| Background and Objective:Chronic kidney disease(CKD)is a serious hazard to human health,trimethylamine N-oxide(TMAO)is an important component of intestinal flora metabolites,which is closely related to renal progression in CKD,and has high predictive value for the clinical prognosis of this disease.However,the efficacy of current treatments to improve TMAO damage is not satisfactory,the development of new therapies to improve kidney injury from traditional Chinese medicine(TCM)has broad clinical application potential.Suyin Jiedu granules is an experiential prescription made by professor Enchao Zhou based on the theory of"nephrotoxicity" and clinical experience.Abnormal accumulation of intestinal microflora metabolites such as TMAO in CKD patients is also a kind of "nephrotoxicity".Therefore,is it possible for Suyin Jiedu granules to alleviate TMAO-induced kidney injury in CKD by"relieving nephrotoxicity"?According to the scientific problems,this study aims to clarify the effects and mechanisms of Suyin Jiedu granules and the critical herb Perillae frutescens on alleviating TMAO kidney injury through clinical observation,in vitro and in vivo experiments,improve the theoretical system of treating CKD from "nephrotoxicity",so as to provide effective TCM therapy for kidney injury in CKD patients.Methods:(1)A total of 34 CKD stage 3-4 non-dialysis patients with Shenxu Zhuodu syndrome in the Department of Nephrology of Jiangsu Province Hospital of Chinese Medicine were enrolled.The included patients were randomly divided into treatment group and control group.The treatment group received basic treatment+Suyin Jiedu granules+Baoshen tablets,and the control group received basic treatment+Baoshen tablets.All patients were observed for 12 weeks.The changes of renal function(SCR,BUN,UA,and eGFR),liver function(AST,and ALT),blood potassium and TCM syndrome score were detected before and after treatment,and possible adverse events were observed during treatment.Serum and urine TMAO levels were detected by UPLC-MS/MS,and the association between TMAO levels and renal functions was analyzed.At the same time,the effects of Suyin Jiedu granules on serum and urine TMAO levels was measured its relationship with the protective effect of Suyin Jiedu granules on renal function was analyzed.(2)On the basis of unilateral ureteral obstruction(UUO)induced nephropathy model,combined with oral administration of 1%high choline diet,the hyper-choline diet nephropathy rat model was established.Experimental animals were randomly divided into:Control,UUO,UUO+Choline,UUO+Choline+DMB,UUO+Choline+SYJD low-dose(L),UUO+Choline+SYJD high-dose(H),UUO+Choline+Perillae frutescens aqueous extracts(PFAE)(L)and UUO+Choline+PFAE(H)groups.The changes of renal function(SCR,BUN,Cysc,urinary ACR)were measured.The pathological staining was used to observe the renal tissue structure injury and collagen deposition.Serum inflammatory and fibrosis factors and urine renal tubular injury markers were detected by ELISA.At the same time,the effects of Suyin Jiedu granules on TMAO levels were detected,and its correlation with renal functions was analyzed.(3)In animal experiments,we constructed a hyper-choline diet nephropathy rat model.In cell experiments,TMAO was used to establish the renal tubular epithelial cell(RTEC)injury model.Changes in morphology and viability of RTECs,apoptosis fluorescence staining and expression of apoptosis-related proteins were detected after intervention with PFAE.Immunohistochemistry(IHC),immunofluorescence(IF)and western blot were used to detect the changes of TXNIP-TRX redoxisome,reduced TRXt,ASK1 phosphorylation and MAPK pathway proteins.NRK-52E cells were transfected with TRX overexpression vector lentivirus,TXNIP si RNA,ASK1 si RNA,or interfere with ASKl,p38 and JNK inhibitor.The effects of different levels of TXNIP-TRX,ASK1 and MAPK pathway proteins on TMAO-induced and RTEC apoptosis were observed.Results:(1)A total of 33 CKD stage 3-4 patients completed the final clinical observation,including 17 patients in the treatment group and 16 patients in the control group.The two groups were at the same baseline level.Serum and urine TMAO levels were significantly higher in CKD stage 4 patients than in CKD Stage 3.Serum and urine TMAO levels were positively correlated with SCR and negatively correlated with eGFR.After 12 weeks of treatment,Suyin Jiedu granules could significantly improve SCR and BUN,increase eGFR level and reduce TCM syndrome score.In terms of regulating TMAO metabolism,the results showed that serum TMAO decreased in both groups,but urine TMAO excretion increased only in the treatment group.Moreover,the decrease degree of serum TMAO was positively correlated with the improvement degree of renal function.(2)The results showed that TMAO could increase the levels of BUN,urine ACR and serum Cysc,but no significant effect was observed on SCR and ALB.Renal pathological staining showed that TMAO could promote connective tissue hyperplasia,inflammatory cell infiltration and fibrosis.Suyin Jiedu granules or PFAE could significantly reduce BUN,SCR and urinary ACR,alleviate the shedding and expansion of RTECs,reduce inflammatory cell infiltration and collagen deposition.Serum and urine TMAO detection and correlation analysis showed that both Suyin Jiedu granules and DMB can significantly reduce serum TMAO level and promote urine TMAO excretion.Serum TMAO level was positively correlated with BUN,SCR and urinary ACR,while urine TMAO level was negatively correlated with these indicators.(3)Animal experiments showed that TMAO can promote renal cell apoptosis,and induce the injury of mitochondrial number and structure in RTECs.Combined with PFAE,TMAO-induced renal cell apoptosis was reduced and mitochondrial function was improved.In vitro experiments showed that PFAE could alleviate the TMAO-induced morphological and the impairment of viability,reduce the proportion of apoptotic cells,and down-regulate the expression of pro-apoptotic proteins.IHC,IF and western blot results showed that TMAO could promote the activation of ASK1-JNK pathway.Inhibition of ASK1 activation by Selonsertib,inhibition of JNK activity by SP600125,or down-regulate ASK1 expression by ASK1 si RNA could inhibit TMAO-induced apoptosis.Non-reducing western blot showed that PFAE could reduce the inhibitory effect of TMAO on reduced TRX.In vitro and in vivo experiments showed that TMAO could down-regulate TRX expression and up-regulate TXNIP level,which could be reversed after PFAE intervention.TRX overexpression and TXNIP knockdown NRK-52E cells were constructed by transfection.The results showed that upregulation of TRX or knockdown of TXNIP could reduce TMAO-induced ASK1-JNK pathway activation and RTEC apoptosis.Conclusion:(1)The accumulation of serum TMAO is closely related to the deterioration of renal function.However,the relationship between urine TMAO level and renal function may be inconsistent due to the difference kidney injury types,etc..(2)Suyin Jiedu granules may reduce circulating TMAO level by promoting urine TMAO excretion,thus alleviating renal injury induced by TMAO.(3)TMAO promotes the progression of renal inflammation and fibrosis,aggravates the decline of renal function in nephrotic rats.Both Suyin Jiedu granules and PFAE can improve TMAO-induced renal deterioration,reduce renal inflammation and fibrosis.(4)TMAO induces RTEC apoptosis,and PFAE can inhibit the pro-apoptotic effect of TMAO.(5)PFAE can regulate TXNIP-TRX redoxisome,down-regulate ASK1-JNK pathway,and reduce TMAO-induced RTEC apoptosis.(6)Perillae frutescens is the key herb for Suyin Jidu granules to improve TMAO injury.Regulating redox balance and ASK1 activity to reduce RTEC apoptosis may be the important therapeutic targets for relieving nephrotoxicity Chinese medcine. |
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