The Study Of Combination Therapy With Astragaloside IV And ACEI On Type 2 Diabetic Kidney Disease

ObjectiveDiabetic kidney disease is one of the most frequency comorbidites of diabetes,and is the leading cause of chronic kidney diseases.Beside the strategy of lowering blood glucose,blood pressure and serum lipid,utilizing the renin-angiotensin-aldosterone system blockades,such as angiotensin converting enzyme inhibitor or angiotensin-II type 1receptor blocker,is the cornerstone in reducing proteinuria in DKD treatment.Dual blocking using combination ACEi with ARB was introduced to further reduction of the proteinuria ever.However,a series of trails that investigated the dual blockade of RAAS to prevent DKD progression had provided negative or inconclusive data.These propel the development of additional therapeutic approaches beyond RAAS blockades.In recent years,accumulating evidences had revealed that Chinese herbs could reduce proteinuria and ameliorate the renal injuries independent on RAAS blocking.What is more,some trials demonstrated that the effect on anti-proteinuria and renal protection of Chinese herbs combination with ACEi or ARB seemed to be better than ACEi or ARB alone.These provide us clues to explore more effective add-on therapy to RAAS blockades in treatment of DKD.Astragaloside IV(AS-IV)is the derivate of Huangqi(Radix Astragali Mongolici)and it has a wide range of biological activities,including anti-inflammation,anti-viral,and anti-neoplasm.Some studies indicated that AS-IV could decrease the urinary albumin excretion rate and could protect against diabetic renal injuries.Oxidative stress had been linked to proteinuria and renal injuries.Evidences demonstrated that several antioxidants could reduce inflammation and fibrosis in diabetic kidney.Previous study had shown that the protection of AS-IV on glucose-induced renal cells injury might be associated with reactive oxygen species reduction.However,effect of AS-IV combined with ACEi or ARB on renal protection has not been investigated.Therefore,we reviewed the published literatures of clinical trials about the combination therapy with Chinese herbs formulae and ACEi/ARB on diabetic nephropathy,and mined the Chinese and western medicine prescription regularity.Then we investigated the effect of AS-IV combined with ACEi in type 2 diabetes mice and explored whether its underlying mechanism was dependent on anti-oxidation.Methods1.Data mining4872 published journal articles were retrieved from CNKI,VIP and Wanfang databases according to the search strategy.197 literatures about clinical trials of combination therapy with Chinese herb formulae and ACEi/ARBs on diabetic nephropathy were included for data mining.Then database was build and data were extracted from the literatures manually.The data fields include formulae title,ACEi or ARB used in the control group,Chinese herbs,and the herb dose etc.The data were cleansed and standardized.Script programming language Python and SPSS were used to data analysis.Frequency distribution,and Association rules analysis were applied to explore the prescription regularity.2.Animal experimentsSpecific pathogen free 8-week-old male db/db mice and lean wild type control mice were randomly assigned to five groups:lean wild type(wt)group,db/db group,both group were fed a regular diet;db/db+astragaloside IV(db/db+AS-IV)group,db/db+enalapril(db/db+ACEi)group,mice from these groups were fed a regular diet supplement with5g/kg AS-IV,0.8g/kg enalapril respectively;db/db+combination therapy with AS-IV enalapril(db/db+Combined)group,fed a regular diet supplement with 5g/kg AS-IV and0.8g/kg enalapril.The treatment lasted for 12 weeks.The 24-hour urine was collected using metabolic cages at 0,2,6,9,12 week post treatment.At the same time 24-hour water consumption,urine volume,food intake and feces production were also collected.Urinary albumin excretion rate and fasting blood glucose were also measured at 0,2,6,9,12 week post treatment.The body weight was measured every 2 weeks.After 12 weeks treatment,blood and urine samples were collected,Urine and serological indices(urine glucose and serum ALT,AST,TG)were detected.Blood pressure was measured via tail cuff using MRBP system.Serum H₂O₂ was measured by using Amplex Ultra Red reagent.Paraffin-embedded kidney sections were stained with PAS and scanned to measure glomerular mesangial matrix areas,tubular lumen cross-sectional area(CSA),tubular CSA,tubular cell nucleus count.The tubular cell CSA was calculated.Glomerular and tubular photographs in each sample were taken using electron microscopy to measure the glomerular basement membrane(GBM),foot process width(FPW),and tubular basement membrane(TBM)thickness.The content of Catalase,and SOD₂ in renal cortex tissue were detected by Western blot.Results1.Data mining(1)Herbs for tonifying qi,invigorating blood circulation and eliminating stasis and inducing diuresis to alleviate edema were used most frequently in DKD,and the cumulative propotion of these three categories is almost 48.15%.Huangqi,Danshen,Shanyao,Shanzhuyu and Fuling were the commonest herbs.Conventional dose of the herbs were observed in prescription for DKD treatment.The dose ranges of Huangqi,Shudihuang and Danshen were larger.The maximium dosage of Huangqi was 90 grams,while the minium was 6g.The mean dose of Huangqi was 28.96±13.04g and the median was 30g.