Study On The Mechanism Of DUSP1 Regulating Glomerular Mesangial Cell Proliferation In IgA Nephropathy And The Intervention Effect Of Huaier

Background and Objective: IgA Nephropathy(IgAN),as the most common primary glomerular disease leading to Chronic kidney disease(CKD),has been paid more and more attention.The slow and insidious progression of the disease initially led to the assumption that the prognosis was benign,but it is estimated that up to 40% of IgAN patients will progress to end-stage renal disease(ESRD).Although more than 50 years have passed since the disease was first reported by Jacques Berger,its pathogenesis remains largely unknown.In addition,the non-parallelism between the IgA deposition density and the degree of injury in the glomerular of patients and the significant difference in clinical manifestations result in no clear treatment plan at present.In the pathogenesis of IgAN,the theory of "multiple hits" has been recognized by most nephrologists.However,only the last "hit" in the "multiple hits" occurs in the glomerulus,that is,the polymeric IgA1 immune complex containing galactose deficiency binds to the transfertin receptor(Tf R)on the surface of human glomerular mesangial cells(MC)and activates the MC,resulting in abnormal cell proliferation.The proliferating MC synthesizes too much cell matrix and releases inflammatory factors and chemokines to affect podocyte and renal tubule cell functions though crosstalk.Therefore,MC is the first glomerular intrinsic cell in the occurrence and development of IgAN,and inhibiting or even reversing the proliferation of MC is one of the effective ways to treat IgAN.Existing studies have shown that in IgAN,excessive activation of mitogen-activated protein kinase(MAPK)signaling pathway in the glomerulus promotes the proliferation of MC and aggravates the progression of the disease,and it has also been reported that inhibition of excessive activation of MAPK signal pathway in the glomerulus can play a therapeutic role.Huaiqihuang is a kind of traditional Chinese medicine compound composed of Trametes robiniophila murr(Huaier,HR),wolfberry fruit and Polygonatum.Several clinical studies have shown that IgAN can be treated alone or in combination with other drugs.Huaier,as one of the main components of huaiqihuang,has been proved to regulate and repair a variety of target genes.It has also been shown that Huaier can inhibit the proliferation of tumor cells by regulating MAPK signaling pathway.The above background suggests that in IgAN,Huaier may regulate MAPK signaling pathway in MC through its target genes and thus play a role in inhibiting cell proliferation.High-throughput expression profiling has become a widely used technique to identify key genes in diseases,which can be used to search disease biomarkers,explore pathogenesis and find therapeutic targets.The purpose of this study was to explore the key genes in the occurrence and development of IgAN through bioinformatics analysis technology,verify the expression of the key gene DUSP1,and explore the new mechanism of DUSP1 regulating mesangial cell proliferation and Huaier intervention inhibiting mesangial cell proliferation.In order to provide a new theoretical basis for the pathogenesis of IgAN and clinical treatment.Methods:Part One: Bioinformatics analysis was used to screen the key genes in the development of IgAN and verify DUSP1 in vivo: 1.IgAN data sets GSE93798,GSE99339,GSE50469 and GSE37460 were selected from the public database GEO.Data sets GSE93798,GSE99339 and GSE50469 were combined as training sets,and key genes were screened through a series of analyses.Key genes were verified in the validation set(dataset GSE37460).2.The classical IgAN model of SD rats was established by using bovine serum protein + carbon tetrachloride + lipopolysaccharide.(2)Model group(IgAN group).3.Histopathological morphology of renal tissue was observed by HE and PAS staining,fluorescence intensity in glomerular was observed by IgA direct immunofluorescence,and 24 urinary protein was measured by BCA method to verify IgAN rat model.4.The m RNA and protein expressions of DUSP1 in glomerulus were detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR),Western Blot and IHC.5.The expression and localization of DUSP1 in glomerulus were detected by immunofluorescence double-staining DUSP1 and Integrin-α8.Part Two: Study on the mechanism of DUSP1 regulating the proliferation of human mesangial cells: 1.IgAN mesangial cell disease model was established by polymeric IgA1(p IgA1).They were divided into :(1)normal culture group(Control group);(2)Disease model group(p IgA1 group).2.Cell immunofluorescence Ki67 staining was used to verify cell disease model.3.Protein and m RNA expression of DUSP1 in Control group and p IgA1 group were detected by RT-q PCR and Western Blot method.4.Human glomerular mesangial cells(HMC)was transfected with DUSP1 overexpression lentivirus and divided into(1)Negative overexpression control group(Oe-Con group).