered the serum by centrifuge and detected the content of the blood glucose and the glucosylated hemoglobin (GHb), the excretion of the 24 hour urine protein (Upro), the levels of serum creatinine (SCr), blood urea nitrogen (BUN), total serum cholesterol (TC),triglyceride (TG),high density lipoprotein (HDL-c),low density lipoprotein (LDL-c),.And we took out the kidney of the rats, observed the size and volume of them and measured the kidney weight and relative kidney weight. Then we investigated their renal pathological changes by optical microscope with the coloration method of Hematoxylin and Eosin (HE)Results:46 rats were used to established the DM models and the rate of successful models was 78.9%(30/38). And the death rate of DM model rats was 4.67%(2/30). During the treating eight weeks, The model rats blood glucose were all over 16.7 mmol·L-1, the uric glucose positive experiment were exceeded+++. And they appeared the symptom of the DM such as the quantum of drinking, eating and urinating increased. Compare with the normal rats, their following indexes including GHb,24 h Upro, Scr, BUN, TC, TG, and LDL-c were increased distinctly (P<0.01); and HDL-c were decreased distinctly (P<0.01). Their kidney weight and relative kidney weight increased distinctly and came forth about the 3 stages renal pathological lesions according to the stages of Mogensen. Compare with the rats in model group, Shenkangwan could ameliorate the rat’ general state, amelioration the above indexes markedly and mitigate the renal pathological lesion.Conclusions:Shenkangwan could treat DN rats and had a certain protective effect on the kidney of DN rats.ChartⅡMicroRNA-192 in the kidneys of DN rats and its function in TGF-β1 induced collagen expressionObjective:To explore the effect of the Shenkangwan on TGF-β1 induced collagen expression via miR-192 signaling pathway in the kidneys of the DN rats.Methods:The kidneys used of this study were from the rats in chartⅠ. We detected the protein expression of TGF-β1; Ets-1 with the method of immune histochemistry, and observed the expression of TGF-β1; Ets-1 and miR-192 mRNA in the renal tissue of the rats in every group with the method of the quantitative real-time PCRResults:The renal tissue expression of TGF-β1, Ets-1 and miR-192 mRNA in the rats of DN model group were increased markedly than those in the rats of normal control group (P<0.01). And the expressions of TGF-β1, Ets-1 protein were increased distinctly in these rats’ kidneys too (P<0.01). Compare with those in the rats of the DN model group, the expression of TGF-β1, Ets-1 and miR-192 in the rats of Shenkangwan and micardis group were decreased markedly (P<0.01)Conclusions:The TGF-β1 could effect the miR-192 by control the expression of the Ets-1. Shenkangwan could restrain the activation of collagen expression via miR-192 signaling pathway in the renal tissue of DN rats and reduced the synthesizing and secretion of the ECM, which would conduce to staying the course of the DN.ChartⅢMicroRNA-192 in the MC cultured by high glucose and its function in TGF-β1 induced collagen expressionObjective:To explore the effect of Shenkangwan on TGF-β1 induced collagen expression via miR-192 signaling pathway in the the MC cultured by high glucose.Methods:MC cultured in high glucose in vivo was divided into five groups: high glucose control group, normal rats’serum control group, micardis group and Shenkangwan group (high dose and low dose).A normal glucose control group was used too. Cultured for 24 hours, the secretion of TGF-β1 of MC were detected by using enzyme linked immunosorbent assay (ELISA), the expression of TGF-β1、Ets-1、collagen 1、SIP1、miR-192mRNA were determined by using RT-PCR method and the expression of its protein were detected by Western blot method.Results:Compare with the normal glucose, high glucose could accelerate the secretion of TGF-β1 of MC, increase their expression of TGF-β1、Ets-1、collagen 1、miR-192 mRNA (P<0.01), enhance their expression of its protein (P<0.01), and depress the expression of SIP1 mRNA and protein in the cultured MC in vivo (P<0.01). While intervening and treating with Shenkangwan and micardis could reduce the secretion mRNA and the protein expression of TGF-β1、Ets-1、collagen 1、miR-192 in the cultured MC induced by high glucose (P<0.01), and increase the mRNA and the protein expression of SIP1 (P<0.01)Conclusions:High concentrations glucose could activate the miR-192 signaling pathway and stimulate the secretion of ECM in the cultured MC in vivo. Shenkangwan could restrain the activation of miR-192 signaling pathway in the cultured MC induced by high glucose and reduce their synthesizing and secretion of the ECM.Conclusions of the whole paper:We carried out the experiment on animal and the MC cultured by high glucose, and proved again that Shenkangwan could treat DN and had a certain protective effect on the kidney of DN. Its therapeutic mechanism was related to restraining the activation of collagen expression via miR-192 signaling pathway and reducing the synthesizing and secretion of the ECM. And those provided the molecular and genic therapeutic mechanism and laboratorial evidence for its clinic treatment on DN.