| The prevalence of diabetes is increasing year by year.Diabetic kidney disease(DKD)is one of the most common microvascular complications of diabetes,and its incidence and number of patients are also increasing year by year.The main cause of the disease brings a heavy burden to the country,society and patients,However,the pathogenesis of DKD is complex and not yet fully understood.It is imperative to further understand the pathogenesis of DKD,find new diagnostic biomarkers,new therapeutic targets,and more effective treatments.More and more studies have shown that LncRNA MALAT1 is closely related to the progression of DKD.MALAT1 can be expressed in podocytes,endothelial cells,and renal tubular epithelial cells.It participates in different pathological links of DKD and promotes the occurrence and development of DKD by exerting different molecular functions in different renal cell types.Qihuang Yishen Granule(QHYSG)is a new prescription developed by Professor Zhang Ning from Wangjing Hospital,which based on the core pathogenesis of diabetic kidney that deficiency of both qi and yin,and water dampness and blood stasis as the target.This recipe is composed of Astragalus,Taizishen,Rehmannia glutinosa,Cornus officinalis,Poria,Alisma,and Salvia miltiorrhiza.Clinical trials have confirmed that Qihuang Yishen granules can reduce glomerular damage marker proteins and tubular damage marker proteins in DKD patients,delay the process of DKD,and improve clinical symptoms,which is safe and effective.This study continued to explore the role and mechanism of Qihuang Yishen granules in podocyte injury on the basis of previous studies,and to explore whether MALAT1 could be a diagnostic biomarker and therapeutic target for DKD.Therefore,the following two parts are designed.First,the cross-sectional study.To observe the expression of serum MALAT1 in DKD patients and the correlation between MALAT1 and other indicators,to explore the clinical significance of MALAT1;2.Taking the mouse podocyte injury model induced by glycolipid toxicity as the research object,Qihuang Yishen granule was used as the research object to observe the effect of Qihuang Yishen granule on the expression of EMT and MALAT1 in podocytes,and to explore the mechanism of Qihuang Yishen granule inhibiting EMT and protecting podocyte injury by regulating MALAT1/wntl/β-catenin pathway.Part I:The expression level of LncRNA MALAT1 in serum of patients with diabetic kidney disease and its clinical significanceObjective:To investigate whether LncRNA MALAT1 can be used as a biomarker for the diagnosis and treatment of DKD.Methods:A cross-sectional study was conducted,including 30 patients with type 2 diabetes,30 patients with DKD,and 10 healthy non-diabetic subjects as the normal control group.The expression level of serum lncRNA MALAT1 was detected by qRT-PCR,and the correlation between LncRNA MALAT1 and blood glucose,glycosylated hemoglobin,urine albumin-creatinine ratio and renal function damage-related indicators was statistically analyzed,and whether MALAT1 could be used as a biomarker for the diagnosis of DKD.Results:Compared with the healthy and diabetic groups,the expression of IncRNA MALAT1 in the serum of patients with diabetic kidney disease was significantly increased.Pearson correlation analysis showed that the expression level of MALAT1 in serum was positively correlated with fasting blood glucose,glycosylated hemoglobin,serum creatinine,Cys-C,β 2MG,BUN and UACR,(r=0.431,0.322,0.573,0.619,0.643,0.487,0.645,P<0.001 or P<0.01),which was negatively correlated with eGFR(r=-0.575,P<0.001).Logistic regression analysis showed that Cys-C,UACR,serum creatinine and Malatl were independent risk factors for diabetic kidney disease(P<0.05 or P<0.001).The ROC curve was used to analyze the diagnostic value of UACR,Malatl expression levels,Cys-C,and serum creatinine for DKD.The results showed that the areas under the ROC curve(AUC)were 0.929、0.810、0.844 and 0.826,respectively.Combined detection of the ROC curve of DKD The lower area AUC was 0.951,and the sensitivity and specificity for diagnosing DKD were 0.967 and 0.90,respectively(with>0.52 as the diagnostic cut-off point).Conclusion:The lncRNA MALAT1 is highly expressed in the serum of DKD patients,and MALAT1 may be a biomarker for DKD.Part 2:Study on the mechanism of Qihuang Yishen Granules to alleviate podocyte injury based on LncRNA MALAT1/wntl/β-cateninObjective:To observe and analyze the regulation of Qihuang Yishen granule on LncRNA MALAT1 and wntl/β-catenin signaling pathway and the effect on podocyte EMT,and to explore the protective mechanism of Qihuang Yishen granule on podocytes.Methods:The mouse podocyte injury model induced by high-sugar and high-fat solution was used as the research object,intervened with the freeze-dried powder of Qihuang Yishen Granules,and the positive control drug was losartan potassium.(1)The proliferation activity of podocytes was detected by CCK-8 method;the cytoskeleton was observed by rhodamine phalloidin fluorescence staining;the expression level of IncRNA MALAT1 was detected by qRT-PCR;the podocyte-related proteins nephrin,podocin,and the pathway proteins wntl,β-catenin,and the expression of EMT marker proteins desmin,FSP1,and MMP-7 were detected by WB.To observe the effects of Qihuang Yishen Granules on podocyte proliferation activity,podocyte cytoskeleton,wntl/β-catenin signaling pathway and podocyte EMT.(2)Add the wntl/β-catenin pathway inducer LiCL,the wntl/β-catenin signaling pathway inhibitor DKK-1,and use WB to detect the expression of podocyte-related proteins nephrin,podocin,pathway proteins wntl,β-catenin,and EMT marker proteins desmin,FSP1,MMP-7,to verify whether Qihuangyishen granules inhibit EMT and protect podocytes by regulating the wntl/β-catenin pathway.