Study On The Protective Effect Of Astragaloside Ⅳ On Cadmium-induced Diabetic Nephropathy In Mice

Diabetic nephropathy(DN)is one of the most serious comorbidities of diabetes and its incidence increasely year by year.It has become the second most important factor in the occurrence of end-stage renal disease.Because of high disability and mortality,the healthy life of people has been affected seriously,so studying of DN is great significance in the prevention and treatment of diabetic nephropathy and delaying treatment.The causes of diabetic nephropathy are very wide,most of which are related to human genetics and living standard habits.Related studies have reported that the heavy metal cadmium is related to the occurrence of DN,but its specific mechanism of action has not yer been clarified.Astragaloside IV is one of the important active ingredients in Scutellaria baicalensis.It has biological activities such as promoting metabolism,lowering blood sugar,scavenging free radicals,and reducing podocyte damage.However,the protective effect of scutellarin on cadmium-induced diabetic nephropathy has not been reported.The research in this subject mainly predicts the mechanism and path ofastragaloside IV in the treatment of diabetic nephropathy through network pharmacology,the common target of astragaloside IV and diabetic nephropathy has been screened out and the basic mechanism and process of astragaloside IV has been found.then construction of DN model is to research the protective effects of astragaloside Ⅳ with histomorphology,metabolomics,and molecular biology.The effects of cadmium and high-fat and high-sugar diets on DN were determined by constructing a DN model.in the later period,different doses of astragaloside IV were protected and the dose-effect relationship was studied to determine the optimal dose.Based on the metabolomics technology,the potential biomarkers of the blank group,the DN model group,and the astragaloside protection group were compared,and the protection mechanism of astragaloside was explored in combination with molecular biological analysis.Molecular biology technology studies the expression of corresponding pathway proteins and changes in genes,and studies the pathogenesis of DN and the possible protective mechanism of astragaloside Ⅳ.Therefore,this project aims to explore the drug effect of astragaloside Ⅳ on cadmium-induced diabetic nephropathy and its possible mechanism,so as to further provide theoretical basis and data reference for the prevention and treatment of DN.The relevant content is as follows:1.Network pharmacology predicts the mechanism and approach of astragaloside Ⅳ in the treatment of diabetic nephropathy(1)Screening of targets for diabetic nephropathy and baicalinThrough database analysis and retrieval,2573 targets for diabetic nephropathy and 102 targets for astragaloside Ⅳ has been found.By removing false positive genes and duplicate genes,59 common targets for astragaloside Ⅳtreatment of diabetic nephropathy can be screened.(2)Construction of common protein interaction network diagramAnalysis of the STRING database showed that a protein interaction network diagram can be constructed to reflect the inherent relationship between proteins.(3)Biological process and pathway analysisAccording to DAVID database analysis,the biological process of astragaloside Ⅳ in the treatment of diabetic nephropathy mainly involves biological processes such as ATP binding,RNA polymerase 11 promoter transcription,and endothelial cell proliferation.KEGG enrichment analysis shows that it is mainly involved in the pathway There are PI3K/AKT pathway,mTOR pathway,FOXO pathway and so on.(4)Construction of target-path network diagramBy constructing a protein target-pathway diagram,the relationship between related targets and pathways can be seen intuitively,with PI3K/AKT pathway as the main one.2.Construction of a mouse model of diabetic nephropathy and the protective effect of astragaloside Ⅳ(1)Establishment and verification of cadmium-induced diabetic nephr opathy injury modelPhysiological index research showed that compared with the Con group,the fasting blood glucose level in the DN group increased by 30%(p<0.05).The diet and water consumption also showed an upward trend,but the body weight decreased significantly.The initial characteristics of diabetes are more consistent.At the level of glucose and lipid metabolism,the TG content in the DN group also increased compared with the Con group(p<0.05).HDL showed a significant reduction of 30%(p<0.01),while the LDL content increased relatively(p<0.05).0.01).The study of renal function indicators found that compared with the Con group,the NAG and BUN levels in the DN group were significantly increased(p<0.01,p<0.01),and the CER and renal coefficients also showed consistent trends(p<0.01,p<0.05).It has been shown that the combined application of cadmium chloride and high-fat and high-sugar diets has a damaging effect on mice,and when combined,the damaging effects have a additive effect.