The Relationship Between Qi Deficiency And Blood Stasis Syndrome And Tubulopathy In Diabetic Kidney Disease And The Protective Effect Of Rhodiola Crenulata

Objective 1.Clinical study: first,approach the relationship between proximal renal tubulopathy markers(ua1-MG/u CRE)and distal renal tubulopathy marker(u EGF/u CRE)and u ACR in type 2 diabetes mellitus patients.Secondly,explore the relationship between tubulopathy markers and Qi deficiency and blood stasis syndrome.2.Experiment 1: Based on the pathogenesis of qi deficiency and blood stasis of DKD,the therapeutic effect of Rhodiola renulata water extract(RCE)on renal fibrosis of DKD rats was studied.3.Experiment 2: Investigate the protective effect of salidroside on renal injury in diabetic rats.Based on network pharmacology approach,the anti-apoptosis mechanism of salidroside in proximal tubular cells induced by high glucose was studied.Methods 1.Clinical study: 125 hospitalized patients with type 2 diabetes mellitus were enrolled.Clinical data and blood biochemical indicators were collected.ua1-MG/u CRE,u EGF/u CRE and u ACR were detected.TCM syndrome differentiation was carried out on the 125 patients.Based on u ACR,the patients were divided into diabetes group(n=75,u ACR<30 mg/g)and DKD group(n=50,u ACR>30-300 mg/g).Then,based on TCM syndrome differentiation,they were divided into Qi deficiency and blood stasis group(n=61)and non-qi deficiency and blood stasis group(n=64).The changes of clinical dataand renal tubulopathy markers were analyzed,respectively.2.Experiment Ⅰ: Thirty-two Wistar rats were divided into sham uninephrectomy(unx),diabetes with uninephrectomy(DKD)and DKD treated with RCE(DKD+RCE).RCE was gavage administered for 20 weeks.24-h urinary protein was measured.The glomerular mesangial matrix expansion index and renal tissue fibrosis area were measured using pathological staining.The expression of collagen I,collagen IV,fibronectin,a-SMA and TGF-β1 were detected by Immunostaning.The m RNA and protein expression of collagen I,collagen IV,fibronectin and TGF-βwere detected by RT-PCR and western blot.3.Experiment Ⅱ: Tweenty-eight rats were divided into sham,uninephrectomy(unx),diabetes with uninephrectomy(DKD)and DKD treated with salidroside(DKD+SAL).SAL was gavage administered for 8 weeks.24-h albuminuria was measured.The glomerular mesangial area and renal tubular damage score were detected using athological staining.Immunostaning revealed the expression of NGAL in diabetic rat kidney.Network pharmacology predicted the potential mechanism of salidroside in the treatment of DKD.Apoptosis was determined by TUNEL and Annexin V-FITC/PI in vitro and in vivo.The expression of BIM,BAX,and cleave caspase-3 were evaluated by western blot and immunostaning in vitro and in vivo.Results 1.Clinical study(1)ua1-MG/u CRE in DKD group was higher than that in diabetes group,P<0.01.and u EGF/u CRE was lower than that in diabetes group,P<0.05.(2)Spearman analysis showed that ua1-MG/u CRE was highly positively correlated with u ACR,while u EGF/u CRE was moderately negatively correlated with u ACR,P<0.05.(3)Multivariate logistic analysis: After adjusting for risk factors,there was a positive correlation between ua1-MG/u CRE and abnormal u ACR in type 2 diabetes mellitus patients,[OR 1.053(95% CI1.017-1.091),P=0.004].u EGF/u CRE was negatively correlated with abnormal u ACR in type 2 diabetic patients,[OR 0.903(95% CI0.824-0.990),P=0.030].(4)The proportion of Qi deficiency and blood stasis syndrome in DKD patients was higher than that in diabetes group,P<0.05.