The Protective Effect Of Berberine On Podocytes Of Diabetic Nephropathy And The Preliminary Regulation Of Long Noncoding RNA

Diabetic nephropathy(DN)is one of the main complications of diabetes and the most common cause of chronic kidney disease,accounting for more than 40% of end-stage renal disease(ESRD).Podocytes is the main component of glomerulus with complex cells form,can guarantee the glomerular basement membrane(GBM)of normal structure and function,prevent loss of protein abnormalities,as the main part of glomerular filtration barrier,is very important in maintaining glomerular podocyte function,its damage and loss will further aggravate the glomerular injury and filtration barrier.Berberine(BBR)is the material basis of rhizoma coptidis in the treatment of thirst reduction.It has a variety of pharmacological effects,including lowering blood glucose,blood lipid and blood pressure,improving insulin resistance,anti-inflammatory and anti-oxidative stress,etc,and can improve kidney injury,and has a certain therapeutic effect on DN.Long noncodingRNAs(LncRNAs)are a class of non-protein-coding transcripts that regulate gene expression at multiple levels.Research evidence suggests that LncRNA may play an important role in the pathogenesis of DN and podocyte injury.Epidermal growth factor(EGF)is widely present in submandibular gland,pancreatic islet and kidney tissue,and it affects cell proliferation,apoptosis and oxidative stress by binding to EGF receptor.As an agonist of EGF receptor,EGF is related to the regulation of podocyte function in DN,and EGF is an important protein of podocyte apoptosis.Previous studies have found that LncRNA LOC102549726 in DN kidney tissues and cells are anomalies rise,bioinformatics to predict its target protein for EGF,but how to function and adjust podocytes function between LncRNA LOC102549726 and EGF is still unclear.Studied in this paper through the experiments in vivo and in vitro to elucidate the pathogenesis of DN,clear DN related LncRNA and the mechanism of action of berberine,explore the LncRNA LOC102549726 whether in DN rat renal cortex and under the environment of HG abnormal expression in cells,and whether its affect targeted EGF,and tries to through the study of LncRNA and related function axis control clarify BBR on the prevention and control mechanism of DN.OBJECTIVE Establish DN model in SD rats.Differentially expressed LncRNAs were screened and podocytes injury was observed.Combined with the differential expression of LncRNAs in kidney tissues and podocytes,the functional related LncRNA of podocytes was identified as LncRNA LOC102549726.The involvement of LncRNA LOC102549726 in podocytes apoptosis was confirmed.BBR inhibits podocytes injury by regulating the abnormal expression of LncRNA LOC102549726.It is clear that LncRNA LOC102549726 regulates the injury of podocytes by targeting EGF in DN,and it is clear that BBR plays a role in the treatment of DN by regulating LncRNA LOC102549726.METHODS DN model of SD rats was established by intritoneal injection of STZ combined with high glucose and high fat feed.Fasting blood glucose and urinary protein were detected to confirm the success of DN model.Hematoxylin and eosin(HE)staining was used to determine the glomerular injury,and the expression of Podocin,WT-1 and Desmin was detected by Western Blot.High glucose(HG)stimulated podocytes as an in vitro model,and Western Blot was used to detect podocyte-related protein expression.Flow cytometry was used to detect the change of apoptosis rate of podocytes,which confirmed the success of the podocyte model in vitro.Complete transcriptome sequencing and qRT-PCR were used to screen out the abnormal expression of DN-related LncRNAs in kidney tissues and podocytes of DN rats.Combined with bioinformatics,LncRNA LOC102549726 was found to have the greatest difference in upregulation,which had potential target EGF.LncRNA LOC102549726 siRNA or overexpressed plasmid was transfected,and EGF expression in each group was detected by qRT-PCR,Bim and podocyte-related protein expression were detected by Western Blot.EGF small infereningRNA(siRNA)was transfected with,and the expression of EGF in each group was detected by qRT-PCR,the expression of apoptosis protein Bim and podocyte-related protein in each group was detected by Western Blot,the change of apoptosis rate in each group was detected by flow cytometry,and the change of cell adhesion ability in each group was detected by Transwell.The podocytes stimulated by HG were then treated with BBR at 5 concentration gradients,LncRNA LOC102549726 expression in each group was detected by qRT-PCR.Flow cytometry was used to detect the change of apoptotic rate of podocytes,and the optimal BBR concentration was selected.LncRNA LOC102549726 siRNA,overexpressed optimal and BBR was given.EGF expression was detected by qRT-PCR,Bim and podocyte-related protein expression was detected by Western Blot,WT-1 expression was detected by immunofluorescence,apoptosis rate was detected by flow cytometry,and cell adhesion ability of each group was detected by Transwell.RESULTS1.Establishment of DN model of SD rats and podocyte model in vitro The results showed that there was no obvious renal structure lesion in the normal group,while the glomerular volume of the model group was significantly atrophy,extracellular matrix deposition,basement membrane thickening,and partial glomerular sclerosis.Immunofluorescence showed that the expression level of WT-1 