Study On The Mechanism Of Sericin Regulating Podocyte Injury Induced By High Glucose Through MiR-346

Diabetic nephropathy(DN)is one of the most common microvascular complications of diabetic(DM).The pathogenesis of DN is related to the imbalance of glucose metabolism,oxidative stress,inflammation and other factors,but it is not completely clear now.Podocyte is an important component of glomerular filtration barrier.Podocyte damage can lead to proteinuria and play an important role in the pathogenesis of DN,so many scholars consider that DN is "the disease of podocyte".MicroRNA(miRNA)is a kind of endogenous non-coding single-stranded small molecule RNAs.Mi RNAs are widely involved in the development,cell differentiation,cell proliferation,cell apoptosis and immune response of eukaryotic organisms.In recent years,it has been found that the abnormal expression of miRNAs are involved in the occurrence and development of DN.Among these miRNAs,miR-346 is a newly discovered miRNA,which involves in many diseases,not only plays important role in the inflammatory response,but also its expression abnormal changes in DN.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 and its molecular weight is 10-300 kd.Moreover,the sericin is biocompatible and biodegradable.According to the records of Compendium of Materia Medica,combined with the prescription of folk cocoon soaking in boiling water to control blood glucose,our research group found that sericin can effectively reduce blood glucose,regulate the disorder of blood lipid metabolism and protect kidney damage in type 2 diabetic mellitus rats,but the mechanisms of sericin protecting kidney is still not clear now.In this study,the high glucose-induced mouse podocytes model was established and intervened with sericin.The miR-346 and inflammatory factorIL-6 expression were observed in injured podocytes and the podocytes were transfected with miR-346 inhibitor,so to comprehensively analyze whether sericin has protective effects on podocytes injury by regulating miR-346 and provide new ideas for DN prevention and control.PART ONEEffects of sericin on the expression of Nephrin,IL-6 and miR-346 inpodocytes stimulated by high glucoseObjective:The high glucose-induced mouse podocytes model was established and intervened with sericin.To investigate whether sericin has protective effects on podocytes injury by observing changes of Nephrin protein,miR-346 and IL-6 expression;And determine the optimal dose of sericin for protection.Methods:1 Conditional immortalized mouse podocytes were cultured and proliferated in 33?,5% carbon dioxide incubator.Then,the podocytes were cultured in 37?,5% carbon dioxide incubator to logarithmic growth period.The mice podocytes were cultured with 30mmol/L glucose for 48 h were taken as high glucose control group(HG group).Meanwhile,this study set up normal control group(NG group: 5.5mmol/L glucose),hyperosmotic control group(MG group: 5.5mmol/L glucose + 24.5mmol/L mannitol);and sericin intervention group(LS group: 30mmol/L glucose + 150?g/ml sericin,MS group: 30mmol/L glucose + 300?g/ml sericin,HS group: 30mmol/L glucose +600?g/ml sericin).The podocytes in each group were treated synchronously for 12 h and then treated with different drugs for 48 h respectively.2 The morphological structure of podocytes cultured at 33°C,37°C,as well as the morphological structure of podocytes in each group were observed under inverted microscope.3 Western blot was used to detect the expression of podocyte surface marker Nephrin protein.4 Real time PCR was used to detect the expression of miR-346.5 ELISA and real time PCR were used to detect IL-6 protein and m RNA expression respectively.Results:1 Morphological structure of podocytes1.1 Morphological structure of podocytes at different culture temperatures:Podocytes cultured at 33? were in the state of proliferation and in the shape of "paving stone",they grew faster with small size and small intercellular space,and the foot process could not be observed.The podocytes cultured at 37? were in the state of differentiation.Compared with the podocytes at 33?,the cell body gradually enlarged and formed foot process,which connected to each other between neighbouring podocytes in the form of "tree branches".1.2 Morphological structure of podocytes in each group:The morphological structure of podocytes in NG group was the podocytes morphology at 37°C culture condition.The morphological structure of podocytes in MG group was similar with the podocytes in NG group.In HG group,the podocytes shrank and became round,the foot processes decreased and even disappeared,the intercellular space enlarged,even the podocytes fell off and died.Compared with the podocytes in HG group,the podocytes in LS group,MS group and HS group became larger,foot processes became obvious and the intercellular space became smaller;the morphological structure of podocytes in theses 3 groups were closer to the morphological structure of NG group.2 The expression of Nephrin in podocytes of each group:There was no significant difference in the expression of Nephrin protein between MG group and NG group(P>0.05).The expression of Nephrin protein in HG group was significantly lower than that in NG group(P<0.05).Compared with HG group,the expression of Nephrin protein in podocytes of LS group,MS group and HS group were significantly higher,of which HS group was the highest(P<0.05).3 The expression of miR-346 in podocytes of each group:There was no significant difference in the expression of miR-346 among NG group,HG group and MG group(P>0.05).The expression of miR-346 in LS group,MS group and HS group was significantly higher than that in NG group,HG group and MG group,of which HS group was the highest(P<0.05).4 The expression of IL-6 in podocytes of each group:There was no significant difference in the expression of IL-6 protein between MG group and NG group(P>0.05).The expression of IL-6 in HG group was significantly higher than that in NG group(P<0.05).Compared with HG group,the expression of IL-6 in podocytes of LS group,MS group and HS group were significantly lower,of which HS group was the lowest(P<0.05).The change trend of IL-6 m RNA expression in podocytes was the same as that of IL-6 protein in each group.PART TWOWhether sericin protects podocytes injury induced by high glucoseby regulating miR-346Objective:The miR-346 inhibitor was transfected into podocytes and the podocytes were intervened with 600?g/ml sericin.To comprehensively analyze whether sericin has protective effects on podocytes injury induced by high glucose by regulating miR-346.Methods:1 The podocytes were grouped as following: normal glucose group(NG group: 5.5mmol/L glucose),high glucose control group(HG group: 30mmol/L glucose),sericin intervention group(SI group: 30mmol/L glucose + 600?g/ml sericin),Sericin + miR-346 inhibitor group(miR-346 inhibitor group:30mmol/L glucose + 600?g/ml sericin + Micr OFF mmu-miR-346-5p inhibitor)and sericin + miR-346 control group(miR-346 control group: 30mmol/L glucose + 600?g/ml sericin + Micr OFF mmu-miR-346-5p control).2 ELISA and Real time PCR were used to detect IL-6 protein and m RNA expression respectively.Results:1 The expression of IL-6 in podocytes of each group:There was no significant difference in the expression of IL-6 protein among NG group,SI group and miR-346 control group(P>0.05).There was no significant difference in the expression of IL-6 protein between HG group and miR-346 inhibitor group(P>0.05).The expression of IL-6 protein in NG group was significantly lower than HG group and miR-346 inhibitor group(P<0.05).Compared with HG group and miR-346 inhibitor group,the expression of IL-6 protein in podocytes of SI group and miR-346 control group were significantly lower(P<0.05).The change trend of IL-6 m RNA expression in podocytes was the same as that of IL-6 protein in each group.Conclusions:1 High glucose can cause podocyte injury,which is related to inflammatory reaction.2 Sericin has protective effects on podocytes injury by high glucose,and the effects of 600?g/ml sericin is the best;the mechanism may be related to sericin can increase the expression of miR-346 and inhibit inflammatory reaction.3 Sericin can reduce inflammatory reaction by upregulating the expression of miR-346,thus protect podocytes from injury induced by high glucose.