Research On Regulation Of Podocyte EMT And Apoptosis In Diabetic Nephropathy By LncRNA LOC102549726 And The Effect Of Berberine

Diabetic nephropathy（DN）is a serious complication of diabetes,which can be caused by hyperglycemia,abnormal metabolism of blood lipids,microvascular circulation disorders and inflammatory reactions.The main pathological manifestations of DN were renal tubular fibrosis,glomerular basement membrane thickening,the proliferation of mesangial cells,extracellular matrix（ECM）proliferation and podocyte injury.Podocyte injury is a significant marker of DN.Studies have found that epithelial-mesenchymal transition（EMT）and apoptosis of podocyte play an important role in DN podocyte injury,and the occurrence of EMT and apoptosis of podocyte can dramatically reduce the number of podocytes.The function of podocyte was significantly reduced,resulting in the damage of glomerular filtration barrier,decreased glomerular filtration ability,proteinuria,aggravating DN.Long noncoding RNA（lncRNA）is a class of RNA with more than 200 base pairs in length and no protein-coding function,which can enhance or inhibit the expression of target genes through various ways and affect cell growth,apoptosis,migration and invasion.Epidermal growth factor（EGF）is a low molecular peptide,and activated EGF can stimulate cell proliferation,migration and EMT.Forkhead box O1（FOXO1）is an important transcription factor that regulates cell function.It regulates metabolism,oxidative stress,apoptosis,and inflammation by affecting the expression of multiple downstream transcription targets.Berberine（BBR）is a kind of quaternary ammonium alkaloid,which has the effects of lowering blood sugar,regulating blood lipids,inhibiting inflammation,anti-oxidation and kidney protection,and can treat DN,hypertension and other diseases.Our previous study found that lncRNA LOC102549726 was abnormally upregulated in DN renal tissues and podocytes,which may cause podocyte injury by upregulation of EGF expression.However,the mechanism of lncRNA LOC102549726 causes podocyte injury through EGF and the effect of BBR are still unclear,these need to be further explored.This study intends to clarify the specific mechanism of lncRNA LOC102549726/EGF signaling pathway in podocyte injury and the regulatory effect of BBR on it through in vitro and in vivo experiments.To investigate whether lncRNA LOC102549726 affects the expression of FOXO1 signaling pathway through EGF targeting,thus affecting EMT and apoptosis of DN podocytes and causing podocyte damage.And whether BBR alleviates podocyte EMT and apoptosis and alleviates podocyte injury by regulating the expression of lncRNA LOC102549726 and its related signaling pathways.OBJECTIVE1.To investigate the effects of lncRNA LOC102549726 and its related signaling pathways on EMT and apoptosis of DN podocytes;2.To investigate the regulation of BBR on lncRNA LOC102549726 and EMT and apoptosis of DN podocytes;3.To investigate whether BBR plays a protective role in DN rats by regulating lncRNA LOC102549726 related signaling pathways.METHODS1.QRT-PCR was used to detect the expression of lncRNA LOC102549726 in podocytes after podocytes were stimulated at different times with high glucose（HG）,and the optimal time point of up-regulation was selected for HG stimulation.2.Podocytes were divided into control group and HG group.The expression of lncRNA LOC102549726 in podocytes of each group was detected by FISH.The location of lncRNA LOC102549726 in podocyte was determined.3.After transfection of lncRNA LOC102549726 overexpressed plasmid and siRNA,qRT-PCR was used to detect the expression of lncRNA LOC102549726.Western Blot was used to detect the expression of EMT-related proteins（α-SMA,nephrin,vimentin）and apoptosis protein（Bim）in podocytes of each group.In each group of podocytes,transwell and scratching assay were used to detect the migration ability of podocytes,and flow cytometry was used to detect the apoptosis of podocytes.Use these ways to study the effect of lncRNA LOC102549726 on EMT and apoptosis of DN podocytes.4.After the siRNA of EGF was transfected,in each podocyte group,western Blot was used to detect the expression of EMT-related proteins（α-SMA,nephrin,vimentin）and apoptosis protein（Bim）,qRT-PCR was used to detecte the expression of EGF,transwell and