Study On The Renoprotective Effects Of Isorhamnetin In Diabetic Rat And Its Mechanism

DN, as a kind of diabetes-associated microvascular complication, is the leading cause of end-stage renal disease in many western countries. In recent years, the incidence of DN in our country has increased dramatically and become the second cause of renal replacement therapy, which seriously harm human health and cause a huge economic burden.At present, DN is widely regarded as a result of interaction of environmental,genetic and autoimmune mediators, while the exact mechanisms have not been well elucidated. Elements including advanced glycation end products(AGEs) increasing,activation of the polyol pathway, abnormal renal hemodynamics, excessive activation of inflammation mediators and cytokine interact and promote the glomerular hypertrophy, expansion of the mesangial matrix, thickening of glomerular and tubular basement membranes, ultimately resulted in proteinuria, glomerulosclerosis, and tubulointerstitial fibrosis.Disorder of glucose metabolism is the first most important factor in the pathogenesis of diabetic nephropathy. High blood sugar can cause increased AGEs production,activation of the polyol pathway and enhanced activity of protein kinase C, then promote the development of DN via increasing Ang II, increasing cell permeability, up-regulating many cytokines and oxidative stress; Hemodynamic changes is also an important element in the development of DN. Glomerular in earlier stage of DN has suffered "three high" state, which resulted in damage of endothelial cells, podocytes and filtration barrier. Meanwhile, activation of RAS system in kidney caused by mechanical stress and high glucose also promote glomerulosclerosis through upregulation of TGF-β, decreasing the activity of collagenase, etc;Inflammation act throughout the development and progression of DN, FFA and high blood sugar can activate NF-κB signaling pathway by PKC and ROS, promote downstream inflammatory mediators such as TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1 which can cause vascular permeability changes, mesangial matrix proliferation and glomerular sclerosis; Other disorders such as lipid metabolism,genetic mediators also play roles in DN.NF-κB is a special protein extracted from the nucleus of B cells, which plays a crucial role in immune inflammatory response, cell survival, and stress response by regulating the expression of many genes. NF-κB in cytoplasm binding with its inhibiting protein I-κB is inactive,when cells were stimulated with hyperglycemia,cytokines,endotoxin,peroxide and other extracellular elements,I-κB is degraded and NF-κB activates entering the nucleus and regulates the transcription of some genes.Oxidative stress and PKC activation in high glucose can induce NF-κB activation and increase the expression of downstream inflammatory cytokines in DN,which plays an important role in DN. Cumulative evidence suggests that inhibition of degradation of I-κB and activation of NF-κB can help to preserve renal function and prevent or slow the progression of DN.Isorhamnetin is a plant flavonoid abundant in Ginkgo biloba, sea buckthorn and various plant flowers, fruits and leaves. Previous studies show that isorhamnetin can play anti-inflammatory, antitumor effect and protect endothelial cells by inhibiting NF-κB signaling pathway. In addition, isorhamnetin has obvious concentration dependent antioxidant activity and can scavenge free radical and protect myocardial cells from oxidative stress. At present isorhamnetin related research are mainly focused on antitumor and heart protection, few on kidney disease, and mainly in vitro cell and animal experimental, rarly in clinical trials. More research is needed to further study the mechanism of its action from the molecular level.The present study was designed to test the hypothesis that isorhamnetin presents renoprotective effects in type 2 diabetic rats and to detect its possible mechanisms.We made diabetic nephropathy rat model with high glucose feeding and low-dose STZ injection,then the rats were given two doses of isorhamnetin for 12 weeks orally.Renal injury index,renal pathological changes,oxidative stress and expression of NF-κB and inflammatory mediators in rats were tested to observe the effects of isorhamnetin on diabetic rats.Part I Renoprotective Effects of Isorhmnetin in Type 2 Diabetic RatsObjectiveTo assess the renal injury situation in diabetic rats, and to investigate the effects of Isorhmnetin intervention on renal damage biochemical markers, pathological changes in type 2 diabetic rats.MethodsAnimal Model and groupingForty-five healthy male SD rats were divided randomly into model group (n = 35)and normal control group (NC group, n = 10). NC group were given normal diet, and diabetes was induced in the model group by high fat diet for 4 weeks and a single intraperitoneal injection of streptozotocin. Rats with peripheral blood glucose for three consecutive days higher than 16.7mmol/l were considered to be diabetic,and randomly divided into diabetic (DM) group, isorhamnetin 50mg/kg (ISO-50) group and