| This study from the theory of traditional Chinese medicine to explore the origin of Qing method theory,the etiology and pathogenesis of children hematuria and syndrome differentiation treatment,and further related experimental research.Objective: Using metabonomics nuclear magnetic resonance technology,we dynamically monitor the relationship between urine metabolite changes and blood biochemistry,renal histopathology and cytokines in Ig AN damp-heat syndrome model rats at different time points,so as to screen out the landmark metabolome,and finally determine the biomarker of hematuria damp-heat syndrome by observing the difference of intervention effects of Jinshuiqing on the model at different time,providing a basis for non-invasive clinical judgment of renal histopathological changes of children with hematuria and determine treatment timing.Method: 70 SPF SD rats aged 4 weeks in the first stage were randomly divided into normal group and model group to make Ig A nephropathy model,Urine was collected every week to detect erythrocyte,protein and metabolic urine,By the end of 12 th week,pathological changes of kidney were observed by HE and PAS staining,and blood NF-?B p65,TGF-?1 and biochemical indexes of BUN,SCr,Chol and TG were detected by ELISA.The second stage 115 SPF SD rats aged 4 weeks were randomly divided into normal group,model group,Jinshuiqing early treatment group,middle treatment group and late treatment group,and the Ig A nephropathy model of rats was built,On the basis of the model group,the treatment group was given Jinshuiqing mixture once a day in the 5th week,7th week and 9th week respectively.Until the 12 th weekend,and the pathological changes of kidney were observed by electron microscope at the same stage,The expressions of TGF-?1,CTGF and VEGF protein in kidney tissue were detected by Western-blot and the expressions of TGF-?1,VEGF and CTGF m RNA were detected by RT-PCR.Results:Stage ?1 The rats in the model group appeared damp-heat symptoms in the 4th week.Compared with the control group,the growth rate of body mass in the model group slowed down from the 5th week,and slowed down from the 7th week(P < 0.05),especially in the 11 th week(P < 0.01).2 Urine analysis showed that hematuria appeared from the 6th week(P < 0.05)and proteinuria appeared from the 8th week(P <0.05).3 Compared with the control group,the biochemical indexes showed that the renal function and blood lipid were increased,and the serum creatinine(Scr),triglyceride(TG)and cholesterol(Chol)in the model group were significantly increased(P < 0.05).4 Cytokines showed that nuclear factor ?B(NF-?B)in model group increased from the 6th week(P < 0.05),transforming growth factor-? 1(TGF-? 1)increased from the 10 th week(P < 0.05).5 Renal histopathology showed that mesangial cells and mesangial matrix proliferated obviously in the model group,which gradually increased in the 8th,10 th and 12 th week of the experiment.Immunofluorescence showed Ig A deposition(++)in the mesangial area of the model group at the 12 th week.6 Potential biomarkers related to the damp-heat syndrome model were screened out from urine metabolites,which were differentially expressed compared with the control group from the 5th week(P <0.05),Through the analysis of pathway enrichment and the construction of metabolic network,it was found that the abnormal expression of relevant metabolites in 6 pathways involving tricarboxylic acid cycle,urea cycle,amino acid,carbohydrate metabolism,intestinal flora and choline metabolism in model rats gradually increased.Stage ?1 The performance of damp-heat syndrome in rats improved after treating,especially in the early group,and the weight of rats was catching up,In the 11th-12 th week,the weight of the early group increased compared with that of model group(P<0.05).2 Urine analysis showed that urine red blood cells began to rise from the 6th week of modeling,In the early group red blood cells decreased in the 8th week after treatment,which was statistically significant in the 10 th week compared with the model group(P<0.05).In the middle group red blood cells decreased from the 10 th week,which was statistically significant in the 12 th week compared with the model group(P < 0.05).There was no statistical significance between late group and model group(P>0.05).In the early group urine protein began to increase from the 8th week after treatment.It began to decrease in the 9th week in early Group,and it was statistically significant compared with the model group at the 11 th week(P<0.05),but there was no statistical significance between middle Group and late Group(P>0.05).3 Biochemical indicators showed that urea nitrogen in each treatment group was lower than that in the model group after treatment,but there was no statistical significance(P>0.05).At the 8th week of modeling,the serum creatinine of rats increased compared with