To Explore Hyperoside Ameliorates Diabetic Nephropathy Induced By STZ Via Targeting MiR-499-5p

Part ?:Clinical effect of Qikui granule on patients with diabetic nephropathyObjective:Through Qikui granule to treat patients with diabetic nephropathy(DN),confirm the clinical efficacy of Qikui granule,and provide clinical data support for follow-up research.Method:The patients who were diagnosed with type 2 diabetes and selected as the research object in our hospital from March 2016 to January 2020.All the patients were diagnosed with OGTT diagnosis of type 2 diabetes and had complete medical history data and complete collection of clinical samples.Through urine albumin excretion rate And clinical information diagnosis is divided into diabetes(DM)group and DN group.The DM was treated with conventional hypoglycemic therapy as the control group.The DN group was treated with conventional hypoglycemic treatment and oral Qikui granules as the treatment group.Before and after treatment,blood samples and urine samples were collected from patients in the treatment group.Compare the indicators of gender,age,systolic blood pressure,diastolic blood pressure,hypertension incidence,FPG,HBA1c,TG,PBG,TC,Scr,UACR,24h-UTP and EGFR before and after treatment in DM group and DN group.Result:Before treatment,there were no significant differences in general information between the two groups,such as male to female ratio,age,systolic blood pressure,diastolic blood pressure and incidence of hypertension(P<0.05).Compared with diabetic group(DM group),FPG,HbAlc and TG in DN group were significantly higher than those in DM group,the difference was statistically significant(P<0.05).There were no significant differences in PBG and TC levels between 2 groups(P>0.05).Compared with diabetic group(DM group),SCR,UACR and 24h-UTP levels in DN group were significantly higher and EGFR levels was significantly lower than those in DM group,with statistical significance(P<0.01).Compared with before treatment,the levels of UACR and 24h-UTP in DN group were significantly decreased after treatment,with statistical significance(P<0.01).SCR was slightly increased and EGFR was slightly decreased,but there was no significant difference compared with that before treatment(P>0.05).Compared with the data in Table 3,it can be found that although the renal metabolic indexes of DN group were improved after treatment,there was still a significant difference compared with DM group(P<0.01).Conclusion:Qikui granule can improve the functional treatment of Scr,UACR,24h-UTP and eGFR in patients with DN,and have clinical therapeutic effects on DN.The follow-up pharmacological study of hyperoside,the effective component of Qikui granule,has clinical support.Part ?:Hyperoside protects HK-2 cells against high glucose-induced apoptosis and inflammation via the miR-499-5p/NRIP1 pathwayObjecitve:To observe the effect of hyperoside on HK-2 cells induced by high glucose,and to explore the effect of hyperoside on the apoptosis and inflammation of HK-2 cells intervened by high glucose and related mechanisms.Methods:The HK-2 cell line was used as a model of normal renal tubular cells and cultured in high-glucose conditions(containing 45.0 mmol/L glucose medium)to establish an in vitro DN model.TUNEL method was used to detect cell apoptosis,and flow cytometry was used to detect cell apoptosis.ELISA kit was used to detect inflammation.Western blotting detects the protein levels of apoptosis-related genes and inflammatory cytokines.Mechanism analysis includes luciferase report experiment and RNA pulldown experiment to detect the binding relationship between molecules.Results:First,CCK-8,TUNEL and flow cytometry were used to determine the survival of HK-2 cells in the control group(Control),high glucose model group(HG),and treatment group(HG+Hyperoside).The data shows that high-sugar treatment can induce cell apoptosis,and hyperoside can reduce cell apoptosis in a dose-dependent manner.Western blot analysis showed that hyperoside can increase the expression of Bax and decrease the expression of Bcl-2 induced by HG.The protein levels of IL-10,IL-6 and IL-1? in HK-2 cells treated with HG were detected.HG can up-regulate the protein levels of IL-6 and IL-1?,and down-regulate the protein level of IL-10.Hyperoside can antagonize the effects of HG on IL-10,IL-6 and IL-1?.ELISA kit was used to detect the concentration of these