Study On The Mechanism Of Yishen Huashi Granule In Improving Diabetic Kidney Disease Based On Co-expression Of Metabolism-gene

Objective:After observing body weight and blood glucose changes of diabetic kidney disease（DKD）rats under different treatments,the renal function indexes were detected,and ultra performance liquid chromatography tandem mass spectrometry（UHPLC-MS/MS）coupled with high-throughput transcriptome sequencing（RNA-seq）were used to screen the endogenous differential metabolites and renal differential m RNAs of rats for beneficial kidney dampness particles regulating the serum metabolites and renal m RNA expression changes,explore its mechanism of action to improve DKD.Methods:40 SD rats were randomly divided into normal group,model group,Yishen Huashi granule low-dose group,Yishen Huashi granule high-dose group and valsartan group,with 8 rats in each group.The rat model of DKD was established by intraperitoneal injection of low dose streptozotocin（STZ）.Yishen Huashi low-dose Granule group was given 2.27g·kg^-1·d^-1 by gavage,Yishen Huashi high-dose Granule group was given5.54 g·kg^-1·d^-1by gavage,valsartan group was given 7.38mg·kg^-1·d^-1 by gavage,and the normal group and the model group were given the same amount of normal saline for 6 weeks.During the experiment,the body weight and blood glucose of rats were monitored,and the rats were anesthetized by intraperitoneal injection of 3%pentobarbital sodium(40mg·kg^-1)24 hours after the last administration,blood was taken from the inferior vena cava,the serum was separated,and then the renal function,blood lipid,and inflammatory indicators were detected.Renal tissue of rats was fixed in neutral paraformaldehyde,and stained with hematoxylin eosin（HE）,masson and periodate Schiff reaction（PAS）to observe the pathological changes of the kidney,UHPLC-MS/MS and RNA seq were used to identify the changes of serum metabolism and the differences of renal m RNA expression.q RT-PCR and Western blot were used to detect the differential m RNA and protein expression in renal tissue to explore the common expression mechanism.Results:（1）Compared with the model group,body weight of rats in the treatment group increased,and blood glucose,UACR,blood urea nitrogen（BUN）,cystatin C（Cys C）,β₂-Microglobulin（β₂-MG）,interleukin-6（IL-6）,triglyceride（TG）and total cholesterol（TC）were significantly decreased,and total superoxide dismutase（T-SOD）was significantly increased（P<0.01 or P<0.05）;（2）Compared with the model group,the proliferation of mesangial matrix,fibrous tissue and tubular vacuolar degeneration of rats in the treatment group were alleviated;（3）14 target metabolites of Yishen Huashi Granules were identified,which were mainly concentrated in 20 KEGG pathways including sphingolipids metabolism,glycerol phospholipid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis;（4）96 target m RNAs of Yi-Shen-Hua-Shi granule were identified,and the TOP20 pathway further enriched by KEGG was closely related to glucose and lipid metabolism,and a total of 21 differential m RNAs were expressed in the TOP20 pathway;（5）A total of 6 pathways,glycerophospholipid metabolism,arachidonic acid metabolism,purine metabolism,primary bile acid biosynthesis,ascorbic acid and uronic acid metabolism,and galactose metabolism,were enriched by differential metabolites and differential m RNA,Among them,there are 7 differential metabolites such as phosphatidylethanolamine and 7 differential genes such as Adcy3.（6）7 differential metabolites have high predictive value of AUC,q RT-PCR and Western blot were used to detect the expression of 7 differential genes and their proteins,and the verification results were also highly consistent with the sequencing results.Conclusions:Yishen Huashi Granule can reduce the UACR,BUN and other blood and urine indicators of DKD rats,correct the disorder of glycolipid metabolism,and improve renal function.It can regulate the level of serum metabolites such as phosphatidylethanolamine,correct the disorder of expression of renal Phospho1 and other m RNA,improve the glycolipid metabolism of DKD rats,reduce renal injury,and protect renal function by regulating six metabolism-gene co-expression pathways such as glycerophospholipid metabolism.