the control group(P<0.05),and decreased in Jinzao group compared with the model group after treatment(P<0.05).At the 8th week,TG and Chol of model rats increased(P<0.05),and after treatment,early and middle groups were lower than those of model group(P < 0.05).4 Cytokine:Blood ELISA showed that NF-?B in model group began to increase from the 6th week,but stopped rising at the 8th week and decreased from the 10 th week in early group after treatment(P<0.05),Compared with model group,NF-? B in middle and late group decreased from the 12 th week,has no statistical significance(P> 0.05),TGF-?1 in the model group began to increase from the 8th week,TGF-?1 in the early group decreased from the 10 th week after treatment compared with the model group,which has statistical significance until the 12 th week(P<0.05),But decrease in middle and late group has no statistical significance(P>0.05)compared with the model group,Immunohistochemistry of renal tissue showed that NF-?B was only slightly expressed in the cytoplasm of glomerular epithelial cells in the control group,while NF-?B was obviously expressed in the nuclei of glomerular and renal interstitial cells in the model group,The expression site of NF-?B in each treatment group was the same as that in the model group,but its expression level was lower than that in the model group,and it was obvious in early Group and middle Group,and its optical density was statistically significant(P < 0.05).TGF-?1 has scarcely any slight brown positive expression in glomerulus and renal tubules of rats in control group,but has significant positive expression in mesangial area,proximal convoluted tubule and renal tubule epithelial cells of rats in model group,The positive expression degree of each treatment group is less than that of model group,especially in early group,and its optical density value is statistically significant compared with model group(P<0.05).The expression of TGF-?1,CTGF and VEGF protein in kidney tissue detected by Western-blot showed that the expression of TGF-?1,CTGF and VEGF protein in model group increased(p <0.05),Compared with the model group,the expression of TGF-?1,CTGF and VEGF protein in the treatment group decreased,and there was significant difference between early group and the model group(P < 0.05).The expression of TGF-?1,VEGF and CTGF m RNA detected by RT-PCR showed that the expression of TGF-?1,CTGF and VEGFm RNA in model group was higher than that in control group(p < 0.05),Compared with the model group,the expression of TGF-?1,CTGF and VEGF protein in the treatment group decreased,and there was significant difference between Jinzao group and the model group(P < 0.05).5 Renal histopathological light microscopy showed typical Ig A nephropathy in the model group,which improved from the 10 th week of the experiment in early group,improved slightly in the 12 th week of the experiment in middle group,and did not improve significantly in late group.Electron microscopy showed that the foot processes in the model group fused extensively,mesangial cells and mesangial matrix proliferated,and small pieces of electronic compact were deposited in some mesangial areas.In the treatment group,Early group improved obviously,but late group had no obvious change.6 Urine metabonomics showed that there were 13 kinds of substances with down-regulated expression and 1 kinds of substances with up-regulated expression in the model group,and 14 kinds of metabolites could be recalled in the three treatment groups,among which 13 metabolites could be significantly recalled after early intervention in the early Group(5th week),7 metabolites could be significantly recalled after mid-term intervention in middle Group(7th week),and 4 metabolites could be significantly recalled after late intervention in late Group(9th week)(P < 0.05).Hematuria and damp-heat syndrome involves seven metabolic pathways disorder,which has a tendency of callback after Jinshuiqing treatment.7 The ability of metabolites to predict hematuria was analyzed by ROC curve,and it was found that citric acid,hippuric acid and4-hydroxyphenylacetate had a high correlation with them,and the AUC reached the highest of 0.848(sensitivity 81.6%,specificity71.1%,P<0.001),and the positive likelihood ratio was 2.82.Conclusion: It is concluded that citric acid,hippuric acid and4-hydroxyphenylacetate can be used as biomarkers to judge the changes of early hematuria.Early intervention with Chinese medicine can improve the prognosis of isolated hematuria in children.Urine metabolite screening can be used as a good method to predict kidney diseases and judge the treatment node of hematuria,Early intervention according to metabolite changes can improve the curative effect and prognosis,and provide animal experimental basis and theoretical basis for accurately grasping the treatment timing of hematuria in children. |
PDF Full Text Request