isorhamnetin 150mg/kg (ISO-150) group. Rats in the ISO-50 group and the ISO-150 group were orally given isorhamnetin (50 and 150 mg/kg/day, respectively)after grouping for consecutive 12 weeks, On the last day of isorhamnetin treatment,24 h urine was collected and kidney was removed for test.Renal damage biochemical markers and pathological examination Fasting blood glucose (FBG) was measured using OneTouch(?)Ultra machine.Levels of urinary albumin were measured using an automatic biochemistry analyzer and ELISA kits were used to measure urinary osteopontin and KIM-1. Weigh kidneys and calculate the kidney hypertrophy index (KI), then take renal cortex for pathological examination under light microscope and electron microscope to observe the histological changes in the kidney of rats.Statistical AnalysisStatistical analyses were carried out using SPSS software 14.0. Data were reported as mean±SD and were analyzed using one-way ANOVA followed by Students- Newman-Keuls (SNK) test for multiple comparisons. For all tests, thedifferences were considered significant at a value of P less than 0.05.Results1. General condition of the experimental rats There were 3 rats failed to be diabetic model and 2 rats died during the experiment,a total of 40 rats were into the experimental results analysis,including NC group,DM group, ISO-50 group and ISO-150 group each 10.2. Comparation of KICompared with control group, KI of rats in DM, ISO-50 and ISO-150 groups increased significantly (P<0.05). Compared with DM group, KI of rats in ISO-50 and ISO-150 groups decresed observably (P<0.05). However, there is no significant difference showed between KI of the two isorhamnetin groups (P>0.05).3. Comparation of FBGCompared with NC group, FBG in DM, ISO-50 and ISO-150 groups increased remarkably (P<0.05),while there is no significant difference among this three experimental groups (P>0.05).4. Renal damage biochemical markersThe DM group had higher levels of urinary osteopontin, KIM-1 and albumin than the control group (respectively P<0.05). The ISO-50 group and the ISO-150 group had lower levels of these renal damage markers than the DM group (respectively P<0.05),and those levels of the ISO-150 group were lower than the ISO-50 group(respectively P<0.05), indicating isorhamnetin dose-dependently improved the renal function in the diabetic rats.5. Pathological changes of renal tissue in rats5.1 Light microscopy resultsUnder light microscope, the glomerular morphology of NC group was normal, the structure was clear and there is no capillary basement membrane thickening,mesangial cells hyperplasia and mesangial matrix expansion. Kidney in DM group rats showed glomerulus hypertrophy, part of renal cysts obvious stenosis, capillary basement membrane thickening, mesangial area widened, tubular basement membrane thickening and part of the renal tubular epithelial cells vacuolization.Compared with DM group, the renal pathological changes in ISO-50 and ISO-150 group were significantly lighter, and the improvement in renal pathology of ISO-150 group was more obvious.5.2 Electron microscopy resultsUnder electron microscope, the structure of glomerular capillary basement membrane was clear, and there was no obvious proliferation of mesangial cells and matrix, no GBM thickening and foot process fusion showed. In DM group, the glomerular basement membrane showed diffuse thickening, and there were obvious foot process broadening or fusion. The above changes in ISO-50 and ISO-150 group were lighter than that in DM group, GBM structure is clearer and there were foot process segment fusion, Pathological changes in ISO-150 group improved more significantly.Part Ⅱ Inhibitory effect of Isorhmnetin on oxidative stress activity and NF-κB pathwayObjectiveTo observe the oxidative stress, activation of NF-κB signaling pathway and the expression of inflammatory mediators in diabetic rats, and to observe effects of two doses of isorhamnetin intervention on them; In vitro, meanwhile,to observe the effects of isorhamnetin on LPS induced oxidative stress and NF-κB activation in rat glomerular mesangial cells.MethodsAnimal model and groupingThe same to Part ⅠMesangial cell culture and groupingRat glomerular mesangial cells (GMCs) were randomly divided into the following 4 groups and cultured for 72h in DMEM: normal control group (NC group),LPS (with the presence of 10 nmol/ml LPS in medium), ISO-5 group (with the presence of 10 nmol/ml LPS and 5 μM isorhamnetin in medium), and ISO-10 group(with the presence of 10 nmol/ml LPS and 10μM isorhamnetin in medium).Oxidative stressAntioxidant activity was assessed by measuring levels of total superoxide dismutase (T-SOD) in cell culture medium and in renal homogenate using Xanthine oxidase method, and oxidative stress activity was assessed by measuring levels of malondialdehyde (MDA) using colorimetric method.NF-κB pathway in renal tissue and mesangial cellsELISA method was used to detect the expression of NF-κB p65, phospho-NF-κB p65, IκBα and phospho-IκBα in the kidney homogenate and cell lysis solution. The expression of NF-κB p65 mRNA in renal and mesangial cells