| Diabetic nephropathy(DN)is a kind of chronic progressive nephropathy,which is one of the most important renal microvascular lesions caused by diabetes mellitus(DM).It will eventually develop into end-stage renal disease(ESRD).Microalbuminuria is the main clinical manifestation of DN patients in the early stage.Many studies have confirmed that under the stimulation of hyperglycemia,oxidative stress and the release of inflammatory factors,glomerular podocytes will initiate the process of epithelial-mesenchymal transdifferentiation(EMT).At this time,the podocytes attached to the outer surface of glomerular basement membrane will fall off,and the integrity of glomerular filtration membrane will be destroyed,resulting in albuminuria.Therefore,it is suggested that reducing podocyte EMT can delay the progress of DN.Micro-ribonucleic acid(microRNA,miRNA)is a kind of non-coding small molecule RNA,with the length of about 18～22bp,which mainly regulates gene expression by binding to the 3’untranslated region(3’-UTR)of the target mRNA.The abnormal expression of miRNA plays an important role in the occurrence and development of DM and DN.Some studies have found that the miR-30 family(miR-30a～e)is significantly down-regulated in podocytes of DN patients.Up-regulation of miR-30 expression can target coupling protein 2 to inhibit EMT in renal tubular epithelial cells.Sericin is a high molecular weight water-soluble protein coated on the periphery of silk fibroin.It is mainly composed of 18 kinds of amino acids with a molecular weight of 10-300kd.It has medical effects such as antioxidation and anticancer.Previous studies of our group found that sericin can effectively reduce blood glucose and protect kidney damage during DM in type 2 diabetic rats,but the specific mechanism of sericin protecting kidney is not clear.PART ONEestablishment of mouse podocyte model injured by high glucose Objective:Identify whether the cultured mouse podocytes are mature or not,and determine whether the mouse podocyte model of injury induced by high glucose is established successfully by detecting the expression level of Nephrin on the surface of podocytes in each group.Methods:1 Identification of podocyte maturation at 37℃:podocytes cultured at33℃were cultured at 37℃for about 10-14 days without IFN-γ,the morphological changes of podocytes before and after treatment were observed under inverted microscope,and the localization and expression of Nephrin protein in podocytes cultured at 37℃for 3 days and 14 days were observed under positive fluorescence microscope to judge whether podocytes were mature or not.2 After the podocytes were differentiated and mature,the cells in the logarithmic growth phase were taken for experiment.1×10~5 podocytes per well were seeded in six-well plate,synchronized for 12 h,and treated with different media for 48 h.The podocytes are grouped as follows:(1)NG group(Normal group:5.5mmol/L D-glucose)(2)MG group(Hypertonic control group:5.5mmol/L D-glucose+24.5mmol/L mannitol)(3)HG group(High glucose injury group:30mmol/L D-glucose)3 Western blot was used to detect the expression of Nephrin protein in the podocytes of the above three groups.4 Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the expression of Nephrin mRNA in podocytes of the above three groups.Results:1.Morphological structure of podocytes1.1 Morphological structure of podocytes at different culture temperaturesIn the proliferative state at 33℃,the active podocytes were medium-sized,polygonal and without protruding processes,and the cells grew rapidly to the fusion state with a paving stone-like appearance,while at 37℃,the podocytes continued to grow after digestion and passage,and their cell bodies and nuclei were significantly larger than those in the proliferative state,with obvious dendritic processes protruding from the self-cell bodies,often binuclear,and a connection was formed between the adjacent cells.1.2 Changes of localization and expression of Nephrin protein in immature and mature podocytesAfter cultured at 37℃for 3 days,Nephrin protein was distributed in perinuclear and cytoplasmic podocytes,while Nephrin protein was distributed in perinuclear and cell membrane at 37℃for 14 days.2.Expression of Nephrin in podocytes of each groupThe results of Western blot and qRT-PCR showed that the expression of Nephrin protein and mRNA in podocytes in HG group was significantly lower than that in NG group(P<0.05).Summary:The proliferative podocytes cultured at 33℃were cultured at37℃for 14 days after IFN-γwas removed,and the mature podocytes differentiated and matured after being cultured at 37℃for 14 days,and the injury model induced by high glucose could be successfully established when mature podocytes were cultured in high glucose environment for 48 hours.PART TWOThe optimal concentration of sericin in protecting podocytes from mouse podocyte injury induced by high glucose.Objective:The activity of podocytes and the expression of Snai1 and miR-30a-5p in each group