scar assay were used to detect the migration ability of podocyte,and flow cytometry was used to detect podocyte apoptosis.Use the above methods to study the effect of EGF on EMT and apoptosis of DN podocyte.5.EGF-related signaling pathways were analyzed and verified by GO and KEGG pathway analysis software.In each group of podocytes,the protein expression of FOXO1,p-FOXO1,FOXO3,p-FOXO3,FOXO4 and p-FOXO4 was deteccted by western Blot,and the expression of EGF and FOXO1 was detected by qRT-PCR.6.After BBR administration at different concentrations,in each group of podocytes,CCK-8 was used to detect podocyte activity,qRT-PCR was used to detect the expression of lncRNA LOC102549726,western Blot method was used to detect the expression of EMT-related proteins（α-SMA,nephrin,vimentin）and apoptosis protein（Bim）,transwell and scratch assay were used to detect the migration ability of podocytes,and the apoptosis of podocytes was detected by flow cytometry.Use the above methods to select the optimal concentration of BBR.7.After optimal concentration BBR administration and silencing of lncRNA LOC102549726 expression,qRT-PCR was used to detect the expression of lncRNA LOC102549726,EGF and FOXO1 in podocytes of each group.Western Blot was used to detect the expressions of EMT-related proteins,apoptotic proteins and pathway proteins in podocytes of each group.In each group of podocytes,the migration ability of podocytes was detected by transwell and nicks assay,and flow cytometry was used to detect the apoptosis of podocytes.The effect of BBR on EMT and apoptosis of DN podocytes and lncRNA LOC102549726 and its related signaling pathy was finally studied.8.DN rat model was established,and 8 weeks after BBR administration,body weight,fasting blood sugar,FBG value,renal function index,renal pathological changes,glomerular ultrastructure changes and the expression of nephrin,α-SMA,vimentin,Bim,FOXO1,p-FOXO1,EGF and lncRNA LOC102549726 in renal cortex,to study the therapeutic effect of BBR on DN rats.RESULTS1.Selection of optimal time point for up-regulation of lncRNA LOC102549726 stimulated by HGQRT-PCR results showed that lncRNA LOC102549726 was most significantly up-regulated at 24h HG stimulation compared with the normal group,which could be used as the optimal time point to stimulate lncRNA LOC102549726.2.Localization of lncRNA LOC102549726 in podocyte FISH assay results showed that lncRNA LOC102549726 was highly expressed in podocyte compared with the control group,and was mainly expressed in the cytoplasm of podocyte.3.Effects of lncRNA LOC102549726 on DN podocyte EMT and apoptosisIn western Blot results,the protein expression of vimentin,α-SMA and Bim in the overexpressed lncRNA LOC102549726 group and HG group were increased,but decreased in the low interference lncRNA LOC102549726 group.The protein expression of nephrin was decreased in the overexpressed lncRNA LOC102549726 group and HG group,but increased in the low interference lncRNA LOC102549726 group.Compared with the control group,the scratch healing rate increased in the HG group and the overexpressed lncRNA LOC102549726 group.Compared with the HG group,the low interference lncRNA LOC102549726 group could reduce the abnormally increased scratch healing rate.Transwell results showed that the number of podocyte migrating to the lower ventricle increased in the HG group and the overexpressed lncRNA LOC102549726 group compared with the control group,while the number of podocyte migrating to the lower ventricle decreased in the low interference lncRNA LOC102549726 group compared with the HG group.And the apoptosis rate of podocyte increased in the HG group and the overexpressed lncRNA LOC102549726 group,and decreased in the low interference lncRNA LOC102549726 group.4.Effects of EGF on DN podocyte EMT and apoptosisWestern Blot results showed that the protein expression of α-SMA,vimentin and Bim in the HG group were increased,but decreased in the low interference EGF group.The nephrin expression in the HG group was decreased,but increased in the low interference EGF group.The results of scratch experiment showed the scratch healing rate increased in the HG group,and decreased in the low interference EGF group.Transwell results showed the number of podocytes migrating to the lower ventricle increased in the HG