| Diabetic Nephropathy(DN)is one of the common complications of Diabetes Mellitus(DM).Its occurrence and progression are related to many factors,such as stimulation of high glucose environment,changes in hemodynamics,and involvement of inflammatory factors.Our team's previous study found that the expression of inflammatory factors IL-6 and IL-18 was up-regulated by detecting blood,urine and kidney tissue samples from patients with DN.It was speculated that inflammation participates in the development of DN;by testing blood,urine and kidney tissue samples of model animals and culturing immortalized mouse podocytes in high glucose environment,it was verified in vivo and in vitro that inflammatory factors were involved in the process of podocyte injury.MicroRNAs(miRNAs)are non-coding RNAs that are post-transcriptional endogenous regulators that target complementary mRNA sequences,promote mRNA degradation or interfere with translation.In order to find new biomarkers related to the development of DN,our team used gene chip technology to screen miRNAs in kidney tissue and found that the expression of miRNA138(miR-138)was significantly increased.It has been reported in the nervous system that miR-138 binds to the 3'UTR region of SIRT1 and regulates the expression of SIRT1.Our team used the dual luciferase reporter gene,and the results showed that miR-138 binds to SIRT1in kidney tissue samples from DN patients,and it is concluded that miR-138 may exert biological effects through SIRT1.Mitogen-Activated Protein Kinase(MAPK)is a type of enzyme widely present in mammals.Extracellular signals or stimuli may activate MAPK,including ERK,JNK,p38,etc.wherein phosphorylation of p38 plays a key role in cellular activities.Some scholars have found that the use of lipopolysaccharide(LPS)to stimulate rat macrophages leads to phosphorylation of p38 in macrophages,and inhibition of phosphorylation of p38 can alleviate or even completely block the production of TNF-?in macrophages.This indicates that the phosphorylation level of p38 is closely related to its inflammatory response.The results of our team's previous experiments showed that the expression of SIRT1 in podocytes cultured in high glucose decreased,and the phosphorylation level of p38 increased;the phosphorylation level of p38 was also increased after treatment with SIRT1 specific siRNA in podocytes cultured at normal glucose concentration.Tristetraprolin(TTP)is an AU-enriched region binding protein that mediates the degradation of mRNA and regulates the expression of post-transcriptional proteins.While degrading inflammatory factor mRNA,TTP protein itself is also degraded.Therefore,TTP can be considered as an anti-inflammatory protein.Decreased expression of TTP leads to an increase in the expression of inflammatory factors,which causes the podocytes to remain in an inflammatory state for a long time,losing normal morphology and function.P38 is one of the important enzymes currently known to phosphorylate TTP and affect the expression level of TTP protein.Based on the above theoretical and experimental results,we hypothesized that high glucose in diabetic nephropathy promotes the expression of miR-138,affects the expression of Sirt1,and then affects the expression of TTP through P38-MAPK,and participates in the inflammatory reaction of diabetic nephropathy and then causes podocyte injury.To validate the hypothesis proposed,this project intends to use molecular biology,cell biology experimental techniques,through the use of biological specimens of patients with diabetic nephropathy,cultured immortalized mouse podocytes and diabetic nephropathy animal model db/db mice,from the organization level,cell level,and overall animal level,explored the mechanism by which miR-138 regulates TTP via Sirt1/p38 and participates in the pathogenesis of diabetic nephropathy.Part I Expression of miR-138 and IL-18 in blood samples of patients with diabetic nephropathyObjectiveThe expression of miR-138 and IL-18 in the blood samples of DN patients and the morphology of renal tissue samples from DN patients were observed.MethodsA total of 88 subjects were selected from March 2016 to September 2017 in our hospital.Divided into four groups,diabetes group(DM),UAER<30mg/24h,a total of 18 cases;DN microalbuminuria group(DN1),30 mg/24 h?UAER<0.5 g/24 h,13 cases;DN macroproteinuria group(DN2),UAER>0.5g/24h,17 cases;a healthy control group with age and sex matched,40 cases.Collect blood and urine samples from patients.In the same period,30 cases of renal tissue specimens were collected,including 18 cases of DN group(15 cases of massive proteinuria,3 cases of microalbuminuria),3 cases of DM group and 9 cases of healthy control group(Control,Con).Blood and urine specimen collection:The 24-hour urine protein excretion rate,serum creatinine and other clinical indicators were compared among different groups.The pathological changes of the kidney and the swelling of ultrastructural mitochondria were observed under light microscope and electron microscope.The expression of miR-138 and SIRT1 in renal tissue samples from DN patients was detected by dual luciferase reporter gene.Results1.Compared with DM and Con group,UAER,Scr and Glu in DN1 group and DN2 group were higher,the difference was statistically significant(P<0.05).2.The results of qRT-PCR showed that IL-18,Desmin and miR-138 were higher in DN1 group and DN2 group than in DM group and Con group,the difference was statistically significant(P<0.05).3.PAS results showed that compared with DM and Con group,DN1 group and DN2 group