Study On Apple Polyphenols Alleviating Type 1 Diabete And Diabetic Nephropathy Effects And Mechanisms

ObjectiveApple polyphenol(AP)is a general term for phenolic compounds in apples,which have strong antioxidant,anti-allergic,anti-inflammatory and other effects.In recent years,studies have reported that AP can improve insulin resistance and other diabetic conditions in diabetic mice.This study mainly explored the effects of AP on typical diabetic symptoms,glucose and lipid metabolism,gut microbiota structure,intestinal microbial metabolites of short-chain fatty acids(SCFAs),and diabetic nephropathy in type 1 diabetic mice,and explained the relevant mechanisms.MethodThe components of apple polyphenols were identified by liquid chromatograph-mass spectrometer(LC-MS)method to clarify the main chemical components and the relative percentages of each component.Animal experiment grouping: 42 male C57BL/6 J mice were selected as healthy control group,and the other 36 mice were injected with 130 mg/kg streptozotocin(STZ)by intraperitoneal injection to establish type 1 diabetes mouse model.The fasting blood glucose of mice was greater than 11.1 mmol/L,and the model was determined to be successful.Then they were grouped according to the average blood sugar,and divided into model group(Model),low-dose apple polyphenols group(L-AP,100 mg/kg),medium-dose apple polyphenols group(M-AP,200 mg/kg),High-dose apple polyphenol group(H-AP,400 mg/kg).Animal experiment process: All mice were fed with basal feed and administered by gavage,once a day,for 14 consecutive days.During this period,the water intake,urine output,food intake and body weight of the mice were recorded daily,and the fasting blood glucose(FBG)was measured once a week.One day before the end of the animal experiment,fresh feces of the mice were collected for the detection of intestinal flora and levels of SCFAs.At the end of the animal experiment,the venous blood of the mouse eye frame was collected,and the liver and kidney were harvested.Serum biochemical tests(BUN,ALT,AST,TG)were subsequently performed;histopathological staining(HE staining was used to observe the inflammatory infiltration of mouse liver and kidney,PAS staining was used to observe the glycogen accumulation in mouse liver,and Masson staining was used to observe mouse kidney collagen.Deposition,immunohistochemistry to detect the expression of collagen in mouse kidney);fluorescence quantitative PCR to detect the relative expression of ACE2,GDF15,G6 pc,GPAT genes in the liver;Western blot to detect the AMPK signaling pathway and renin-angiotensin system(RAS)in the liver system),renal collagen and fibrosis factor protein expression.Based on the material basis and protein level data,molecular docking technology was used to explore the interaction between the core components of apple polyphenols and some proteins in the RAS system.Result(1)The composition of AP was analyzed by LC-MS,and the results showed that AP mainly contains caffeoylquinic acid(isomers of 831,833,834),procyanidin B2 M-H,quercetin-3-Ogalactoside,quercetin The proportions of retin-3-O-rhamnoside and phloretin were 14.63 %,7.79 %,5.92 %,5.83 % and 5.83 %,respectively.(2)The apparent data of animal in vivo experiments showed that the M-AP and H-AP groups could significantly reduce the water intake,urine output,food intake,FBG and serum triglyceride(TG)content in type 1 diabetic mice.HE staining and PAS staining showed that AP could improve liver cell necrosis and glycogen accumulation.At the hepatic transcriptional level,H-AP upregulated the relative expression of ACE2,decreased the expression of G6 pc and enhanced adaptive responses.At the level of liver proteins,H-AP can activate the hepatic AMPK signaling pathway,including up-regulation of p-AMPKα(Thr172)and the expression of fat oxidationrelated proteins PPAR-α,PPAR-γ and CPT-1α,and up-regulation of appetite suppressant in the RAS system.Expression of the proteins angiotensin-converting enzyme 2(ACE2)and enkephalinase(NEP).The results of molecular docking between the core components of AP and the RAS system showed that the docking of quercetin-3-O-rhamnoside with NEP and quercetin-3-O-galactoside with ACE2 had high affinity.In addition,high-throughput sequencing analysis found that high-dose apple polyphenols can modulate the structure of the gut microbiota in diabetic mice,increase the relative abundance of probiotics Akkermansia and Odoribacter,and reduce the level of short-chain fatty acids in the feces of diabetic mice.(3)H-AP can improve collagen deposition in the kidneys of diabetic mice,including inhibiting the expression of Collagen III,TGF-β1,p-ERK(Thr202/Tyr204)proteins.In addition,H-AP can significantly inhibit the expression of NLRP3 and TXNIP proteins in the kidney,suggesting that it can prevent renal oxidative stress and inflammation caused by hyperglycemia.ConclusionAP can alleviate the typical symptoms of hyperglycemia,polydipsia,polyphagia,and polyuria in STZ-induced type 1 diabetic mice.The mechanism involves activation of the hepatic AMPK signaling pathway,upregulation of ACE2 and NEP protein expression levels in the RAS system,and increased abundance of probiotics in the gut.At the same time,AP is effective in preventing diabetic nephropathy,and the mechanism involves down-regulating the expression of renal TXNIP,NLRP3 and Collagen III,TGF-β1,p-ERK(Thr202/Tyr204)proteins.