group,but decreased in the low EGF group.In results of flow cytometry,the HG group had a higher rate of podocyte apoptosis,and the low EGF group had a lower rate of podocyte apoptosis.5.Pathways involved in lncRNALOC102549726 and its target protein EGFGO analysis combined with KEGG pathway analysis showed that FOXO signaling pathway was associated with EGF.Western Blot results showed that p-FOXO1 protein expression increased in HG group,while p-FOXO3 and p-FOXO4 protein expression did not change significantly,while p-FOXO1 protein expression decreased in low interference EGF group,while p-FOXO3 and p-FOXO4 protein expression did not change significantly.qRT-PCR results showed that EGF mRNA expression was increased in the HG group and the overexpressed lncRNA LOC102549726 group,and FOXO1 mRNA expression was not significantly changed in the HG group,but decreased in the overexpressed lncRNA LOC102549726 group,while EGF mRNA expression was decreased and FOXO1 mRNA expression was increased in the low interference lncRNA LOC102549726 group.In western Blot results,the protein expression of p-FOXO1 was increased in HG group and the overexpressed lncRNA LOC102549726 group,and decreased in the low-interference lncRNA LOC102549726 group.6.Selection of the optimal concentration of BBR administrationCCK-8 showed that podocyte activity was decreased in HG group,and BBR（30μM）,BBR（60μM）and BBR（90μM）groups could effectively increase podocyte activity.QRT-PCR results showed that the expression of lncRNA LOC102549726 was increased in HG group,and the expression of lncRNA LOC102549726 was decreased in BBR（60μM）and BBR（90μM）groups.From western Blot results,it is known that in HG group,the protein expression of α-SMA,vimentin and Bim was increased,while the protein expression of nephrin was decreased.BBR（90μM）group can decrease the protein expression of α-SMA,vimentin and Bim,and increase the protein expression of nephrin.BBR（60μM）group can decrease the protein expression of Bim and increase the protein expression of nephrin,BBR（30μM）group can only decrease the protein expression of Bim.The results of scratch experiment showed the scratch healing rate was increased in HG group,and the scratch healing rate was decreased in BBR（30μM）,BBR（60μM）and BBR（90μM）groups.Transwell results showed that the number of podocytes migrating to the lower compartment increased in HG group,and decreased in the BBR（60μM）and BBR（90μM）groups.Flow cytometry results showed that the apoptosis rate of podocyte was increased in HG group,and only BBR（90μM）group could reduce the apoptosis rate of podocyte.7.BBR may be associated with lncRNA LOC102549726/EGF/FOXO1 signaling pathway in alleviating HG-induced podocyte EMT and apoptosisQRT-PCR results showed that BBR（90μM）group and small interference lncRNA LOC102549726 group could decrease the expression of lncRNA LOC102549726 and EGF mRNA,and increase the expression of FOXO1 mRNA.Western Blot results showed BBR（90μM）group and lncRNA LOC102549726 group could decrease the protein expression of α-SMA,vimentin,Bim and p-FOXO1,and increase the protein expression of nephrin.The results showed that BBR（90μM）group and lncRNA LOC102549726 group could reduce the scratch healing rate of podocytes.Transwell assay showed that BBR（90μM）group and lncRNA LOC102549726 group could reduce the number of podocytes migrating to the lower ventricle.Flow cytometry results showed that BBR（90μM）group and lncRNA LOC102549726 group could reduce the apoptosis rate of podocyte.8.Therapeutic effect of BBR on DN ratsThe results showed that BBR could decrease FBG,decrease renal function index,improve renal pathological changes,increase the protein expression of nephrin in renal cortex,decrease the protein expression of α-SMA,vimentin,Bim and p-FOXO1,and increase the mRNA expression of FOXO1 in renal cortex,decreased lncRNA LOC102549726 expression and EGF mRNA expression.CONCLUSIONS1.Abnormal upregulation of lncRNA LOC102549726 can affect EMT and apoptosis of DN podocytes.2.LncRNA LOC102549726 regulates EMT and apoptosis of DN podocytes by targeting EGF and affecting FOXO1 signaling pathway expression.3.BBR may relieve EMT and apoptosis of DN podocytes and improve podocyte injury by inhibiting lncRNA LOC102549726/EGF/FOXO1 signaling pathway,thus playing a protective role in kidney of DN rats.