(2)The association rules analysis showed that herbs,including Huangqi,Danshen,Fuling,Shendihuang,Shudihuang,Shanzhuyu,Shanyao,Zexie and Mudanpi,might combine in one prescription.The combination of herbs for tonifying qi,invigorating blood circulation and eliminating stasis and inducing diuresis to alleviate edema,consisting of Huangqi,Danshen and Shendihuang,was more likely.(3)ARB combined with chinese herbal formula more frequently than ACEi in DN.Among the included literatures,about 58.88%of that used ARB and about 38.08%of that used ACEi.Chinese herbal fourmulae more likely combined with RAAS blockades,including irbesartan,valsartan or benazepril.(4)In the early stage of DKD,herbs used frequently includes Huangqi,Danshen,Shanyao,Shanzhuyu,Shendihuang,Fuling,Zexie and Dahuang etc.Huangqi and Danshen were still used the most frequently.According to the classification of effects,herbs for tonifying qi,invigorating blood circulation and eliminating stasis,inducing diuresis to alleviate edema,clearing heat and cooling blood,secure essence and reduce urination and tonifying yin were frequently used to combine with RAAS blockades.(5)The dose of Huangqi used for treating DKD in early stage were normally distributed about its value.49 literatures reported that Huangqi prescripted in dose of 30g.Large dose of Huangqi were used in the early stage of DKD commonly.(6)In the early stage of DKD,Huangqi were more likely to combine with Danshen,Shanzhuyu,Shanyao or Shengdihuang.Herbs for tonifying qi,invigorating blood circulation and eliminating stasis were more likely to combine with others.(7)In the early stage of DKD,both ACEi and ARB were more likely to combine wiht Huangqi,Shendihuang or Danshen.(8)In the clinical stage of DKD,Huangqi,Fuling and Danshen were used frequently.ACEi was likely to combine with Huangqi and Danshen,and ARB might combine with Huangqi,Danggui,Baizhu,Fuling,Zexie.Huangqi combined with RAAS blockades might be the important strategy in clinical stage of DKD.2.Animal experiments(1)Compared with db/db group,all treatment groups showed a significant decrease in UAER.Importantly,the effects of combination therapy on UAER were much more obviously than group treat with ACEi or AS-IV alone.(2)Combination therapy with AS-IV and ACEi had no effect on the kidey weight index in db/db mice.(3)Combination therapy with AS-IV and ACEi could improve the foot process width,glomerular base membrane thickness,glomerular tuft cell proliferation,tubular cell atrophy,tubular base membrane thickness,and improved tubular cell proliferation.(4)Compared with db/db group,all therapies did not show effects on improving blood glucose,urinary glucose,Hb A1c and some other diabetic symptoms including polydipsia,ployuria and increased feces production.(5)Combination therapy with AS-IV and ACEi group showed lighter body weight compared to db/db group which was significant in statistics and the effects continued through the end of study.(6)Compared with that in wt group,serum H₂O₂ concentration was elevated in db/db group significantly.AS-IV therapy,including AS-IV only or combined with ACEi,reduced serum H₂O₂ concentration.Combination therapy with AS-IV and ACEi reduced the serum H₂O₂concentration further.(7)The Catalase content decreased in db/db group.Compared with db/db group,all therapies increased the catalase content of renal cortex.There is no change of SOD₂ in all groups.(8)ALT and AST were higher in db/db mice group than in wt group,all therapy groups were show no significant change compared with db/db group.Conclusion1.In the early stage of DKD,herbs of supplementing qi and nourishing yin,including Huangqi,Shendi,Shanzhuyu,were combined with ACEi/ARB to delayed the progression of the disease.However,in the clinical stage of DKD,the treatment was quite different from that in the early stage.Supplementing qi was used,and Fuling,Baizhu and Zexie,herbs of invigorating the spleen and inducing diuresis for removing edema were added to the ACEi/ARB.These results suggested that the pathogensis of DKD might be deficiency of both qi and yin in early stage and deficiency of vital energy of spleen and kidney with edema in clinical stage.Qi deficiency with blood stasis has a key role in the progression of the DKD.To tonify qi,activate blood and remove blood stasis might be the standard therapy for DKD.2.Huangqi is used commonly for treating DKD.Utilizing the ACEi or ARB in addition with Huangqi or Chinese herbs formula mainly contained Huangqi might improve the therapeutic effects on DKD.3.This study firstly indicated that combination with AS-IV and ACEi alleviated the proteinuria and delayed the progression of renal injuries of DKD in db/db mice better than using AS-IV or ACEi alone.The mechanisms of the renal protective effect of combination therapy with AS-IV and ACEi might be associated to the enhanced anti-oxidative effects dued to their combination.