Curcumin Derivatives(FM0807) Improve Diabetes-induced Kidney Damage And Its Mechanism

Objective:Diabetic kidney disease（DKD）is one of the most serious complications of diabetes.The incidence in my country is getting higher and higher,and it has become the second cause of end stage renal disease（ESRD）,second only to various glomerulonephritis.Many studies have shown that long-term high blood sugar caused by diabetes can cause inflammatory infiltration of kidney cells and aggravate kidney tissue damage.The curcumin derivative FM0807 developed by the question group in the early stage has good anti-inflammatory and antioxidant effects.Therefore,this article aims to explore the therapeutic effect of FM0807 on kidney injury in diabetic mice and its possible mechanism,and provide a new therapeutic basis for improving DKD.Methods:In vivo experiment:Using 8-week-old SPF wild-type db/m mice and original db/db diabetic mice,db/m mice and db/db mice were randomly divided into 6 groups:normal group（control）,model group（model）,positive drug Benazepril group(model+Benazepril 2 mg·kg^-1),FM0807 low-dose group(model+25 mg·kg^-1),FM0807 middle-dose group(model+50 mg·kg^-1)and FM0807 high-dose group(model+100 mg·kg^-1).The control group was given normal saline,and the positive drug group was given benazepril hydrochloride（dissolved in normal saline）,FM0807 was given by gavage every two days at the established dose,body weight and blood glucose levels were monitored regularly,and the activity of the mice was recorded.After 8 weeks of continuous administration,the mice were sacrificed,and the kidney tissues were taken for hematoxylin-eosin（HE）,PAS,Masson and hexaamine silver staining to observe the pathological morphology,collagen fibers,glycogen and basement membrane thickness of the mouse kidney tissue;q RT-PCR was used to detect the expression of renal inflammatory factor-related proteins（JAK2,STAT3,etc.）and their m RNA;plasma was taken to detect changes in renal tissue biochemical indicators（CRE,BUN,TG,etc.）;urine was taken to detect 24 h urine protein in mice.In vitro experiment:Using rat glomerular mesangial cells（HBZY-1）as an in vitro model,D-glucose（25 mmol·L-1）was used to induce a high glucose model.The cell groups are as follows:normal group（control）,model group（model）,positive drug group（JAK2 specific inhibitor model+AG490 50μM）,FM0807 low-dose group（model+25μM）,FM0807middle-dose group（model+50μM）,FM0807 high-dose group（model+100μM）.MTT method was used to observe the effect of different concentrations of FM0807 on the viability of HBZY-1 cells;high-content cell imaging analysis method was used to detect the effect of HBZY-1 cell apoptosis,flow cytometry was used to determine the level of HBZY-1 cell apoptosis;reactive oxygen species（Reactive oxygen species,ROS）detection kit to detect changes in cell oxidation levels;Western blot determination of HBZY-1 cell proliferation（Bcl-2,Bax）and inflammatory signal pathway（JAK2,STAT3,IL-1β）related protein expression levels;q RT-PCR detection of related inflammatory factors（JAK2,STAT3,IL-18）expression level of m RNA.Result:In vivo experiment:（1）FM0807 can reduce blood sugar,glycosylated hemoglobin（Ghb）and 24h urine protein levels in db/db mice;reduce triglycerides（TG）,low-density lipoprotein cholesterol（LDL-C）,creatinine（CRE）and Urea Nitrogen（BUN）levels.It can increase the level of high-density lipoprotein cholesterol（HDL-C）and is dose-dependent with FM0807.（2）The results of HE staining showed that the glomeruli in the model group were significantly enlarged,with irregular shapes,and bowman-like cysts.After the intervention of FM0807,the glomeruli decreased in volume and became regular in shape.Masson staining results showed that the collagen fibers in the kidney tissue of the model group increased,and the increase in kidney collagen fibers was significantly reduced after the intervention of FM0807.The PAS staining results showed that the accumulation of glycogen in the model group increased significantly,and the accumulation of glycogen in the kidney tissue decreased after the intervention of FM0807.The results of hexammine silver staining showed that the glomerular mesangium and basement membrane were significantly thickened in the model group.The thickening of the glomerular mesangium and basement membrane was significantly reduced after the intervention of FM0807.The staining results showed that various pathological damages decreased with the increase of the dose of FM0807.