(2)Protective effect of astragaloside IV(Ast)on DN miceAnalysis of physiological indicators showed that the weight of mice in the protection group increased significantly compared with the DN group,and the amount of water and diet decreased,and the fasting blood glucose was similar to and decreased in the Con group.The performance of the ASTH group was the most obvious.At the same time,in the level of glucose and lipid metabolism,the TC content in the ASTH group was significantly lower than that in the DN group(p<0.05),while the HDL content was significantly increased(p<0.05).It was found through renal function indicators that the levels of NAG,CRE and BUN in the ASTH group were significantly lower than those in the DN group(p<0.01,p<0.01,p<0.05).It shows that astragaloside IV has a linear relationship in the treatment of DN mice,and high-dose protection is better.(3)Observation and analysis of histomorphologyH&E,Masson,and PAS triple staining can directly detect the intrinsic pathological changes in mouse kidney tissues.Among them,DN mice have obvious infiltration of mesenchymal inflammation cells,mesangial hyperplasia and interstitial edema.In the ASTH group,the glomerular structure of the mice was basically intact and the damage was restored,and the tissue morphology was close to the normal state of the mice.Observation of renal transmission electron microscopy showed that hypertrophy of the foot process and abnormal mitochondria appeared in the kidneys of DN mice.The morphological structure of the mice in the ASTH group was regularly arranged,and the above injuries were recovered.It shows that astragaloside Ⅳ can protect kidney damage in DN mice.3.The protective effect of astragaloside Ⅳ on diabetic nephropathy based on metabonomicsThe study of mouse serum samples by LC/MS and GC/MS showed that the Con group,DN group and AST group showed better aggregation degree in each group and greater separation between groups.According to the metabolic database,various types of differential metabolic markers such as LysoPI(18:0/0:0)and bile acids and amides were screened.And analysis found that glycerol phospholipid metabolism,bile acid metabolism and other metabolic pathways have changed significantly.It is concluded that the pathogenesis of DN may be related to mitochondrial metabolic disorders.4.Study on the protective effect of astragaloside Ⅳ on DN mice based on podocyte functional protein Desmin and LC3 protein and AMPK/PI3K/AKT/mTOR pathwaya.Podocyte function Desmin and LC3 proteinCompared with the Con group,the Desmin protein content in the DN group increased significantly(p<0.01);the expression of LC3II/LC3-1 protein was increased(p<0.05).Compared with the DN group,the expression of Desmin protein in the protection group decreased significantly(p<0.01);the expression of LC3II/LC3-1 protein decreased significantly(p<0.05).This shows that LC3Ⅱ-associated autophagy protein in podocytes of DN mice is increased,and mitochondrial damage is severe.Astragaloside Ⅳ can activate the ubiquitination of LC3-1 protein on mitochondrial damage,resulting in the normal occurrence of mitochondrial autophagy.b.AMPK/PI3K/AKT/mTOR pathwayCompared with theblank group,the AMPK protein expression in DN mice was significantly up-regulated(p<0.01);p-PI3K/PI3K protein expression was down-regulated(p<0.01);p-Akt/Akt protein expression was significantly down-regulated(p<0.01).0.01);mTOR protein expression increased significantly(p<0.01).Compared with the DN group,the expression of AMPK protein in the protection group was significantly reduced(p<0.01);the expression of p-PI3K/PI3K protein was increased(p<0.01);the expression of p-Akt/Akt protein was significantly increased(p<0.01);MTOR protein expression was significantly down-regulated(p<0.01).This shows that the AMPK/PI3k/AKT/mTOR pathway of DN mice is inhibited.Astragaloside Ⅳ can activate AMPK,promote PI3k phosphorylation expression,activate AKT,reduce mTOR expression,and promote mitochondrial autophagy.The results indicate that the occurrence of DN is related to the disruption of mitochondrial homeostasis.Astragaloside Ⅳ has a corresponding protective effect by regulating mitochondrial biogenesis and mitochondrial autophagy.