Mechanism Research Of Astragalus-Angelicathe On The Improvement Of Renal Oxidative Damage In Diabetic Mice By Regulating The Nrf2 Pathway

Objective: 1.To systematically comb the clinical cases of the treatment of early diabetic nephropathy by professor Zhao Quan-lin,and summarize the prescription rule and academic thoughts of the prescription of the disease.2.To establish a mice model of type 2diabetes mellitus and to observe the effects of different ratio of Astragalus-Angelica(Qigui)couplet medicines on kidney injury indexes,pathological changes and the influence of the Nrf2 pathway in mice,and to explore the protective effect of Qigui couplet medicines on the kidney of diabetic mice and the mechanism.Methods:1.Clinical research: collected and screened the prescription of early diabetic nephropathy in the affiliated hospital of Shandong University of TCM from October 2015 to January 2018,and established the database after standardized treatment.Using SPSS Statistics 22.0and SPSS Modeler 18.0 software,to carry on the drug use frequency,drug categories,sexual flavour,clustering analysis and association rules analysis,summarize the rule and academic thought of the tutor in treating early diabetic nephropathy,and to explore the drug characteristics of the treatment of DN.2.Experimental research: The model of type 2 diabetic mice was established by using a high-fat and high-sugar diet combined with STZ intraperitoneal injection.The modelmice were randomly divided into the model group and 5 different Qigui couplet medicines groups(5:1,2:1,1:1,1:2,1:5)and valsartan group,C57BL/6 mice were treated as normal control group.The treatment group was gavaged the corresponding concentration of drugs,the model group and the normal control group were given the equal volume purified water.The general situation,weight and FPG were observed regularly.After 12 weeks of drug intervention,blood,urine and kidney tissue were collected.The serum renal function indexes and urine ACR of each group were examined,and the indexes of oxidative stress,inflammation and fibrosis injury in kidney tissues were measured.The renal morphological changes were observed by HE and PAS staining.At the same time,RT-PCR,Western Blot,immunohistochemistry and immunofluorescence were used to detect the Nrf2,HO-1m RNA and protein levels of mice kidney,and the tables of antioxidant enzymes T-SOD,CAT and GSH-PX were further detected.Results:1.Clinical research: Among the 316 prescriptions collected and collated,the top 10 of the high frequency drugs were Astragalus,Angelica,Poria,Salvia miltiorrhiza,Atractylodes,Ligusticum wallichii,Radix rehmanniae,Licorice,Fructus corni,and Achyranthes.It involves 18 kinds of drugs,in which the supplement of deficiency drugs is the most widely used,followed by the medicine for activating blood and removing stasis,heat-clearing drugs and damp-clearing drugs.The taste of drugs used is mainly sweet,bitter,and acrid.The medicine properties is mainly warm,cold and flat.Drugs are often attributed to the liver,spleen and kidney.The results of association rule analysis showed that the most commonly used drug pairs were Astragalus-Angelicae,followed by Astragalus-Salvia miltiorrhiza,Astragalus-Atractylodes,Astragalus-Ligusticum wallichii,and Poria-Yam,and the most commonly used three drugs and four combinations were Astragalus-Angelicae-Ligusticum wallichiiand and Astragalus-Angelicae-PaeonyLigusticum wallichiiand.2.Experimental research:(1)The protective effect of Astragalus-Angelica on kidney in diabetic mice: After themodel is successful,the weight of mice increases and FPG decrease obviously.The levels of serum Cys-C,?2-MG,urinary ACR,kidney tissue MDA,NF-?B,TNF-?,TGF-?1 and CTGF increased significantly,yet T-AOC level significantly decreased,which indicate diabetes mice kidney damage,and there is an obvious oxidative stress,inflammation,and fibrosis damage.After the intervention,all the mice in the group except the body weight are improved of different degrees.HE and PAS staining results show: In the model group,the glomerular volume increases,the basement membrane is thickened,the mesangial matrix is increased,and the basement membrane of the renal tubule is thickened,and some renal tubules atrophy,and the vacuolar sample changes.After the use of the drug,the group of 5:1 group,1:1 group and valsartan group were significantly improved.(2)Effects of Astragalus-Angelica on Nrf2 pathway in diabetic mice: Compared with the normal group,Nrf2,HO-1m RNA and protein expression in the model group increased significantly,and T-SOD,CAT and GSH-PX decreased significantly.After the drug intervention,the Nrf2 m RNA and protein in the group were further increased by the group of5:1,1:2 and valsartan groups,HO-1m RNA and protein levels were also significantly increased in the 5:1 group and valsartan group.The results of immunohistochemistry and immunofluorescence in detecting Nrf2 and HO-1 were similar to those of Western Blot.There was a significant increase of T-SOD,CAT and GSH-PX in the 5:1 group and 1:1group.Conclusion:1.Tutor proposed "deficiency of Qi and blood stasis obstructing collaterals " was the basic pathogenesis of early DN,and the principle of treatment was "reinforcing the primordial Qi and promoting blood circulation to remove meridian obstruction ".2.Astragalus-Angelica was the core couplet medicines for the treatment of early diabetic nephropathy by professor Zhao Quan-lin.3.Astragalus-Angelica had obvious protective effect on the kidney of type 2 diabetic mice,mainly related to lower FPG,reducing proteinuria,improving renal function,reducing oxidative stress,inflammation,and fibrosis damage.4.Astragalus-Angelica could improve the renal injury of diabetic mice by activating Nrf2 pathway,and the comprehensive efficacy analysis showed Astragalus-Angelica1:1group was the best.