were detected by real-time RT-PCR.NF-κB p65 DNA binding activityNuclear protein was extracted from kidney and rat GMCs. NF-κB DNA binding activity was measured using the NF-κB p65 transcription factor ELISA assay kitsInflammatory mediators in renal tissue and mesangial cellsELISA and real-time RT-PCR assay were used to detect the expression of NF-κB’s downstream inflammatory mediators TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1 in protein and mRNA levels in cell culture medium and renal homogenate.Statistical AnalysisStatistical analyses were carried out using SPSS software 14.0. Data were reported as mean±SD and were analyzed using one-way ANOVA followed by Students-Newman-Keuls (SNK) test for multiple comparisons. For all tests, the differences were considered significant at a value of P less than 0.05.Results1 Effects of isorhamnetin on oxidative stress1.1 T-SOD activity and MDA level in renal tissues of ratsDM group had higher levels of MDA and lower levels of T-SOD than the control group (respectively P<0.05). Isorhamnetin(50mg and 150mg) dose-dependently revered the changes of MDA and T-SOD if compared to the DM group (respectively P<0.05).1.2 T-SOD activity and MDA level in GMCsLPS group showed higher levels of MDA and lower levels of T-SOD than the normal control cells (respectively P<0.05). Isorhamnetin (5μM and 10μM)dose-dependently revered the changes of MDA and T-SOD if compared to the LPS group (respectively P<0.05).2. Effects of isorhamnetin on NF-κB signaling pathway2.1 Effects of isorhamnetin on NF-κB signaling pathway in ratsThe high-fat diet plus STZ injection activated the renal NF-κB signaling by increasing levels of NF-κB p65 (protein and mRNA), phospho-NF-κB p65 and phospho-IκBα in the DM group if compared to the NC group (respectively P<0.05).The NF-κB activation was inhibited by isorhamnetin if compared to the DM group(respectively P<0.05), and the inhibitory effects were dose-dependent in the ISO-50 group and the ISO-150 group (respectively P<0.05). The IκBα level showed no significant differences among this four groups (P>0.05).2.2 Effects of isorhamnetin on NF-κB signaling pathway in GMCsIn the in vitro study, LPS increased the generation of NF-κB p65 (protein and mRNA), phospho-NF-κB p65 and phospho-IκBα in GMCs if compared to the normal control cells (respectively P<0.05). Isorhamnetin dose-dependently (5 μM and 10 μM)inhibited the over-production of NF-κB p65 (protein and mRNA), phospho-NF-KB p65 and phospho-IκBα if compared to the LPS group (respectively P<0.05). No significant differences in levels IκBα were observed among the groups in vitro studies(respectively P>0.05).2.3 Effects of isorhamnetin on NF-κB p65 DNA binding activityIn the in vivo study, the DM group had much higher NF-κB p65 DNA binding activity than NC group (P<0.05). Isorhamnetin (50 mg and 150 mg) dose-dependently decreased the NF-κB p65 DNA binding activity if compared to the DM group(respectively P<0.05). In the in vitro study, the LPS group showed higher NF-κB p65 DNA binding activity than the normal control cells (P<0.05), and isorhamnetin dose-dependently (5μM and 10 μM) inhibited the NF-κB p65 DNA binding activity if compared to the LPS group (respectively P<0.05).2.4 Effects of isorhamnetin on inflammatory mediators in renal tissues In the in vivo study, there were higher levels (protein and mRNA) of inflammatory mediators TNF-α,IL-1β,IL-6, ICAM-1 and TGF-β1 in the DM group than the NC group (respectively P<0.05). Isorhamnetin (50mg and 150mg) dose-dependently decreased these inflammatory mediators if compared to the DM group (respectively P<0.05).2.5 Effects of isorhamnetin on inflammatory mediators in GMCs In the in vitro study, there were higher levels (protein and mRNA) of inflammatory mediators TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1 in the LPS group than the normal control cells (respectively P<0.05). Isorhamnetin (5μM and 10μM)dose-dependently decreased these inflammatory mediators if compared to the LPS group (respectively P<0.05).Conclusion1. Isorhamnetin slowed progressive kidney lesions through reducing the renal hypertrophy index, decreasing the excretion of urinary albumin and lightening the renal pathological damage in type 2 diabetic rat model.2. Isorhamnetin significantly improved renal oxidative stress in diabetic rats in vivo, meanwhile obviously relieved oxidative stress in mesangial cells induced by LPS in vitro.3. There were excessive activation of NF-κB signaling pathway and over-expression of its downstream inflammatory mediators in diabetic rat kidney;LPS could induce NF-κB pathway activated and inflammatory mediators increased in mesangial cells,which could simulate inflammation state.4. In vivo, isorhamnetin could negatively regulate the NF-κB signal pathway and inhabit over-expression of inflammatory mediators TNF-α,IL-1β,IL-6, ICAM-1 and TGF-β1 dose-dependently in diabetic rat model; In vitro, isorhamnetin could dose-dependently inhibit excessive activation of NF-κB signal pathway and decrease the levels of mediators TNF-α, IL-1β, IL-6, ICAM-1 and TGF-β1 in rat mesangial cells induced by LPS.