Protective Effects Of SOCS3 On High Glucose-induced Injury In Lung Epithelial Cells Through The JAK2/STAT3 Pathway

BackgroundDiabetes mellitus was an epidemic diseases threatening the present world.Both microvascular and macrovascular disorder with debilitating a lot of organs were prevalent in diabetic patients.The alveolar-capillary network in the lung was a large microvascular unit and might be affected by microangiopathy.Evidences suggested that inflammation might play a crucial role in pathogenesis of diabetes.Therefore,the inhibition of inflammation could be a therapeutic strategy for treatment of diabetes.A number of inflammatory cytokines were shown to play an important role in the progression of diabetic lung injuries.Cytokines regulated many biological processes through the activation of intracellular signaling pathways.Most cytokines relayed biological signal to target cells by activating the Janus kinase(JAK)/signal transducers and activators of transcription(STAT)pathway.It had been reported that the JAK/STAT pathway regulates a wide range of genes involved in cell proliferation,inflammation,and fibrosis.It mightbe an important mechanism that hyperglycemia contributes to lung injuries associated with diabetes.This pathway was negatively regulated by various mechanisms including the suppressor of cytokine signaling(SOCS)proteins,which had been identified as classical inducible feedback inhibitors of cytokine receptors and had been shown to be of crucial importance for the limitation of inflammatory responses.SOCS was one of the most studied factors of recent years.The mammalian CIS/SOCS family was consists of 8 members,which included CIS and SOCS1 to SOCS7.Each of them shared a central SH2 domain,an amino-terminal domain of variable length and sequence,and a C-terminal SOCS box.There were more SOCS3 than SOCS1 in the lung.The SH2 domain of SOCS3 did not have a high affinity to the activation loop of JAKs yet the KIR of SOCS3 had a higher affinity to the kinase domain of JAK2 than that of SOCS1.Because the receptors to which SOCS3 bound mostly activate STAT3,SOCS3 was also an inhibitor that was relatively specific to STAT3.The influence of SOCS proteins seemed broader,which were involved in complications related with diabetes.Moreover,SOCS-modulating properties that regarding the treatment of diabetes have already been described.ObjectiveTo study the protective effects of SOCS3 proteins in cells of the respiratory system and the relationship with the JAK2/STAT3 signal pathway.Methods1.The A549 cells were divided into three groups randomly:(1)the normoglycemic group(NG group);(2)the hyperglycemic group(HR group);(3)the high osmosi group(OG group);All the cells were incubated at 37? in 5%CO2 for 48 h.When the A549 cells grew to appropriate density,they were incubated with NG DMEM medium without FBS for 24 h synchronously.The CCK-8 and LDH cytotoxicity test was adopted to detect the activity of cells.Then,the content of IL-6 and TNF-? were measured.The Western blot was adopted to detect the expression of SOCS3,JAK2,STAT3,p-JAK2 and p-STAT3 proteins.2.The A549 cells were divided into six groups randomly:(1)the normoglycemic group(NG group);(2)the hyperglycemic group(HR group);(3)the high osmosi group(OG group);(4)the hyperglycemic+AG490 group(HG+AG490 group);(5)the hyperglycemic+low glucose medium with transfection of vector plasmid(HG+SOCS3-group);(6)the hyperglycemic+low glucose medium with transfection of pcdna3.1 SCOS3 plasmid(HG+SOCS3+group).All the cells were incubated at 37? in 5%CO2 for 48 h.When the A549 cells grew to appropriate density,they were incubated with NG DMEM medium without FBS for 24 h synchronously.The CCK-8 and LDH cytotoxicity test was adopted to detect the activity of cells.Then,the content of IL-6 and TNF-? were measured.The Western blot was adopted to detect the expression of SOCS3,JAK2,STAT3,p-JAK2 and p-STAT3 proteins.Results1.A549 cells cultured in normal control medium exhibited shuttle shape,longer and fine cell body.Exposure to 25 mM high glucose for 48 h lead to dramatic changes in A549 cells morphology,displaying short and less extended cell body,with nuclear condensation and vacuole in cytoplasm,which indicated that high glucose induced A549 cells to typical apoptotic changes after 48 h exposure.The effect of growth inhibition was enhanced along with the 25mM high glucose concentration compare with NG groups(p<0.05).LDH activity was further increased under the condition of HG as compared with NG groups(p<0.05).However,there is no significant effects of hyperosmolarity were seen in OG groups.The IL-6 and TNF-? levels were further increased under the condition of HG as compared with NG groups(p<0.05).SOCS3 were increased in the A549 cells at protein level after HG exposure(P<0.05).At the same time,western bolt showed weak signals for p-JAK2 and p-STAT3 proteins in A549 cells of NG groups.In contrast,significant increases in protein expression for p-JAK2 and p-STAT3 were found in the HG groups(P<0.05).2.When 25 mM high glucose groups were given the AG490,the cells grew better and presented increase of density than in NG groups.It seemed the same as the groups that overexpression the SOCS3 after HG exposure.However,there were not significantly changes between the HG groups and the groups which vector plasmid were transfected after HG exposure.The viability rate of HG+AG490 and HG+SOCS3+groups was higher than that in HG groups(p<0.05).There were also statistically significant differences in the viability rate between HG+SOCS3-and HG+SOCS3+ groups(P<0.05).LDH activity was further increased under the condition of HG as compared with NG groups(p<0.05).In.contrast,the LDH activity of HG+AG490 and HG+SOCS3+groups dropped further as compared with HG groups(p<0.05).Compare with the HG+SOCS3-,the LDH activity was also reduced in the HG+SOCS3+groups(p<0.05).Compare with HG groups,the IL-6 and TNF-? levels of HG+AG490 and HG+SOCS3+groups were significantly lower(P<0.05).Both of them were also statistically significant in comparison with the HG+SOCS3" and HG+SOCS3+groups(P<0.05).When given the AG490,it greatly reduced the p-JAK2 and p-STAT3 release compared with those in the HG groups(P<0.05).SOCS3 overexpression significantly inhibited HG-induced tyrosine phosphorylation p-JAK2 and p-STAT3(P<0.05).Conclusion1.HG induced JAK2/STAT3 pathway with an increase of SOCS3 expression and inflammatory cytokines in A549 cells.2.SOCS3 overexpression and JAK2/STAT3 inhibitor AG490 significantly reduced lung cell lesions associated with hyperglycemia.These findings indicate therapeutic potential of modulating JAK2/STAT3 in alleviating diabetic lung injuries.