| Objective: The incidence and mortality of diabetes mellitus are increasing year by year in China.Diabetic Peripheral Neuropathy(DPN)is the most common chronic complication of diabetes.Schwann cells are the main supporting cells of peripheral nerves which the autophagy function are inhibited in DPN.However,the exact mechanisms is still unclear.Rab5(Ras-related protein 5)protein is a kind of regulatory small molecule GTP-binding protein in plasma membranes and organelle membranes,which can regulate cell autophagy.In order to investigate whether Rab5 is involved in the autophagy inhibition of diabetic Schwann cells,diabetic mice and human Schwann cell HSC(Human Schwann cell line)cultured in vitro were used to investigate the effect of Rab5 on Schwann cell autophagy.Methods:1.The expression of Rab5 in sciatic nerve of diabetic miceThe male CD1 mice were randomly divided into normal control group(Control group)and diabetes mellitus group(diabetic mellitus,DM group).Diabetic mice models were induced by an intraperitoneal injection of streptozotocin(STZ,150mg/kg).At the age of eight weeks,all animals were sacrificed.The sciatic nerve was isolated and fixed in 4% paraformaldehyde for immunohistochemistry.Immunohistochemistry was used to detect Rab5 expression in sciatic nerve.2.The effect of Rab5 expression on autophagy in vitro-cultured Schwann cellsHuman Schwan cells HSC were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO2 incubator at 37?.(1)In order to determine the effects of high glucose on the Rab5 expression and autophagy : HSC cells were divided into three groups: normal glucose group(N),mannitol group(D-Mannitol,M)and high glucose group(H).Cells were collected after 24 h,48 h and 72 h stimulation respectively.The expression of Rab5,P62 and LC3 in HSC cells were detected by Western blot and immunofluorescence.(2)To investigate the direct effect of Rab5 knockdown on autophagy: HSC cells were transfected with p Genesil-1-Rab5 or p Genesil-1 using Lipofectamine 2000.HSC cells were randomly grouped: normal control group(normal glucose,Blank),negative control group(normal glucose+p Genesil-1,Vehicle)and Rab5 sh RNA transfection group(normal glucose+p Genesil-1-Rab5,Rab5 sh RNA).After 48 h of transfection,the transfection efficiency was observed by fluorescence microscopy.Western blot was used to detect the expression of LC3 and P62.3.Effects of NF-?B signaling pathway on Rab5 expression and autophagy in high glucose-stimulated HSC cells(1)To detect the effect of inhibition of NF-?B pathway on autophagy of HSC cells induced by high glucose: cells were randomly divided into high glucose control group(high glucose,H),high glucose+DMSO group(high glucose+DMSO,H+DMSO)and high glucose+SN50 group(high glucose+SN50,H+SN50).After 48 h of SN50 stimulation,cells were collected.Western blot and immunofluorescence were used to detect the expression of Rab5,LC3 and P62.(2)To detect the effect of NF-?B pathway on autophagy of high glucose-stimulated HSC cells after Rab5 knockdown: HSC cells were divided into normal glucose(N),high glucose(H),high glucose+SN50 group(high glucose+SN50,H+SN50)and high glucose+SN50+p Genesil-1-Rab5 group(high glucose+SN50+p Genesil-1-Rab5,H+SN50+Rab5 sh RNA).Western blot was used to detect the expressions of Rab5,LC3 and P62 protein.Results:1.The expression of Rab5 in sciatic nerve of diabetic miceThe results of Immunohistochemistry showed that Rab5 was mainly expressed in the cytoplasm of Schwann cells which formed myelin sheath of sciatic nerve.Compared with normal mice,the expression of Rab5 in sciatic nerve tissue of diabetic mice was decreased;2.The effect of Rab5 expression on autophagy in vitro-cultured Schwann cells(1)The results of Western blot showed that Rab5 protein in the high glucose group was decreased by 57.76% and 62.43% respectively compared with the mannitol group at 48 h and 72 h(P<0.05).Immunofluorescence results showed that Rab5 was highly expressed in the cytoplasm of HSC cells in the normal control group,but the expression of Rab5 was markedly reduced in the cytoplasm of HSC cells under high glucose stimulation.Under the stimulation of high glucose,the ratio of LC3?/LC3 ?was down-regulated,and the expression of P62 was increased.The results of Western blot showed that compared with the mannitol group,the ratio of LC3?/LC3 in the high ?glucose group were decreased by 55.92% and 52.85% respectively at 48 h and 72 h,and the expression of P62 were up-regulated by 2.37 times and 2.86 times(P < 0.05).The results of immunofluorescence showed that: under the stimulation of high glucose,the fluorescence intensity of LC3 was decreased in HSC cells and the fluorescence intensity of P62 was increased.(2)Western blot results showed that the expression of Rab5 protein in HSC cells transfected with Rab5 sh RNA was decreased.Compared with the negative control group,the expression of Rab5 in Rab5 sh RNA transfected group was reduced by 64.58%,the ratio of LC3?/LC3?was decreased by 74.38%,but the expression of P62 was up-regulated by 2.98 times(P <0.05);3.Effects of NF-?B ignaling pathway on Rab5 expression and autophagy in high glucose-stimulated HSC cells(1)The results of western blot showed that SN50 could significantly reverse the down-regulation of Rab5 expression induced by high glucose.The expression of Rab5 in the high glucose + SN50 group was 2.13 times higher than that in the high glucose + solvent control group.Inhibition of NF-?B signaling pathway increased autophagy.The ratio of LC3?/LC3 in the high ?glucose+SN50 group was 1.65 times higher than that in the high glucose + solvent control group,while the expression of P62 was decreased by 65.82%.The difference was statistically significant(P<0.05).Immunofluorescence results showed that Rab5 and LC3 expression were increased,while P62 expression was decreased in the high glucose + SN50 group.(2)To detect the effect of NF-?B pathway on autophagy of HSC cells stimulated by high glucose after Rab5 knockdown.Western blot results showed that compared with the high glucose +SN50 group,Rab5 expression in high glucose + SN50 + p Genesil-1-Rab5 group was reduced by 45.62%.The ratio of LC3?/LC3 ?was inhibited by 51.63% and the expression of P62 was increased by 3.44 times.The difference was statistically significant(P <0.05).Conclusions:1.The expression of Rab5 in sciatic nerve of diabetic mice and high glucose-cultured HSC cells was decreased,accompanied by the decrease of autophagy.It is suggested that Rab5 may be involved in cell damage caused by decreased autophagy of diabetic Schwann cell.2.Downregulation of Rab5 by sh RNA in normal glucose-cultured HSC cells lead to autophagy reduction in Schwann cells.It is suggested that high glucose possibly induce HSC cells autophagy reduction by reduce the expression of Rab5.3.Sn50 reversed the decrease of Rab5 expression and autophagy in HSC cells cultured in high glucose by inhibiting the activation of NF-? B signaling pathway;meanwhile,knockdown of Rab5 gene expression in HSC cells under high glucose condition could inhibit the enhancement of autophagy in HSC cells stimulated by sn50,It is suggested that NF-? B signaling pathway may be involved in cell damage caused by decreased autophagy of Schwann cells stimulated by high glucose by inhibiting the expression of Rab5. |
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