pro-inflammatory cytokines.The results showed that hyperoside reversed the stimulating effect of HG on the inflammatory response of HK-2 cells.Next,we found that miR-499-5p was down-regulated by HG in HK-2 cells and was further up-regulated by hyperoside in a dose-dependent manner.RT-qPCR was used to detect the gene knockout effect of miR-4995p on HK-2 cells treated with HG.The expression of miR-4995p was effectively down-regulated by miR-499-5p inhibitors.miR-499-5p inhibitor can rescue the anti-apoptotic effect of hyperoside on HK-2 cells treated with HG.Western blot and ELISA results showed that inhibition of miR-499-5p can rescue the hyperoside-induced increase in IL-10 levels in HK-2 cells,as well as the decrease in IL-6 and IL-1? levels.According to Starbase predictions,NRIP1 was identified as the downstream target of miR-499-5p.HG induced up-regulation of NRIP1 expression in HK-2 cells,and hyperoside treatment decreased NRIP1 expression in a dose-dependent manner.The miR-499-5p inhibitor promoted NRIP1mRNA and protein levels in HK-2 cells,independent of HG treatment.The binding sequence of miR-499-5p and NRIP1 was predicted from Starbase.The binding site of NRIP1 is mutated and used for the next step of the luciferase reporter analysis.Transfection of miR499-5p inhibitor enhanced the luciferase activity of pmirGLO-NRIP1-Wt plasmid in HK-2 cells,but had no significant effect on the luciferase activity of pmirGLO-NRIP1Mut plasmid.The RNA pulldown experimental data show that compared with bio-miR-499-5p Mut,BIO-miR-499-5p Wt pull-down products are rich in NRIP1 content,indicating that miR-499-5p interacts with NRIP1.Rescue experiments showed that inhibiting NRIP1 can rescue the pro-apoptotic effect of miR-499-5p on HK-2 cells.The stimulating effect of silent miR-499-5p on IL-10,IL-6 and IL-1? protein levels can be restored by down-regulating NRIP1.Conclusion:Hyperoside reduces the apoptosis and inflammation of HK-2 cells induced by HG through the miR-499-5p/NRIP1 axis.Part ?:Hyperoside ameliorates diabetic nephropathy induced by STZ via targeting miR-499-5p/APC axisObjective:The aim of this study was to explore the mechanism of hyperoside targeting miR-499-5p/APC axis to improve STZ-induced DN.Method:As a model of normal renal tubular cells,the HK-2 cell line was cultured in high-glucose conditions(containing 45.0 mmol/L glucose medium)to establish an in vitro DN model.TUNEL method was used to detect cell apoptosis.Flow cytometry was used to detect cell apoptosis.ELISA kit was used to detect inflammation.Western blot was uesd to detect the protein levels of apoptosis-related genes and inflammatory cytokines.Mechanism analysis includes luciferase report experiment and RNA pull-down experiment to detect the binding relationship between molecules.Using SPF grade C57BL/6 male mice,high-fat and high-sugar diet and STZ induction,a mouse model of DN was established.Different doses of hyperoside were administered for treatment,and urine protein and blood sugar,body weight,systolic blood pressure,blood creatinine and glomerular filtration rate of mice in each dose group were measured.RT-qPCR was used to detect the levels of miR-499-5p and APC,and the effects of miR-499-5P and APC in mice were evaluated on blood glucose.Histological immunostaining was used to observe the structure of kidney tissue,measure the glomerular area index and mesangial expansion index of PAS-positive mice,and quantify the collagen deposition in the glomerulus and renal interstitium of mice.The protein levels of Bax,Bcl-2,collagen ?,collagen ? and fibronectin in each group were detected by western blot.The content of cytokines(tumor necrosis factor-?,monocyte chemoattractant protein-1,interleukin-1-?)in each group was detected by ELISA method.The TargetScan(http://www.targetscan.org)database was used to predict potential miRNAs containing APC3' non-coding region binding sites.Result:Podocytes were treated with high glucose(25mmol/L)to simulate cell injury,and normal glucose(5.5mmol/L)was used as control.High glucose significantly up-regulated the expression of extracellular matrix(including collagen ?,collagen ?and fibronectin),while hyperoside inhibited it.In addition,the protein levels of inflammatory cytokines TNF-?,monocyte chemoattractant protein-1 and IL-1?increased in high glucose environment.At the same time,hyperoside can reduce the inflammatory reaction in hyperglycemia