Protective Effect And Mechanism Of Wogonin On Diabetic Kidney Podocyte Injury

Background and purpose: Diabetic nephropathy(DN)is the most common and serious complication of patients with diabetic mellitus(DM).It is also one of the important cause of end-stage renal disease(ESRD).There is currently no effective treatment.With the continuous deepening of DN research,people realize that early improvement of DN's early pathological changes,reducing or preventing the occurrence of proteinuria have important value for the prevention and treatment of DN.Podocytes are an important part of the glomerular filtration barrier,and various mechanisms such as inflammation,oxidative stress,apoptosis,and autophagy can cause podocyte damage.Recent studies have shown that podocyte damage is a key factor for early DN lesions.Therefore,it is of great clinical significance to explore treatment methods aimed at reducing podocyte damage and then intervening in the progression of DN.Wogonin is a flavonoid compound isolated from the roots of Scutellaria baicalensis,Scutellaria barbata,and the oleander of the oleander plant Pueraria lobata.It has a wide range of biological activities and has been confirmed in inflammation,oxidative stress,apoptosis,autophagy and other aspects have clear pharmacological effects.Given that these mechanisms are also involved in podocyte injury,whether wogonin has a preventive and therapeutic effect on DM kidney injury and whether it can alleviate DM kidney injury by inhibiting podocyte injury has aroused our strong interest.The purpose of this study was to explore the protective effect of wogonin on DM kidney podocyte injury and its possible mechanisms to provide new ideas and measures for the prevention and treatment of DN.Methods: Part 1: In vivo: C57/BL mice were intraperitoneally injected with Streptozocin(STZ)50mg/kg to construct a diabetes model as the research object,and wogonin(10,20,40mg/kg)was used for intervention.The experiment was divided into 6 groups: normal control group(NC),single administration group(wog40mg/kg),diabetes model group(DM),low-dose administration group(DM+wog10mg/kg),intermediate-dose administration group(DM+wog20mg/kg),high-dose administration group(DM+wog40mg/kg),8 per group.Successful modeling(blood glucose value ?16.7mmol/L)and reared for 12 weeks.Random blood glucose,kidney weight/body weight,serum creatinine value and 24-hour urine albumin in each group of mice were tested to observe the clinical indicators of wogonin on diabetes kidney influences;PAS staining and electron microscopy were used to observe the renal pathological changes of mice in each group;Western blot to detect the expression of KIM1 and NGAL to determine the effect of wogonin on the degree of diabetic kidney injury;Immunohistochemical method was used to detect the expression of podocyte marker proteins WT1 and Nephrin,Western blot was used to detect the expression of WT1,Nephrin and Podocin,and real-time quantitative PCR was used to detect the expression of WT1 m RNA to determine the effect of wogonin on diabetic kidney podocyte damage.In vitro: The mouse kidney podocytes were used as experimental objects.Firstly,the optimal therapeutic concentration of wogonin was screened by MTT experiment.Then,high-sugar treatment of podocytes was used as the research model,and wogonin(4,8,16?mol/L)was used for group intervention.The experiment was divided into 7 groups: normal control group(NC),single administration group(wog16?mol/L),mannitol group(MG),high glucose group(HG),low-dose administration group(HG+wog4?mol/L),middle-dose administration Medicine group(HG+wog8?mol/L),high-dose administration group(HG+wog16?mol/L).Cellprotein and m RNA were extracted,the expression of WT1,Nephrin and Podocin were detected by Western blot,and the expression of WT1 m RNA was detected by real-time quantitative PCR to clarify the effect of wogonin on renal podocyte damage in high glucose environment.Part 2: In vivo: Western blot was used to detect the phosphorylation of NF-kB-P65,a key protein in the inflammatory pathway,and real-time quantitative PCR was used to detect the expression of TNF-?,MCP-1 and IL-6 m RNA in kidney inflammatory factors to clarify the effect of wogonin on diabetic kidney inflammation influences.Electron microscopy was used to observe the changes in the number of autophagosomes in mouse kidney tissue;Western blot was used to detect the expression of autophagy-related proteins Beclin-1,Atg7 and P62 to clarify the effect of wogonin on autophagy in diabetic renal podocytes.Western blot was used to detect the expression of the key apoptosis protein Cleaved-Caspase3,the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax to clarify the effect of wogonin on the apoptosis of diabetic renal podocytes.Western blot was used to detect the expression of NOX1,NOX2 and NOX4,important members of the NOX family of oxidative stress,to determine the effect of wogonin on oxidative stress in diabetic kidneys.In vitro: This part of the experiment is divided into 6 groups: normal control group(NC),single administration group(wog16?mol/L),high glucose group(HG),low dose administration group(HG+wog4?mol/L),medium dose administration group(HG+wog8?mol/L),High-dose administration group(HG+wog16?mol/L).Cellular protein and m RNA were extracted,and the expression of NF-kB-P65 and P-P65 was detected by Western blot.Real-time quantitative PCR was used to detect the expression of kidney inflammatory factors TNF-? and IL-1? m RNA to observe the effect of wogonin on renal effects of cellular inflammation.Electron microscopy was used to