| Part 1:Clinical study Effect of Modified Huangqi Chifeng Decoction on Serum Klotho Protein and Oxidative stress in patients with IgA NephropathyObjective:To observe the clinical efficacy of MHCD in patients with IgA nephropathy and its effects on serum Klotho,SOD and MDA through a randomized controlled double-blind trial.Methods:A total of 74 patients with IgA nephropathy,with clinical manifestations of mild to moderate proteinuria,CKD1-2 stage,and Chinese medicine syndrome of qi deficiency wind evil blood stasis and toxicity were included.Patients were divided into a control group and a test group using the random number table method,with 37 cases in each group.The control group was treated with telmisartan and basic treatment,while the test group was treated with MHCD + telmisartan and basic treatment,with 24 weeks of medication intervention and 12 weeks of follow-up.The Chinese medicine syndrome scores of each group were recorded at the 0 and 24 weeks,the 24-hour urine protein quantification,liver and kidney function,serum SIgA and IgA/C3 levels were measured at weeks 0,8,16,and 24,and the serum Klotho,SOD and MDA levels were measured at weeks 0 and 24.Results:(ⅰ)24-hour urine protein quantification:After 24 weeks of drug intervention,the 24hour urine protein quantification in both groups decreased compared with that before treatment(P<0.01)and the test group was better than the control group(P<0.01).(ⅱ)Clinical efficacy:after 24 weeks of treatment,the Chinese medicine syndrome scores and syndrome efficacy of patients in both groups improved significantly compared with those before treatment,and the test group was better than the control group(P<0.01,P<0.01);the clinical efficacy of the test group was better than that of the control group(P<0.05).(ⅲ)Liver and kidney function,serum SIgA and IgA/C3 levels:Scr,ALT,AST and TG were not significantly different between the two groups before and after treatment,and there was no significant difference in intra-group comparison;serum SIgA and IgA/C3 both decreased at week 24 compared with those before the treatment(P<0.01,P<0.01),and the test group was better than the control group(P<0.01,P<0.01).(ⅳ)Klotho protein,SOD,MDA:After 24 weeks of treatment,serum levels of Klotho and SOD were significantly increased compared with those before treatment(P<0.01,P<0.01),and the test group was better than the control group(P<0.05,P<0.05),while MDA levels decreased compared with those before treatment(P<0.01),and the test group was better than the control group(P<0.01).(v)Safety evaluation:There was no significant difference in liver and kidney function between the two groups before and after the test,no sudden deterioration of the condition,and no cardiovascular and cerebrovascular diseases occurred in all patients during the whole process,suggesting that the drug was safe and reliable.Conclusions:The MHCD can effectively improve the clinical manifestations,enhance the expression levels of serum Klotho and SOD,and reduce the expression levels of serum SIgA,IgA/C3 and MDA in patients with IgA nephropathy,and no significant liver and kidney function damage was observed during the treatment period,suggesting that the MHCD can improve the oxidative stress status and protect the kidney in patients with IgA nephropathy,and the medication is safe and effective.Part Ⅱ:Experimental study The Mechanism of anti-oxidative stress by modulating Nrf2 in IgA nephropathy rat with the MHCD.Objective:To investigate the intrinsic mechanism of the anti-oxidative stress effect of the MHCD achieved by Klotho protein,Nrf2,SOD and MDA through the adoption of a rat model of IgA nephropathy.Methods:A total of 46 rats were used,33 of which were used to construct a rat model of IgA nephropathy,after model identification to confirm successful modeling,the model group was divided into the model group,the telmisartan group and the MHCD group using a random number table method,with 10 rats in each group.The drug intervention was started from the 13th week of the experiment,and the drug intervention was carried out for 8 weeks,the whole experiment last for 21 weeks.The general condition of the rats was observed daily,and the 24hour urine protein quantification of the rats was measured every three weeks.The rats were anesthetized and executed at the 21 st week to obtain blood from the abdominal aorta and kidney tissue.The renal tissue sections were stained by HE,Masson and PAS staining methods,and the histopathological changes were observed under light microscope and ultrastructural changes of kidney was observed by transmission electron microscope.The serum Klotho,SOD and MDA and SOD MDA in kidney tissues were detected by ELISA,the expression of Klotho protein in rat kidney was detected by immunohistochemistry,and the expression levels of total Nrf2 and nuclear Nrf2 levels in rat kidney tissue were detected by Western Blot.Results:(ⅰ)24-hour urine protein quantification:The proteinuria in the model group increased significantly from the 9th week of the membrane-making phase,and the difference was statistically significant compared with the blank group rats in the same period(P<0.01).At week 15 and 18 of the drug intervention phase,a statistically significant difference was found between the blank group and the other three group(P<0.01),telmisartan and MHCD group have no significant difference;at the end of the 21th week,there was a significant decrease in the 24-hour urine protein quantification in the telmisartan group and the MHCD group compared with the model group(P<0.05,P<0.05).