model.hyperoside could antagonize the increase of Bax,the decrease of Bcl-2 and the decrease of Caspase-3 activity induced by HG in podocytes.DN mice were induced by high-fat diet and streptozotocin(STZ)injection.The blood glucose of DN mice increased and then decreased after treatment with hyperoside or insulin.Based on the results of PAS and Masson' s trichromatic staining,we found that hyperoside treatment could counteract the increase in glomerular area and size mediated by STZ.After injection of streptozotocin,the glomerular surface area of diabetic rats increased significantly,glomerular mesentery dilated,and tubular-mesenteric collagen accumulated.However,this effect was significantly abolished by hyperoside treatment.Western blot results showed that hyperoside could reduce the accumulation of extracellular matrix(collagen ?,collagen ? and fibronectin)in renal tissue of DN mice.In addition,Western blot and ELISA analysis showed that hyperoside treatment could inhibit STZ-induced inflammation.Finally,compared with Sham+DMSO group,the increase of Bax mediated by STZ and the decrease of Bcl-2 protein level were antagonized by hyproside treatment.Compared with Sham+DMSO group,STZ mediated the increase of Bax and the decrease of Bcl-2 protein level.RT-qPCR analysis showed that miR-499-5p was more up-regulated in hyperoside-treated podocytes than other miRNAs.Then,RT-qPCR found that the level of miR-499-5p was decreased by high glucose and then recovered by hyperoside treatment in vivo.The binding sequence between miR-499-5p and APC 3'UTR was inferred by TargetScan.The expression level of miR-499-5p was overexpressed and down-regulated by transfection of miR-499-5p mimic and anti-miR-499-5p,respectively.Luciferase report experiment with podocytes showed that the luciferase activity of wild-type pGLO-APC-3'UTR was significantly decreased by ectopic miR-499-5p,while the luciferase activity of wild-type pGLO-APC-3'UTR silenced by miR-499-5p was significantly increased,but the luciferase activity of mutant pGLOAPC-3'UTR had no significant change.RT-qPCR and Western blot analysis showed that miR-4995p amplification could increase mRNA and protein levels,while miR-499-5p inhibition could decrease mRNA and protein levels.Finally,miR-499-5p inhibition abolished the inhibitory effect of hyperoside on podocyte APC mRNA level.After injection of AAV-miR-499-5p and AAV-APC respectively,the levels of miR-499-5p and APC in the left kidney increased significantly,but there was no obvious differentiation in the right kidney.Overexpression of miR-499-5p and APC had no significant effect on blood glucose,body weight,serum creatinine and blood pressure in DN mice.The inhibitory effect of miR-499-5p on GFR was counteracted by the overexpression of APC.Compared with DN+Mock+vector group,glomerular hypertrophy and glomerular surface area decreased in AAvmir-499-5p group.This effect was counteracted by the overexpression of APC.In DN mice,the inhibitory effect of miR-499-5P on glomerular surface area and mesangial extension and the decrease of tubulointerstitial collagen accumulation were restored by AAV-APC treatment.In DN mice,the inhibitory effect of miR-499-5P on glomerular surface area and Mesangial extension and the decrease of tubulointerstitial collagen accumulation were restored.Similarly,Western blot analysis showed that the decrease of ECM in renal tissue induced by miR-499-5p was also offset by the overexpression of APC.In addition,injection of AAV-miR-499-5p could reduce the content of inflammatory cytokines in serum of DN mice.However,this result was offset by the overexpression of APC.The decrease of Bax and the increase of Bcl-2 induced by AAV-miR-499-5p can be offset by the overexpression of APC in DN mice.Finally,the overexpression of miR-499-5p destroyed the promoting effect of high glucose treatment on urinary protein,which could be reversed by ectopic APC.Conclusion:Hyperoside can inhibit ECM aggregation,inflammation and apoptosis induced by HG in vitro.hyperoside treatment improved the symptoms of diabetes in STZ-induced mice and reduced renal damage and fibrosis in DN mice.Hyperoside can inhibit the level of APC both in vitro and in vivo.APC is directly targeted and negatively regulated by miR499-5p.miR-499-5p attenuates renal dysfunction in DN mice by targeting APC.The overexpression of APC reversed the effect of miR-499-5p on renal injury and fibrosis.