（3）QRT-PCR results showed that compared with the control group,the m RNA expression levels of inflammation and fibrosis related factors JAK2,STAT3,IL-1β,TNF-αand CTGF in the model group were significantly increased,and the m RNA expression levels were down-regulated after FM0807 treatment in a dose-dependent manner,with statistically significant differences.（4）Western blot results show that,compared with the control group,Model group mice JAK2 and STAT3 phosphorylation and its expression form are raised,antiapoptotic proteins the Bcl-2 and promote the higher the ratio of apoptosis proteins Bax,inflammation,and fibrosis of NLRP3,Smad2/3,IL-1β,IL-18 expression increased,given FM0807 after treatment,the protein expression is reduced,the Bcl/Bax ratio decreased,said FM0807 relieve diabetes mice kidney inflammation and fibrosis progression.In vitro experiment:（1）MTT method determined that the D-glucose concentration of HBZY-1 cells induced hyperglycemia model was 25 m M,and the IC₅₀ value of FM0807 to HBZY-1cells was 38.76μM,so the dose of FM0807 was determined to be 25μM,50μM,100μM.Under inverted microscope,high glucose promoted the proliferation of HBZY-1cells,while AG490 and FM0807 inhibited the proliferation induced by high glucose.（2）FCM results showed that the apoptosis rate of HBZY-1 cells in the model group was significantly lower than that in the control group,and the apoptosis rate of HBZY-1 cells was increased after administration of AG490 and FM0807,suggesting that FM0807 and AG490 could inhibit the proliferation of HBZY-1 cells induced by high glucose and promote cell apoptosis.（3）ROS detection kit test results showed that compared with the control group,ROS level in the model group was significantly increased,and ROS expression level in HBZY-1 cells was decreased after treatment with AG490 and FM0807,suggesting that AG490 and FM0807 can reduce the oxidative stress level of HBZY-1 cells induced by high glucose.Mito-tracker Red CMXROS（mitochondrial Red fluorescent probe）was used to detect the mitochondrial membrane potential.The results showed that the mitochondrial membrane potential of the model group increased,while the mitochondrial membrane potential decreased after administration of AG490 and FM0807,suggesting that AG490 and FM0807 could promote the apoptosis of HBZY-1 cells induced by high glucose.（4）The Hoechst 33342/PI staining kit was used to detect the apoptosis level of HBZY-1 cells.The results of the high-content cell imaging analysis system showed that compared with the control group,the PI staining of HBZY-1 cells in the model group was decreased,but the PI staining of HBZY-1 cells was significantly increased after the administration of AG490 and FM0807,suggesting that AG490 and FM0807 could promote the apoptosis of HBZY-1 cells induced by high glucose.（5）RT-PCR and Western Blot results show that high glucose can induce the expression of JAK2,STAT3,IL-1β,TGF-β1 and other inflammatory factors,and also promote the expression of anti-apoptotic protein Bcl-2,and promote the expression of NLRP3 related pathway proteins.Promote the expression of proteins related to the fibrosis pathway aggravate renal tissue fibrosis,and the expression of related pro-inflammatory factors and pro-fibrotic proteins was significantly reduced after the administration of AG490 and FM0807,indicating that AG490 and FM0807 can inhibit the inflammatory response and fibrosis induced by high glucose in HBZY-1 cells.Conclusions:1.FM0807 can improve renal function in diabetic kidney injury mice.2.FM0807 can significantly reduce the expression of JAK2,STAT3 gene level and protein level.3.High glucose can promote the abnormal proliferation and fibrosis of HBZY-1cells,and FM0807 can inhibit the abnormal proliferation and fibrosis of HBZY-1 cells.4.FM0807 mainly regulates the JAK2/STAT3 signaling pathway to relieve inflammation and epithelial to mesenchymal transition to improve kidney damage caused by diabetes.