(ⅱ)Liver and kidney function:Liver and kidney function were tested at the end of the experiment,and it was found that there was no statistical difference between the groups for Scr,ALT and AST(P>0.05).(ⅲ)Comparison of HE,PAS and Masson staining:The glomerular morphology of the rats in the control group was basically normal,and the tubular structure was intact.Compared with the control group,the model group showed compensatory glomerular expansion,partial wrinkling,partial congestion and swelling,proliferation of thylakoid cells,deposition of thylakoid matrix,obvious lobular changes in the glomeruli,disorganized tubular arrangement,and visible tubular dilatation.Lesions were seen to be reduced to different degrees in the telmisartan group and the MHCD group.(ⅳ)Ultrastructural observation of renal tissue under electron microscope:In the control group,the podocytes were arranged in a regular and orderly manner with uniform size and basement membrane;in the model group,the podocytes were arranged in a disorderly manner with unequal size,and the podocytes were widely fused with each other,and the basement membrane was not thickened;in the telmisartan group and the MHCD group,the podocytes were partially fused and arranged in an orderly manner compared with the model group,and the podocyte injury was effectively relieved.(ⅴ)The expression levels of serum Klotho,SOD and MD A:The expression levels of serum Klotho,SOD and MDA were measured by ELISA at the end of the experiment,the serum levels of Klotho and SOD significantly lower(P<0.01,P<0.01)and MDA were significantly higher(P<0.01)in the model group compared with the control group.Compared with the model group,the serum Klotho and SOD levels were increased(P<0.01,P<0.01;P<0.05,P<0.01),and MDA levels were decreased(P<0.05,P<0.01)in different degrees after the intervention with telmisartan and MHCD.(ⅵ)Expression level of Klotho protein in renal tissues:The expression of Klotho protein in the renal tissues of rats in each group was detected by immunohistochemistry at the end of the 21 st week,and it was seen that Klotho showed positive cytoplasmic staining and was mainly expressed in renal tubular epithelial cells.In the normal group,high expression was seen in the renal tubules,and the expression in the model group was significantly decreased compared with that in the control group(P<0.01).Compared with the model group,the expression of Klotho was elevated to different degrees in the telmisartan group and the MHCD group(P<0.01,P<0.01),and there was no significant difference between the two intervention group(P>0.05).(ⅶ)The expression levels of SOD and MDA in the kidney tissues:The expression levels of SOD and MDA in the kidney tissues of rats were detected by ELISA at week 21,and it was found that the SOD level in the model group was lower(P<0.01)and the MDA level was higher(P<0.01)than that in the control group;the SOD level in the telmisartan group and the MHCD group was higher than that in the model group,and there were statistical differences(P<0.01,P<0.01),and MDA was decreased compared with the model group and was statistically significant(P<0.05,P<0.01).(ⅷ)The expression levels of total Nrf2 and neclear Nrf2 in the kidney tissues:The expression levels of total Nrf2 and neclear Nrf2 in the kidney tissues of rats were detected by Western Blot method.The results showed that the expression levels of total Nrf2 and nuclear Nrf2 in kidney tissues were decreased in the model group compared with the control group(P<0.01,P<0.01);the expression levels of total Nrf2 and nuclear Nrf2 were increased to different degrees in the Telmisartan group and the MHCD group compared with the model group,and the two groups both have statistically significant(P<0.05,P<0.01);the expression levels of total Nrf2 and nuclear Nrf2 were increased in the MHCD group compared with the Telmisartan group,but the differences were not statistically significant(P>0.05).It is suggested that the MHCD can promote the dissociation of Keapl from Nrf2 in kidney,effectively increase the total Nrf2 content in cells,and promote the translocation of Nrf2 into the nucleus to activate the expression of downstream antioxidant stress factors,thus playing an antioxidant stress effect.Conclusions:(ⅰ)The MHCD can improve 24-hour urine protein quantification in rats with IgA nephropathy.(ⅱ)The expression levels of Klotho and SOD in serum and kidney tissues of rats with IgA nephropathy decreased,and the expression level of MDA increased,indicated that there was oxidative stress;the MHCD could effectively enhance the content of Klotho protein and SOD and reduce the level of MDA.(ⅲ)The levels of total Nrf2 and nuclear Nrf2 in the kidney tissues of rats with IgA nephropathy decreased;MHCD can promote the dissociation of Keap1 and Nrf2 and increase the level of total Nrf2 and promote the translocation of Nrf2 into the nucleus,so that Nrf2 can accumulate in the nucleus and combine with ARE,thus playing an anti-oxidative stress role,delaying the progression of kidney disease and protecting the kidney.(ⅳ)The renoprotective effect of MHCD on rats with IgA nephropathy may be achieved by upregulating Klotho protein expression,modulates Nrf2 and induces downstream antioxidant enzyme gene expression,elevates the expression level of SOD in the body and reduces MDA.The present trial provides scientific proof for the antioxidant stress effect and the underlying mechanism of the treatment of IgA nephropathy with MHCD. |
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