Effect Of Tongluoyishen Prescription On Mitochondrial Dysfunction In Diabetic Nephropathy Rats

Objective: To observe the changes of mitochondrial ultrastructure and mitochondrial function in early,middle and late DKD rats,and to explore the effect of Tongluoyishen recipe on the renal cortical mitochondrial function in DKD rats during different stages of disease.The clinical application provides sufficient experimental evidence.Methods: A total of 260 healthy male Sprague-Dawley(SD)SD rats were selected and fed with normal diet for one week.Blood glucose and urinary protein were detected.All were negative and were used in the experiment.Fifty rats were randomly selected as the normal control group(No-treatrment gr-oup,NG group),and the remaining 210 rats were modeled.Model rats were treated with right kidney resection and intraperitoneal injection of streptozotocin(STZ)to establish rat DKD model.When 24 h urinary protein ≥ 30 mg DKD rat model was successfully prepared.The success of modeling was taken as the start of the experiment,recorded as 0 weeks.The normal control group was sham-operated.The successful DKD rats were divided into 61 model groups(MG control group),47 valsartan treatment groups(PG group)and Tongluoyishenfang group according to the random number table method.Twenty-seven rats in the traditional medicine early-treatrment group(TEG group)and the traditional medicine middle-treatrment group(TGA group: 9 weeks of gavage administration)were administered with Tongluyi kidney.Twenty-three patients were treated with Traditional Medicine Late-treatrment Group(TLG group: starting at 22 weeks).Dosing lasted 26 weeks.In the NG,MG,PG,and TEG groups,10 rats were sacrificed at the 9th and 22 nd weekends,respectively.The remaining rats were sacrificed on the 26 th weekend.The general state of rats was observed,fasting blood glucose was measured,urinary protein24 h,renal function(BUN,Scr);ultrastructure of renal cortex mitochondria was observed by transmission electron microscope;PGC-1α,TFAM m RNA and mitochondrial DNA copy number were detected by Real-time PCR.The ATP content of renal cortex was measured by luciferase assay.The intracellular ROS activity was detected by flow cytometry combined with DCHF-DA staining,and the glomerular mitochondrial membrane potential was measured by flow cytometry combined with JC-10 staining.Result:1.Tongluoyishen recipe can significantly improve the mental status,coat color,and activity of rats with diabetic nephropathy;it can reduce fasting blood glucose,24 h urinary protein and BUN,Scr in rats,reduce urinary protein,and protect kidney function,and the early intervention of traditional Chinese medicine is better than the middle and late stages.2.Ultrastructure of podocyte mitochondria transmission electron microscopy showed that the glomerular basement membrane was normal at all time points in the NG group,and the mitochondria were round,oval or rod-shaped.The morphology was full,and the structure of mitochondria was clear and tidy.In the MG group,the glomerular basement membrane was significantly unevenly thickened,mitochondria swollen in mitochondrial body weight at the end of the 9th week,the matrix was thin,and the mitochondrial body weight was damaged at the end of the 22 nd and26th weekends.The mitochondria were swollen and ruptured,and the stromal cavitation and iliac crest were disordered or reduced.disappear.Compared with MG group,the degree of mitochondrial damage was lighter in TEG and PG groups.At the end of the26 th week,compared with the TEG group,the degree of mitochondrial damage was heavier in the TMG group and the TLG group.The results showed that the degree of mitochondrial damage was reduced after DKD rats were treated with Tongluoyishen recipe,and the early intervention of traditional Chinese medicine is better than the middle and late stages.3.The results of PGC-1 and TFAM m RNA expression in renal cortex mitochondrial biosynthesis showed that: PGC-1α and TFAM m RNA decreased with time,and DKD rats received intervention of Tongluoyishen recipe and renal cortex mitochondrial PGC-1α.The expression of m RNA and TFAM m RNA was up-regulated,and the effect of early intervention was better than that in the middle and late stages.The results of mt DNA copy number test showed that the mt DNA copy number decreased with time.After intervention of Tongluoyishen recipe,the glomerular mt DNA copy number expression was up-regulated in DKD rats,and the early intervention effect was better than that in the middle and late stages;ROS level test results The results showed that the ROS synthesis increased with time,and the ROS level in DKD rats was significantly down-regulated after intervention with Tongluoyishen recipe The ROS level was significantly lower at 22 and 26 weeks than that of the western medicine group.The effect of early intervention was better than that of the middle and late stages.Membrane potential detection results showed that: glomerular mitochondrial membrane potential decreased over time,Tongluoyishen recipe intervention glomerular mitochondrial membrane potential in DKD rats increased,and 26 weeks better than Western medicine group,early intervention was better than the mid-term,Late stage.The results of mitochondrial ATP content showed that the ATP content decreased with time,and the ATP content in renal cortex of DKD rats increased after intervention of Tongluoyishen recipe.The effect of early intervention was better than that of the middle and late stages.Conclusion: 1.Tongluoyishen recipe can improve the general state of DKD rats,reduce urinary protein to a certain extent,reduce urea nitrogen and serum creatinine,and improve renal function.2.Tongluoyishen recipe reduce podocyte mitochondrial ultrastructural lesions in DKD rats and protect kidneys.3.Mitochondrial dysfunction participates in DKD kidney injury.Tongluoyishen recipe can block renal cortical mitochondrial dysfunction in DKD rats,which in turn reduces DKD glomerular injury.The mechanism may be achieved by regulating mitochondrial biosynthesis,inhibiting mitochondrial oxidative stress,and increasing mitochondrial production.The main manifestations include up-regulation of PGC-1α and TFAM m RNA expression,promotion of mt DNA replication and transcription,and suppression of ROS levels.Promotes ATP production and increases in membrane potential.4.The effect of Tongluo Yishenfang in early-rising administration is better than that of Tongluyiren recipe in the middle and late period.The longer the administration time,the more obvious the effect is.