Clinical And Experimental Study Of "Bushen Huoxue" Prescription In Treating Patients With Diabetic Kidney Disease

ObjectiveTo observe the clinical efficacy of "Bushen Huoxue" prescription in treating patients with diabetic kidney disease and the study on the renal protection mechanism of Astragaloside Ⅳ on type 1 diabetic STZ mice,so as to provide theoretical basis for the prescription and provide new ideas and evidence for the prevention and treatment of DKD with traditional Chinese medicine.Methods1、Clinical researchPatients meeting the diagnostic criteria for stage Ⅲ-Ⅴ diabetic kidney disease were divided into the observation group and control group randomly.On the basis of symptomatic treatment of western medicine,the observation group was treated with a self-designed "Bushen Huoxue" prescription,which was decocted in water with 1 dose per day and taken 30 minutes after meals in the morning and evening.The control group was treated with the hospital preparation "kidney-tonifying and sugar-lowering pill",one packet at a time,and two doses in the morning and evening.Before and after treatment,the clinical symptom scores(main observation indicators),24-hour urine protein quantification,serum creatinine,HbA1c and fasting blood glucose of the patients were recorded,as well as the total treatment rates and the safety and effectiveness.2、Animal experimentsType 1 diabetic mice was induced by a single intraperitoneal injection of streptozotocin into C57BL/6J mice,aged 8 weeks after adaptive feeding.Diabetes model group:one-time intraperitoneal injection of STZ solution(200 mg/kg,soluble in citrate buffer).Normal control group:one-time intraperitoneal injection of isovolumetric citrate buffer.The mice were randomly divided into type 1 diabetes model group(STZ group,n=6),Astragaloside Ⅳ treatment group(STZ+AS-Ⅳ group,n=6)and the Control group(n=6).The STZ group and Control group were fed with standard diet,and STZ+AS-Ⅳ group were given drug diet with AS-Ⅳ.After 8 weeks treatment,fasting blood glucose,urinary albumin,NGA,and serum creatinine were detected.After the mice were sacrificed,the kidneys were dissected and stained with PAS to evaluate the damage of renal tubules.The expression distribution of catalase was observed by immunohistochemical staining,the protein contents of catalase,OPA1,VDAC in renal tissues and catalase,OPA1,VDAC,TFAM in renal mitochondria were determined by western blot.The mitochondrial copy number of each group was determined by qPCR.Results1、Clinical researchThe treatment period lasted for 8 weeks.Before treatment,statistical analysis was conducted on the clinical symptom scores,24-hour urine protein quantification,serum creatinine,HbAlc and fasting blood glucose of the two groups of patients,all of which showed significant differences,indicating that the two groups were comparable.After treatment,the two groups had significantly lower clinical symptom scores and 24-hour urine protein quantification than before,and the observation group was better.HbA1c,fasting glucose and serum creatinine were improved in the observation group and control group,but there was no significant difference between them.2、Animal experimentsAfter 8 weeks treatment,24h urinary albumin excretion rate and NAG in STZ group were significantly higher than Control group and astragaloside Ⅳtreatment decreased them obviously.Compared with the STZ group,the serum creatinine of the Control group increased,but there was no significant difference.After 8 weeks of as-iv intervention,the serum creatinine of the STZ+AS-Ⅳ group significantly decreased.Fasting blood glucose in STZ group was significantly higher than in Control group and weight was significantly lower,while treatment did not show effect on them.PAS staining showed that the renal tubular area in STZ group slightly larger than in Control group,after 8 weeks of intervention with AS-Ⅳ,the pathological changes seems alleviate.The results of immunohistochemical staining suggested that catalase was mainly expressed in renal tubules.Western blot results showed that the catalase in kidney tissues and mitochondria of STZ group was significantly lower than Control group,and the catalase in kidney tissues was not significantly changed after 8 weeks of AS-Ⅳ treatment,while the catalase in mitochondria was significantly increased.Compared to the Control group,the expression of OPA1 and VDAC in renal tissues of STZ group was significantly decreased,and their expression be obviously increased after 8 weeks of intervention of AS-Ⅳ.In mitochondria,there was no difference in the expression of OPA1 and VDAC in each group.TFAM was increased in STZ group compared with Control group,while not significantly change.Real-time fluorescence quantitative PCR indicated that the copy number of mitochondria in STZ group was significantly reduced compared with Control group,and there was an increasing trend after AS-Ⅳ treatment,but without significantly difference between STZ group and STZ+AS-Ⅳ group.Conclusion1、Clinical researchThe curative effect of "Bushen Huoxue" prescription in the treatment of patients with stage Ⅲ-Ⅴ DKD is obviously better than that of the hospital preparation "kidney-lowering sugar pill",providing sufficient evidence-based medicine evidence for the treatment of diabetic nephropathy with traditional Chinese medicine,which is worthy of in-depth study.2、Animal experimentsThis study showed that AS-Ⅳ may improve mitochondrial function and provide renal protection by increasing the expression of catalase in renal mitochondria and OPA1 and VDAC proteins in renal tissues,providing new evidence for the prevention and treatment of DKD by traditional Chinese medicine.