(3)The expression of MALAT1 was knocked down by siRNA,and the expression level of IncRNA MALAT1 was detected by qRT-PCR;the expression of podocyte-related proteins nephrin,podocin,desmin,and pathway proteins wntl and β-catenin were detected by WB.To observe the effect of LncRNA MALAT1 on wntl/β-catenin signaling pathway,and to explore whether Qihuang Yishen granule protects podocytes by regulating LncRNA MALAT1/wntl/βcatenin.Results:(1)Qihuangyishen granules could improve cell proliferation activity.Qihuangyishen granules could protect the cytoskeleton of podocytes.Western blot results showed that compared with the normal group,the protein expression levels of nephrin and podocin in the model group were significantly decreased(P<0.001),while the protein expressions of desmin,FSP1 and MMP-7 were significantly increased(P<0.001);compared with the model group,the expression levels of nephrin and podocin in losartan potassium group and Qihuangyishen granule group were significantly increased(P<0.001),while the protein expression levels of desmin,FSP1 and MMP-7 were significantly decreased(P<0.001).It showed that podocyte injury was induced by glycolipid toxicity,and Qihuang Yishen granule could inhibit Epithelial mesenchymal transition and alleviate podocyte injury induced by high glucose and high fat.qRT-PCR test results showed that compared with the normal group,the mRNA expression of MALAT1 in podocytes in the model group was significantly increased(P<0.001);compared with the model group,the mRNA expression of MALAT1 in podocytes in the traditional Chinese medicine group and losartan potassium group was significantly increased decreased(P<0.001).It suggested that glycolipid toxicity up-regulated the expression of MALAT1,and Qihuang Yishen granules could down-regulate the expression of MALAT1.(2)The results of qRT-PCR showed:the expression of MALAT1 in podocytes:Compared with the normal group,the mRNA expression of MALAT1 in the model group was significantly increased(P<0.001);compared with the NC group,the mRNA level of MALAT1 in the podocytes in the siRNA group was significantly decreased(P<0.001).Compared with the model group,the mRNA level of MALAT1 in the podocytes of the traditional Chinese medicine group and the siRNA group was significantly decreased(P<0.001).The results of Western blot showed:the expression of podocytes now off protein:compared with the normal group,the expression levels of nephrin and podocin proteins in podocytes in the model group were significantly decreased(P<0.001),and the expression levels of desmin were significantly increased(P<0.001);Compared with the NC group,the protein expression levels of nephrin and podocin in the siRNA group increased significantly(P<0.001),and the expression level of desmin decreased significantly(P<0.001).Compared with the model group,the expression levels of nephrin and podocin in the traditional Chinese medicine group and the siRNA group were significantly increased(P<0.001),and the expression level of desmin was significantly decreased(P<0.001).Expression of wntl/β-catenin pathway proteins:Compared with the normal group,the expression levels of wntl/β-catenin pathway proteins wntl and βcatenin in podocytes in the model group were significantly increased(P<0.001);compared with the NC group,wntl/β-catenin in the siRNA group The expression levels of the pathway proteins wntl and βcatenin were significantly decreased(P<0.001);compared with the model group,the expression levels of the wntl/β-catenin pathway proteins wntl and β-catenin in the traditional Chinese medicine group and the siRNA group were significantly decreased(P<0.001).It is suggested that glycolipid toxicity activates the wntl/β-catenin signaling pathway by up-regulating MALAT1.The traditional Chinese medicine Qihuangyishen granules can inhibit the expression of wntl and β-catenin proteins by down-regulating MALAT1,inhibit the activation of the wntl/β-catenin signaling pathway,and protect podocytes.(3)Western blot results showed that compared with the normal group,the expression levels of nephrin and podocin in the LiCL group were significantly decreased(P<0.001),the wntl/β-catenin pathway proteins wntl,β-catenin and the EMT marker proteins desmin,FSP1,MMP-7,the expression levels of their were significantly increased(P<0.001);compared with the LiCL group,the protein expressions of nephrin and podocin in the LiCL+Chinese medicine group were significantly increased(P<0.001),the protein expressions of wntl,β-catenin,desmin,FSP1,and MMP-7 were significantly increased(P<0.001).Compared with the normal group,the expression levels of nephrin and podocin in the model group were significantly decreased(P<0.001),and the expression levels of wntl/β-catenin pathway proteins wntl,β-catenin and EMT marker proteins desmin,FSP1,and MMP-7 were significantly increased(P<0.001);compared with the model group,the expression levels of nephrin and podocin proteins in the DKK-1 group were significantly increased(P<0.001),and the expression levels of the wntl/β-catenin pathway proteins wntl,β-catenin and EMT marker proteins desmin,FSP1,and MMP-7 were significantly increased.It is suggested that Qihuang Yishen Granules can inhibit podocyte EMT and protect podocytes by inhibiting the wntl/β-catenin signaling pathway.Conclusion:LncRNA MALAT1 expression was up-regulated and wntl/βcatenin signaling pathway was activated in podocytes cultured with glycolipid toxin.Epithelial mesenchymal transformation occurred in podocytes.Qihuang Yishen Granules protect podocytes by downregulating MALAT1,inhibiting the expression of Wntl and β-catenin proteins,inhibiting the activation of Wntl/β-catenin pathway,and inhibiting the epithelial-mesenchymal transformation of podocytes. |
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