(2)Overexpression of DUSP1 group(Oe-DUSP1 group).5.The effect of overexpression of DUSP1 on cell proliferation was detected by CCK8 assay.6.Flow cytometry and Western Blot assay were used to detect the effects of overexpression of DUSP1 on cell cycle and apoptosis.7.Western Blot method was used to detect the effects of overexpression of DUSP1 on MAPK signaling pathway and expression of p53 and p21.Part Three: Mechanism of HR regulation of mesangial cell proliferation by DUSP1: 1.The rat model of HR intervention on IgAN was established and divided into :(1)Control group;(2)Model group(IgAN group);(3)Model+HR intervention group(IgAN+HR group).2.HE and PAS staining were used to observe the pathological morphology of renal tissue,IgA immunofluorescence was used to observe the fluorescence intensity in the glomerular,and BCA method was used to measure 24 urinary protein quantitative to verify the intervention effect of HR on IgAN rat model.3.IHC and Western Blot methods were used to observe the effect of HR intervention on the expression of DUSP1 in the glomerulus.4.CCK8 method was used to detect IC50 of HR intervention in HMC.5.The mesangial cell disease model of IgAN with HR intervention was established and divided into :(1)normal culture group(Control group);(2)Disease model group(p IgA1group);(3)Model+HR intervention group(p IgA1+HR group).6.CCK8 and Western Blot methods were used to detect the effect of HR intervention on proliferation of cell disease models.7.Western Blot method was used to detect the effect of HR intervention on DUSP1 expression in cell disease models.8.HMC was transfected with DUSP1 knockdown lentivirus and divided into(1)knockdown negative control group(Sh-Con group);(2)Knockout negative control +HR intervention group(Sh-Con+HR group);(3)Knock out DUSP1+HR intervention group(Sh-DUSP1+HR group).9.Flow cytometry and Western Blot assay were used to determine whether HR intervention regulated cell cycle and apoptosis through DUSP1.10.Western Blot method was used to detect whether HR regulates MAPK signaling pathway and the expression of p53 and p21 through DUSP1.Results:Part One: 1.Two key genes were screened out through analysis of the training set and verification of the validation set,and DUSP1 was selected for subsequent experiments.2.The results of HE and PAS staining,IgA direct immunofluorescence and 24-hour urine protein determination confirmed the successful establishment of IgAN rat model.3.IHC,RT-q PCR and Western Blot results showed that protein and m RNA expression of DUSP1 in glomerular of IgAN group were down-regulated.4.Immunofluorescence double staining of DUSP1 and Integrin-α8 revealed DUSP1 expression and localization in glomerular mesangial cells.Part Two:1.RT-q PCR and Western Blot results showed that both m RNA and protein expressions of DUSP1 were down-regulated in cell disease model.2.CCK8 results showed that overexpression of DUSP1 induced inhibition of HMC cell proliferation.3.Flow cytometry and Western Blot results showed that overexpression of DUSP1 increased the proportion of HMC cells in G0/G1 phase,and down-regulated the expression levels of CDK2 and Cyclin D1.4.Flow cytometry and Western Blot results showed that overexpression of DUSP1 increased the percentage of cell apoptosis,up-regulated expression of Bax and cleaved Caspase3,and down-regulated expression of Bcl2.5.Western Blot results showed that overexpression of DUSP1 down-regulated the expression of p-ERK/t-ERK,p-JNK/t-JNK,p-P38/t-P38 in HMC,while up-regulated the expression of p53 and p21.Part Three:1.After HR intervention,the number of cells and matrix components in the glomerulus of IgAN rats were decreased,IgA deposition was weakened,and 24-hour urinary protein content was decreased.2.IHC and Western Blot results showed that HR intervention up-regulated DUSP1 expression in glomerulus of IgAN rat model.3.CCK8 and Western Blot results showed that HR intervention down-regulated the expression of PCNA in cell disease models and inhibited p IgA1-induced cell proliferation.4.Western Blot results showed that HR intervention up-regulated DUSP1 expression in cell disease models.5.Flow cytometry results showed that DUSP1 knockdown attenuated the increase in the proportion of G0/G1 cells induced by HR intervention;Western Blot results showed that DUSP1 knockdown attenuated the down-regulation of CDK2 and Cyclin D1 induced by HR intervention.6.Flow cytometry results showed that DUSP1 knockdown attenuated the increase in the proportion of apoptosis cells induced by HR intervention;Western Blot results showed that DUSP1 knockdown attenuated up-regulation of Bax and cleaved-Caspase3 expression and down-regulation of Bcl2 expression induced by HR intervention.7.Western Blot results showed that DUSP1 knockdown attenuated the down-regulation of p-ERK/t-ERK,p-JNK/t-JNK and p-P38/t-P38 induced by HR intervention.8.Western Blot results showed that DUSP1 knockdown attenuated the up-regulation of p53 and p21 expression induced by HR intervention.Conclusion:1.Through bioinformatics analysis of IgAN gene chip data set,DUSP1 was identified as the key gene in the development of IgAN.In vivo experiments confirmed that DUSP1 expression was down-regulated in the glomerulus of IgAN rat model,and DUSP1 expression existed in mesangial cells.2.DUSP1 expression was down-regulated in IgAN mesangial cell disease model in vitro.Overexpression of DUSP1 in mesangial cells can induce cell cycle arrest,induce apoptosis and inhibit cell proliferation.In addition,MAPK signaling pathway was inhibited and p53 and p21 expression was up-regulated.3.HR intervention can up-regulate DUSP1 expression in vivo and in vitro,confirming that DUSP1 is a new target gene of HR.In vitro experiments confirmed that HR intervention induced mesangial cell cycle arrest and apoptosis,inhibited MAPK signaling pathway and up-regulated p53 and P21 expression through DUSP1.HR intervention up-regulated DUSP1 expression may inhibit mesangial cell proliferation by inhibiting MAPK signaling pathway and up-regulating p53 and P21 expression.