in the kidney tissue of the DN group was reduced,and the DN model was successfully constructed.Western Blot results showed that compared with the normal group,the expressions of podocyte marker proteins WT-1 and Podocin were significantly decreased in the DN group,while the expression of podocyte injury marker protein Desmin was increased,and podocyte injury occurred in the DN group.Western Blot results showed that compared with the normal group,the expression of WT-1 and Podocin in the HG group was significantly decreased,while the expression of Desmin was increased.Flow cytometry showed that the apoptosis rate of podocytes increased in the HG group compared with the normal group.It indicates that high glucose stimulation can cause podocyte injury.2.Screening of differentially expressed LncRNAs High-throughput sequencing results showed that there were significant differences in a large number of LncRNAs between the normal group and the model group.Twenty LncRNAs with the highest up-regulation ratio and down-regulation ratio were selected,respectively,and 9 cell function-related LncRNAs were identified.The expressions of these 9 LncRNAs in kidney tissues and cells were detected by qRT-PCR,among which the differentially expressed LncRNA ENSRNOT00000087471(i.e.LncRNA LOC102549726)was consistent with that in kidney tissues and significantly increased.3.Prediction and validation of LncRNA LOC102549726 target protein Bioinformatics results indicated that the target protein of LncRNA LOC102549726 was EGF.qRT-PCR results showed that EGF expression decreased after silencing LncRNA LOC102549726,while EGF expression increased after overexpression of LncRNA LOC102549726.The results showed that the target protein of LncRNA LOC102549726 was EGF.4.LncRNA LOC102549726 is associated with podocyte injury LncRNA LOC102549726 siRNA or overexpressed plasmid was transfected.Western Blot result showed that,conpare with the noormal group and the overexpressed group was abnormally increased,while the expression of WT-1 and Podocin was decreased.Transfection of LncRNA LOC102549726 siRNA reversed the abnormal expression of these proteins.Immunofluorescence result showed that the expression of WT-1 decreased when LncRNA LOC102549726 siRNA was transfected.It suggested that LncRNA LOC102549726 was associated podocyte injury.5.LncRNA LOC102549726 target protein EGF is associated with podocyte injury Western Blot results during transfection of EGF siRNA showed that apoptosis proteins Bim and Desmin in podocytes were abnormally increased and WT-1 and Podocin expressions were decreased in the high glucose group compared with the normal group.EGF siRNA transfection could reverse the abnormal expression of these proteins.Flow cytometry showed that the apoptosis rate of podocytes increased in the high glucose group and decreased in the silent group.Transwell results showed that the podocyte adhesion ability of the model group was decreased,and the podocyte adhesion ability was enhanced when transfected with EGF siRNA.LncRNA LOC102549726 can target EGF to affect podocyte injury.When LncRNA LOC102549726 overexpressed plasmid was transfected,Bim and Desmin increased abnormally,while WT-1 and Podocin was decreased.The abnormal expression of these proteins was reversed by transfection of LncRNA LOC102549726 siRNA.Immunofluorescence results showed that the expression of WT-1 decreased in the high glucose group,while the expression of WT-1 increased in the low interference group.LncRNA LOC102549726 targets EGF to affect podocyte function.6.The improvement of podocyte injury by BBR may be related to it impact LncRNA LOC102549726 expression qRT-PCR results showed that different concentrations of BBR could improve the abnormal expression of lncRNA LOC102549726 to varying degrees,and BBR of 90μM had the best inhibition effect.Flow cytometry showed that BBR could inhibit the apoptosis of podocytes,and 90μm BBR had the best effect on improving the apoptosis of podocytes.qRT-PCR results showed that EGF gene expression increased abnormally when LncRNA LOC102549726 was overexpressed,and inference of LncRNA LOC102549726 or BBR administration could reverse its abnormal expression.Western Blot results showed that when LncRNA LOC102549726 was overexpressed,Bim and Desmin were abnormally increased,while Podocin and WT-1 expression were decreased.Silencing LncRNA LOC102549726 or BBR administration could reverse the abnormal expression of these proteins.Immunofluorescence results showed that WT-1expression decreased when LncRNA LOC102549726 was overexpressed,and increased when LncRNA LOC102549726 was inferenced or BBR was administered.Flow cytometry results showed that the apoptosis rate of podocytes increased in the model group and the LncRNA LOC102549726 overexpression group,and decreased in the silent group and the drug administration group.Transwell results showed that the podocyte adhesion ability was decreased in the model group and the LncRNA LOC102549726 overexpression group,while the podocyte adhesion ability was increased in the silent group and the administration group.The improvement of podocyte injury by BBR may be related to the LncRNA LOC102549726.CONCLUSIONS1.LncRNA LOC102549726 was abnormally increased in both renal tissue and HG-induced podocytes of DN rats.2.LncRNA LOC102549726 regulates injury of DN podocytes by targeting EGF.3.BBR may play a protective role in podocytes by regulating LncRNA LOC102549726 and its related target protein.