had increased mesangial matrix and thickened glomerular basement membrane.4.Masson staining results showed that glomerular interstitial fibrosis and sclerosis were higher in DN1 group and DN2 group than in DM group and Con group.5.Electron microscopy showed that DN1 group and DN2 group had glomerular foot process fusion and mitochondrial swelling compared with DM and Con group.6.The dual luciferase reporter gene results showed that miR-138 binds to the3'UTR of SIRT1.Conclusions1.The expression of miR-138 is increased in patients with diabetic nephropathy.2.miR-138 may cause renal fibrosis,cirrhosis and podocyte mitochondrial swelling in DN patients through inflammatory factors.Part II Mechanism of miR-138 in db/db mice induced by glomerular podocyte injury by SIRT1/TTP and its shRNA repair of podocytesObjectiveTo observe the mechanism of miR-138 in the model animal db/db mice to induce podocyte injury by SIRT1/TTP;miR-138 shRNA was injected into the tail vein to observe whether the glomerular podocytes of db/db mice were improved.MethodsWhen db/db mice grow to 5-6 weeks of age,spontaneous blood glucose rises,DM symptoms appear,proteinuria and renal function damage occur at 8-10 weeks of age,and DN symptoms appear.A total of 60 db/db mice were purchased and divided into three groups:db/db group,20 mice;in the db/db+vehicle group,20 mice were given a tail vein injection at 10 weeks of age and 14 weeks of age to give an unrelated sequence of shRNA of miR-138 at a dose of 40 ul and a titer of 2×10~7/per;the db/db+LV group,ie miR-138 shRNA group,20 mice were treated with miR-138shRNA tail vein injection at 10 weeks and 14 weeks of age,with an injection dose of40 ul and a titer of 2×10~7/per.Twelve control db/m mice of the same age were matched,that is,the control group.Blood and urine samples were collected at different time points,and the mice were sacrificed and their kidney tissue samples were collected.Western blot,immunohistochemical(IHC),immunofluorescence(IF),PAS,electron microscopy and other observations were used.Results1.When miR-138 shRNA was not injected,the blood glucose,body weight,urine protein and creatinine in the db/db group were higher than those in the db/m group at the same time,and the difference was statistically significant;compared with the db/m group in the same period,the expression of SIRT1 and Desmin in the db/db+veh-group was lower,and the difference was statistically significant(P<0.05).2.After miR-138shRNA was injected into the tail vein,the expression of nephrin was increased and the expression of Desmin was decreased in the db/db+LV group compared with the db/db+veh-group,and the difference was statistically significant;compared with the db/db group at the same age,the expression of nephrin in the db/db+LV group was increased,and the difference was statistically significant(P<0.05).3.IHC,IF,PAS and electron microscopy showed that compared with the db/db group and db/db+veh-group,the degree of glomerular fibrosis and hardening of db/db+LV group was reduced,and the degree of podocyte swelling was alleviated.Conclusions1.miR-138 may down-regulate the expression of SIRT1,phosphorylate p38 and inhibit TTP,leading to glomerular podocyte injury in db/db mice.2.Intravenous injection of miR-138 shRNA can increase the expression of SIRT1 and improve glomerular fibrosis and mitochondrial swelling of podocytes.Part III High-glucose cultured podocyte miR-138 induces podocyte injury by SIRT1/p38/TTP and its shRNA and SRT1720 repair podocyte mechanismObjectiveTo observe the mechanism of miR-138 in podocytes cultured in high glucose-induced podocyte injury by SIRT1/p38/TTP and the repair of podocytes by miR-138 shRNA and SIRT1 agonist SRT1720.MethodsDifferentiated mature podocytes were divided into three groups:normal glucose concentration group(5.6 mmol/L glucose),hypertonic control group(5.6 mmol/L glucose+44.4 mmol/L mannitol),and high glucose group(30 mmol/L glucose).The expression levels of various indicators under high glucose conditions were measured.The high glucose group was transfected with miR-138 shRNA,the high glucose group was transfected with SIRT1 agonist SRT1720,and the normal glucose concentration group was transfected with SIRT1 inhibitor EX527 to observe podocyte repair.Results1.High-glucose cultured podocytes were transfected with miR-138 shRNA,which increased the expression of podocyte marker proteins Nephrin,TTP and SIRT1,and decreased the expression of podocyte damage proteins Desmin,IL-18 and TNF-?.The difference was statistically significant(P<0.05).2.IF showed a decrease in SIRT1 and TTP expression in the high glucose group;SIRT1 and PI are co-localized in the nucleus and TTP is expressed in the cytoplasm;transfection of miR-138 shRNA can increase the expression of TTP and SIRT1 and promote the translocation of SIRT1;transfection of SRT1720 in the high glucose group increased its expression and promoted the translocation of SIRT1.3.High glucose cultured podocytes transfected with SRT1720 can increase podocyte marker protein Podocin and TTP expression;podocytes cultured at normal glucose concentration,transfected with SIRT1 inhibitor EX527,can reduce the expression of podocyte marker proteins Podocin and TTP.Conclusions1.High glucose causes damage to podocytes.2.The expression of miR-138 in podocytes cultured in high glucose is up-regulated,which may lead to podocyte injury through SIRT1/p38/TTP mechanism.Transfection of miR-138 shRNA and SRT1720 can effectively alleviate injured podocytes. |
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