were detected to determine the best concentration of sericin to protect podocytes from podocyte injury induced by high glucose.Methods:1 The podocytes are grouped as follows:(1)NG group(Normal group:5.5mmol/L D-glucose)(2)HG group(High glucose injury group:30mmol/L D-glucose)(3)LS group(Low dose sericin group:30mmol/L D-glucose+150μg/ml sericin)(4)MS group(Medium dose sericin group:30mmol/L D-glucose+300μg/ml sericin)(5)HS group(High dose sericin group:30mmol/L D-glucose+600μg/ml sericin)2 The morphology of podocytes in the above five groups was observed under inverted microscope.3 The activity of podocytes in the above five groups was detected by CCK8 method.4 The expression of Snai1 protein in podocytes of the above five groups was detected by Western blot method.5 qRT-PCR technique was used to detect the expression of Snai1mRNA and miR-30a-5p in podocytes of the above five groups.Results:1.Morphological changes of podocytes in each groupIn NG group,the podocyte cell body and nucleus were larger,the self-cell body protruded obvious dendritic processes,often binuclear,and the adjacent cells formed connections;In HG group,there was no significant difference compared with NG group;After HG treatment,podocyte shrank and became round,foot process decreased and fused or even disappeared,cell morphology changed and gradually became"fusiform cell-like",and floated and exfoliated.Compared with HG group,the volume of podocytes in LS,MS and HS groups was larger,the foot process was more obvious,the number of exfoliated cells decreased,and the cell morphology tended to be normal.2.The vitality of podocytes in each groupThe podocyte activity in HG group was significantly lower than that in NG group(P<0.05).The podocyte viability in LS group,MS group and HS group was significantly higher than that in HG group(P<0.05),and there was significant difference in podocyte viability among LS,MS and HS groups(P<0.05).3.Expression of Snai1 in podocytes of each groupThe expression of Snai1 protein and mRNA in podocytes in HG group was significantly higher than that in NG group(P<0.05),while the expression of Snai1 protein and mRNA in podocytes in LS group,MS group and HS group was significantly lower than that in LS group,MS group and HS group(P<0.05).Moreover,the expression of Snai1 protein and mRNA in podocytes in HS group was significantly lower than that in LS group and MS group(P<0.05).4.Expression of miR-30a-5p in podocytes of each groupThe results of qRT-PCR showed that the expression of miR-30a-5p in podocytes in HG group was significantly lower than that in NG group(P<0.05),and the expression of miR-30a-5p in podocytes in LS group,MS group and HS group was significantly higher than that in LS group,MS group and HS group compared with HG group(P<0.05).In addition,the expression of miR-30a-5p in podocytes in LS,MS and HS groups was significantly higher than that in LS and MS groups(P<0.05).The expression of miR-30a-5p in podocytes in);HS group was significantly higher than that in LS and MS groups(P<0.05).Summary:1.Sericin has a protective effect on podocytes injured by high glucose.2.According to the activity,Snai1 and miR-30a-5p expression of podocytes in three sericin groups,the best concentration of sericin to protect podocytes against podocyte injury induced by high glucose was 600μg/ml.PART THREESericin retards the EMT process of mouse podocytes induced by high glucose by targeting Snai1 with miR-30a-5pObjective:After the optimal transfection concentration of miR-30a-5p inhibitor was determined,podocytes were transfected with this concentration,and 600μg/ml.Sericin was given to analyze whether sericin delayed the process of podocyte EMT stimulated by high glucose by regulating miR-30a-5p targeting Snai1.Methods:1 The recombinant plasmid and miR-30a-5p mimics/mimics NC were transfected into 293T cells and the double luciferase reporter gene was detected.2 The mature podocytes were transfected with miR-30a-5p inhibitor of0nM,100nM,150nM and 200nM,and the optimal concentration of miR-30a-5p inhibitor was determined by detecting the expression level of miR-30a-5p and Snai1.The number of fluorescent labeled genes on the plasmid of podocytes at this concentration was observed under inverted fluorescence inverted microscope to judge the transfection efficiency.3 The optimal concentration of miR-30a-5p inhibitor(150nM)was used to transfect differentiated mature podocytes for 4 hours,followed by 12hours of synchronization treatment and 48 hours of treatment with different drugs.The cell groups are as follows:(1)NG group(Normal group:5.5mmol/L D-glucose)(2)HG group(High glucose injury group:30mmol/L D-glucose)(3)HS group(Sericin intervention group:30mmol/L D-glucose+600μg/ml sericin)(4)miR-30a-5p inhibitor NC group(Sericin+30mmol/L D-glucose+miR-30a-5p inhibitor negative control group)(5)miR-30a-5p inhibitor group(Sericin+30mmol/L D-glucose+miR-30a-5p inhibitor group)4 Transwell test was used to detect the effect of sericin on the migration