(5)Compared with non-Qi deficiency and blood stasis syndrome,the levels of glycosylated hemoglobin,cystatin,u ACR,and ua1-MG/UCRE were increased in Qi deficiency and blood stasis syndrome group,P<0.05,P< 0.01,P<0.05,P< 0.01 respectively,and the levels of high density lipoprotein and u EGF/u CRE were decreased,both P<0.01.(6)There was a positive correlation between ua1-MG/u CRE and DKD syndrome of qi deficiency and blood stasis [OR 1.145(95%CI 1.021–1.285),P=0.021].There was a negative correlation between u EGF/u CRE and DKD in the occurrence of Qi deficiency and blood stasis syndrome [OR 0.728(95%CI0.571–0.928),P=0.010] 2.Experiment Ⅰ(1)Compared with DKD group,24 h urinary protein in DKD+RCE group,glomerular mesangial matrix expansion index and renal tissue fibrosis were all decreased,P<0.01,P<0.05,P<0.01 P<0.05,respectively.(2)Compared with the DKD group,the expression of collagen I,collagen IV fibronectin and a-SMA were decreased in DKD+RCE group,all P<0.01.The m RNA levels of Collagen I,collagen IV and Fibronectin in the renal cortex of DKD+RCE group were all decreased,P<0.01,P<0.01 and P<0.05,respectively.(3)Compared with the DKD group,the protein and m RNA expression of TGF-β1in the DKD+RCE group were decreased,P<0.05,P<0.01.3.Experiment Ⅱ(1)Compared with the DKD group,SAL treatment reduced 24-h albuminuria,,renal mesangial dilatation area and renal tubule injury score,P< 0.01,P<0.05 P<0.05,respectively.(2)Compared with sham group and unx group,the expression of NGAL in DKD group was increased,both P<0.01,while SAL treatment inhibited the expression of NGAL,P<0.01.(3)Network pharmacology indicated that the mechanism of SAL on DKD may be related to the regulation of apoptosis,inflammatory response and extracellular degradation.(4)In vivo,TUENL staining results showed the amounts of renal tubular cells apoptosis in DKD group was higher than that in sham group and unx group,both P<0.01,which was inhibited by SAL treatment,P<0.01.(5)In vitro,TUENL staining and Annexin V-FITC/PI showed the rate of cells apoptosis in HG+SAL groups for 24 h,48h,72 h were significantly lowere than that in HG group,all P<0.01.(6)Compared with the control group,silenced of BIM by sh RNA in HK-2 cells prevent high gluoces induced apoptosis,P<0.01.(7)In vivo,western blot showed the protein expression of BIM,BAX,cleaved caspase-3 in DKD+ SAL group were decreased than those in DKD group,P<0.05,P<0.01,P<0.01,respectively.(8)In vitro,western blot showed the protein expression of BIM,BAX,cleaved caspase-3 in HG+SAL group was lower than those in HG group,all P<0.01.Conclusions 1.Clinical study:(1)In type 2 diabetes mellitus,the changes of ua1-MG/u CRE and u EGF/u CRE are closely related to the changes of u ACR,which maybe used as potential markers for evaluating the renal injury induced by diabetes.(2)Compared with type 2 diabetes mellitus patients with non-Qi deficiency and blood stasis syndrome,the levels of glycosylated hemoglobin,cystatin,u ACR,ua1-MG/u CRE were increased,and the levels of high density lipoprotein and u EGF/u CRE were decreased in patients with Qi deficiency and blood stasis syndrome.(3)DKD patients represent the Qi deficiency and blood stasis syndrome.ua1-MG/u CREand u EGF/u CRE maybe used as potential markers for evaluating DKD syndrome of qi deficiency and blood stasis.2.Experiment Ⅰ: Rhodiae crenulata water extract can reduce urinary protein of DKD rats and inhibit renal fibrosis of DKD rats.The mechanism may be related to the inhibition of TGF-β1.3.Experiment Ⅱ: Salidroside alleviate renal injury in diabetic rats.And the possible mechanism may be partially attributed to inhibiting apoptosis of proximal tubular cells mediated by BIM...