observe the changes in the number of autophagosomes in renal podocytes.Western blot was used to detect the expression of autophagy-related proteins Beclin-1,Atg7 and P62 to observe the effect of wogonin on autophagy in renal podocytes under high glucose environment.Flow cytometry was used to detect the level of apoptosis.Western blot was used to detect the expression of the key apoptosis proteins,Cleaved-Caspase3,anti-apoptotic protein Bcl-2,and pro-apoptotic protein Bax.Use Tunel staining to observe the effect of wogonin on renal podocyte apoptosis in high glucose environment;Western blot was used to detect the expression of NOX1,NOX2 and NOX4,and DCF and DHE were used to detect intracellular ROS and superoxide levels to observe the effect of wogonin on oxidative stress in renal podocytes under high glucose environment.Molecular docking method was used to detect the active site of the binding of wogonin to Bcl-2.Using software to predict the target of wogonin.Using CETSA test to detect the binding of wogonin to Bcl-2 protein.Using IP experiment to detect that wogonin can regulate autophagy and apoptosis by regulating the binding of Bcl-2 to Beclin-1 and Bax.Results: Part 1: In vivo:(1)Diabetic mice had significantly increased serum creatinine value,kidney weight/weight and 24-hour urine albumin,and wogonin could effectively improve the changes of the above clinical indicators;(2)PAS staining of kidneys in diabetic mice showed an increase in glomerular volume,mesangial matrix hyperplasia,atrophy of renal tubular epithelial cells,and dilation of the lumen.The scores of mesangial expansion index and tubular interstitial injury index were significantly higher than normal mice High,electron microscopic observation of foot process fusion,basement membrane thickening is obvious,the average foot process width(FPW)is significantly increased,and wogonin can effectively improve the above pathological damage of diabetic kidney;(3)Kidney injury marker proteins KIM1 and NGAL in diabetic mice increased significantly,and wogonin could effectively reduce its expression;(4)The results of immunohistochemistry of kidney tissues in diabetic mice showed that the expression of podocyte-specific marker proteins WT1 and Nephrin decreased.Similarly,Western blot detected the expression of WT1,Nephrin and Podocin.Real-time quantitative PCR detects the decrease of WT1 m RNA level.It was confirmed that the above indexes in diabetic mice decreased significantly,and wogonin can effectively reduce the diabetic kidney podocyte damage.In vitro:(1)MTT showed that at a concentration of 4?mol/L,8?mol/L and 16?mol/L,wogonin's cell activity was not affected,and the activity of renal podocytes after high glucose treatment could be significantly restored;(2)Western blot was also used to detect the expression of WT1,Nephrin and Podocin,and real-time quantitative PCR was used to detect the level of WT1 m RNA.The results all confirmed that high glucose can induce the expression of the above podocyte marker protein to be reduced,and wogonin can restore renal podocyte function.Part 2: In vivo:(1)Western blot was used to detect the phosphorylation of NF-?B-P65,and real-time quantitative PCR was used to detect the expression of inflammatory factors TNF-?,MCP-1 and IL-6 m RNA.The results showed that wogonin could reduce the inflammation of diabetic kidneys;(2)Western blot detection of the expression of Beclin-1,Atg7,P62 in renal cell podocyte autophagy,electron microscopy observation of the number of autophagosomes,confirmed that wogonin can activate renal podocyte autophagy and promote the formation of autophagic bodies;(3)Western blot detection of apoptosis-related proteins Cleaved-Caspase3,Bcl-2,Bax,the results found that wogonin can inhibit diabetic kidney podocyte apoptosis;(4)Western blot detection of NOX1,NOX2,NOX4 expression proved that wogonin can inhibit diabetic kidney oxidative stress response.In vitro:(1)Western blot detected the phosphorylation of NF-?B-P65,and real-time quantitative PCR detected the release of inflammatory factors TNF-? and IL-1?,showing that wogonin can alleviate the high glucose-induced inflammation of renal podocyte damage;(2)Western blot detection of podocyte autophagy-related proteins Beclin-1,Atg7,P62 expression,electron microscopy observation of the number of autophagosomes,showed that wogonin can restore high glucose inhibition of renal podocyte autophagy;(3)Flow cytometry,Western blot detection of apoptosis-related proteins Cleaved-Caspase3,Bcl-2,Bax,and Tunel staining confirmed that wogonin can reduce high glucose-induced apoptosis of renal podocytes;(4)Western blot detection of NOX1,NOX2,NOX4 expression,as well as DCF staining and DHE staining,proved that wogonin can reduce high glucose-induced oxidative stress in renal podocytes.(5)Using software to perform target prediction of wogonin and found that the fitting value of Bcl-2 is very high.Both molecular docking method and CETSA experiment confirmed the binding of wogonin and Bcl-2 protein.IP experiment confirmed that wogonin can upregulate autophagy by inhibiting the binding of Bcl-2 and Beclin-1,and promote the binding of Bcl-2 and Bax to inhibit apoptosis.Conclusion:(1)Wogonin can inhibit renal podocyte damage caused by high glucose and improve the clinical and pathological changes of diabetic kidney;(2)Wogonin can reduce the inflammation and oxidative stress of kidney tissues and podocytes,upregulate podocyte autophagy and inhibit apoptosis;(3)Wogonin may regulate the injury of diabetic kidney podocytes through the interaction of Bcl-2 mediated apoptosis and autophagy.