| With the improvements of people’s living standard and changes of life style,the incidence of diabetes mellitus is rising gradually around the world.As one of the major microvascular complications of diabetes mellitus,the incidence of diabetic nephropathy(DN)is also rising.The main manifestations of renal damage in early stage of DN is microalbuminuria,which suggests that the glomerular filtration barrier is impaired.In the three layers of glomerular filtration barrier,the podocytes play important role in maintaining the completeness of glomerular filtration barrier and preventing proteinuria.Impairing of podocytes may involve the development of DN and affect the prognosis of DN patients.c-Jun N-terminal kinase(JNK)signal transduction pathway participates in various physiological processes and pathological processes,such as cell cycle,reproduction,apoptosis and stress.Activation of JNK signal transduction pathway can accelerate injury and apoptosis of podocyte,and damage the integrity of glomerular filtration barrier.As a global health problem,DN brings serious economic burden to the patients’ families and the society.In the view of the toxic and side effects of chemical synthetic drugs,it is necessary to explore a drug that can reduce blood glucose and protect kidney with reliable therapeutic effects,low cost and less adverse reactions.Therefore,researchers try to find a natural substance.Sericin(water extract of silkworm cocoon)is a high-molecular water-soluble protein that wraps the periphery of silk fibroin.According to Compendium of Materia Medica and folk prescription of silkworm cocoon bubble water assistanting hypoglycemic,our research group found that sericin could reduce the blood glucose effectively and could protect kidney injury during diabetes mellitus.However,the specific mechanisms of sericin protecting renal injury in diabetes mellitus needs to be further explored.This study intends to investigate whether sericin has the protective effects on podocytes by regulating JNK signaling transduction pathway using high glucose injuried podocytes’ model.In order to provide data supports for the mechanisms research of sericin to protect renal injury in diabetes mellitus and provide new ideas for prevention and treatment of DN in clinic.Objective:To investigate whether sericin has the protective effects on high glucose injuried podocytes by regulating JNK signaling transduction pathway through observing the expression of JNK signaling transduction pathway correlating factors,such as mitogen-activated protein kinase kinase kinase 1(MEKK1),mitogen-activated protein kinase kinase 4(MKK4)and c-Jun NH2-terminal kinases(c-Jun).Methods:1.Podocyte culture,grouping and treatment: 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 podocytes were divided into the following 6 groups: normal sugar group(NG: 5.5 mmol/L glucose);high sugar group(HG: 30 mmol/L glucose);mannitol hypertonic control group(MG: 5.5 Mmol/L glucose + 24.5mmol/L mannitol);sericin treatment low dose group(LS: 30mmol/L glucose +150μg/L sericin);sericin treatment medium dose group(MS: 30mmol/L glucose + 300μg/L sericin);sericin treatment high dose group(HS: 30mmol/L glucose + 600μg/L sericin).The podocytes in each group were used for experiments after 48 hours of different drug effects.The podocytes in each group were treated synchronously for 12 h and then treated with different drugs for 48 h respectively.2.Observation of morphological structure of podocytes: 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 nephrin,MEKK1,MKK4,JNK1 and c-Jun protein in podocytes.4.Real-time quantitative PCR(Real Time PCR)was used to detect the expression of nephrin,MEKK1,MKK4,JNK1 and c-Jun m RNA in podocytes.5.Apoptosis of podocytes was detected by flow cytometry using Annexin V/PI double staining.Results:1.Morphological structure of podocytes1.1 Morphological structure of podocytes cultured at different temperature At 33°C culture condition,the podocytes was in proliferation state and grew faster;the podocytes was small and "cobblestone-like".At 37°C culture condition,the podocytes turned into the state of differentiation;the cell body gradually enlarged and formed foot process,which connected to each other in the form of "tree branches".The podocytes were fully matured in about 10-14 days at 37°C.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.The podocytes in HG group became small;and the foot process fined,fused and disappeared;the space between adjacent cells became larger;with timing the podocytes gradually shed and suspension cells increased obviously.The podocytes in LS,MS,HS groups were fairly larger compared with the podocytes in HG group;the space between adjacent cells became narrow;the suspension cells and exfoliated became less;the morphological structure of podocytes tended to be normal.2.Nephrin expression in podocytes of each group Compared with NG group,the nephrin protein and m RNA expression in podocytes of HG group decreased significantly(P<0.05);There was no significant difference between about nephrin protein and m RNA expression between MG group and NG group(P>0.05).Compared with HG group,the nephrin protein and m RNA expression in podocytes of LS group,MS group and HS group increased gradually(P<0.05);And the pairwise comparison was statistically significant about the nephrin protein and m RNA expression among the three groups(P<0.05).3.MEKK1 expression in podocytes of each group Compared with NG group,the MEKK1 protein and m RNA expression in podocytes of HG group increased significantly(P<0.05);There was no significant difference between about MEKK1 protein and m RNA expression between MG group and NG group(P>0.05).Compared with HG group,the MEKK1 protein and m RNA expression in podocytes of LS group,MS group and HS group decreased gradually(P<0.05);And the pairwise comparison was statistically significant about the MEKK1 protein and m RNA expression among the three groups(P<0.05).4.MKK4 expression in podocytes of each group Compared with NG group,the MKK4 protein and m RNA expression in podocytes of HG group increased significantly(P<0.05);There was no significant difference between about MKK4 protein and m RNA expression between MG group and NG group(P>0.05).Compared with HG group,the MKK4 protein and m RNA expression in podocytes of LS group,MS group and HS group decreased gradually(P<0.05);And the pairwise comparison was statistically significant about the MKK4 protein and m RNA expression among the three groups(P<0.05).5.JNK1 expression in podocytes of each group Compared with NG group,the JNK1 protein and m RNA expression in podocytes of HG group increased significantly(P<0.05);There was no significant difference between about JNK1 protein and m RNA expression between MG group and NG group(P>0.05).Compared with HG group,the JNK1 protein and m RNA expression in podocytes of LS group,MS group and HS group decreased gradually(P<0.05);And the pairwise comparison was statistically significant about the JNK1 protein and mRNA expression among the three groups(P<0.05).6.c-Jun expression in podocytes of each group Compared with NG group,the c-Jun protein and m RNA expression in podocytes of HG group increased significantly(P<0.05);There was no significant difference between about c-Jun protein and m RNA expression between MG group and NG group(P>0.05).Compared with HG group,the c-Jun protein and m RNA expression in podocytes of LS group,MS group and HS group decreased gradually(P<0.05);And the pairwise comparison was statistically significant about the c-Jun protein and m RNA expression among the three groups(P<0.05).7.Apoptosis of podocytes in each group:The early apoptosis rate of podocytes in NG group was(6.9±0.62)%;the early apoptosis rate of podocytes in HG group and MG group was(37.53±0.49)% 、(11.66±1.11)%,which was significantly higher than NG group(P<0.05).The early apoptosis rate of podocytes in LS group,MS group and HS group were(28.34±1.02)%,(21.17±0.84)% and(16.27±0.45)%respectively,which were all significantly lower than HG group(P<0.05);And the pairwise comparison was statistically significant about the early apoptosis rate among the three groups(P<0.05).Conclusions:Sericin can inhibit activation of JNK signal transduction pathway and reduce apoptosis of podocytes by downregulating the expression of JNK pathway correlating factors MEKK1,MKK4,JNK1 and c-Jun,so to lighten podocyte damaging and has protective effects on podocytes. |
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