ability of podocytes in the above five groups.5 The protein expressions of Snai1,podocin,E-cadherin,ZO-1,FSP-1,α-SMA and Desmin in podocytes of the above five groups were detected by Western blot method.6 qRT-PCR technique was used to detect the expression of Snai1,podocin,E-cadherin,ZO-1,FSP-1,α-SMA,Desmin mRNA and miR-30a-5p in podocytes of the above five groups.Results:1.The results of double luciferase report showed that compared with Snai1-3’-UTR-WT+miR-30a-5p mimics NC group,Snai1-3’-UTR-MU+miR-30a-5p mimics group,Snai1-3’-UTR-MU+miR-30a-5p mimics NC group,pcmv-neo-Bam+miR-30a-5p mimics group and pcmv-neo-Bam+miR-30a-5p mimics NC group,the luciferase activity of Snai1-3’-UTR-WT+miR-30a-5p mimics group was significantly inhibited(P<0.05).2.Determination of the optimal concentration of podocytes transfected with miR-30a-5p inhibitorCompared with 0nM group,the expression of miR-30a-5p in podocytes in 150nM group was significantly lower and the expression of Snai1 protein was significantly higher than that in 200nM group(P<0.05),while the expression of miR-30a-5p and Snai1 protein in podocytes in 200nM group had no significant difference(P>0.05).Compared with 100nM group,the expression of miR-30a-5p in podocytes of 150nM group was significantly decreased and the expression of Snai1 protein was significantly increased(P<0.05).Four hours after the podocyte was transfected with the optimal transfection concentration 150nM miR-30a-5p inhibitor,three visual fields were selected under the inverted fluorescence microscope,and the ratio of the number of fluorescence labeled genes on the plasmid to the total number of cells in the podocytes was counted,and the transfection efficiency was about85%.3.Effect of sericin on podocyte migration in each groupThe results of transwell experiment showed that the number of ventricular podocytes under transwell in HG group was significantly higher than that in NG group(P<0.05),and the number of ventricular podocytes under Transwell in HS group was significantly lower than that in HG group(P<0.05),and the number of ventricular podocytes under transwell in miR-30a-5p inhibitor group was significantly higher than that in HS group(P<0.05).4.Expression of podocyte Snai1,epithelial phenotypic related factors podocin,E-cadherin,ZO-1 and interstitial phenotypic related factors FSP-1,α-SMA and Desmin in each group4.1 The expression of Snai1 in podocytes of each groupThe expression of Snai1 protein and mRNA in podocytes in HG group was significantly higher than that in NG group,the expression of Snai1 protein and mRNA in podocytes(P<0.05);HS group was significantly lower than that in HG group(P<0.05),and the expression of Snai1 protein and mRNA in podocytes in miR-30a-5p inhibitor group was significantly higher than that in HS group(P<0.05).4.2 Expression of podocin,E-cadherin and ZO-1 in podocytes of each groupThe expression of podocin,E-cadherin,ZO-1 protein and mRNA in podocytes in HG group was significantly lower than that in NG group,and the difference was statistically significant(P<0.05).The expression of podocin,E-cadherin,ZO-1 protein and mRNA in podocytes in HS group was significantly higher than that in HG group(P<0.05).The expression of podocin,E-cadherin,ZO-1 protein and mRNA in podocytes in miR-30a-5p inhibitor group was significantly lower than that in HS group(P<0.05).4.3 Expression of FSP-1,α-SMA and Desmin in podocytes of each groupThe expression of FSP-1,α-SMA,Desmin protein and mRNA in podocytes in HG group was significantly higher than that in NG group,and the difference was statistically significant(P<0.05).The expression of FSP-1,α-SMA,Desmin protein and mRNA in podocytes in HS group was significantly lower than that in HG group(P<0.05).The expression of FSP-1,α-SMA,Desmin protein and mRNA in podocytes of miR-30a-5p inhibitor group was significantly higher than that of HS group(P<0.05).Summary:1.Snai1 is one of the target genes of miR-30a-5p,and miR-30a-5p can inhibit the expression of Snai1.2.Sericin can up-regulate the expression of podocin,E-cadherin and ZO-1through miR-30a-5p targeted inhibition of Snai1,and down-regulate the EMT process and migration rate of podocytes stimulated by high glucose by down-regulating FSP-1,α-SMA and Desmin.Conclusions:1.The model of podocyte injury induced by high glucose was successfully established after high glucose treatment for 48 hours,and the best concentration of sericin to protect podocytes from podocyte injury induced by high glucose was 600μg/ml.2.Snai1 is one of the target genes of miR-30a-5p,and miR-30a-5p can inhibit the expression of Snai1.3.Sericin can up-regulate the expression of epithelial phenotype-related factors and down-regulate the expression of interstitial phenotype-related factors through miR-30a-5p targeted inhibition of Snai1,affect the EMT process of podocytes and reduce podocyte migration,thus play a protective role in podocytes injury induced by high glucose. |
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