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Objective To observe the expressing changes of apolipoprotein M (ApoM),tumor necrosis factor-α(TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in human retinal vascular endothelial cells (HRECs) under the high glucose culture condition and investigate the inhibitory effects of ApoM overepression on the expressions of TNF-α and MCP-1.Methods HRECs were cultured in DMEM containing 10％ fetal bovine serum and 5.5 mmol/L D-glucose and assigned to 6 groups.The cells in the normal control group were cultured in above culture medium;the cells in the high glucose group were treated using the DMEM with 30 mmol/L D-glucose;ApoM was transfected into the cells using lentiviral vector in the ApoM transfected group;lentiviral vector without ApoM sequence was transfected in the empty vector group;the cells transfected by empty vector were cultured in high glucose culture medium in the empty vector+high glucose group;the cells in the ApoM transfection+high glucose group were treated by ApoM sequence transfection and high glucose incubation.The relative expression of ApoM,TNF-α and MCP-1 mRNA was detected using real-time quantitative PCR,and the relative expression of ApoM protein was evaluated using Western blot assay.Results Compared with the normal control group,the mRNA expression levels of ApoM,TNF-α and MCP-1 in the high glucose group were significantly increased (t=5.517,3.295,2.555;all P＜0.05).HRECs grew well after infected with lentivirus.The relative expression level of ApoM mRNA in the ApoM transfected group was 236.400±39.270,which was significantly higher than 1.000±0.153 in the empty vector group (t=5.995,P＜0.01).An enhanced protein band of ApoM was seen in the ApoM transfected group,and the protein band was absent in the empty vector group.The relative expression band in the ApoM transfected group was 1.000± 0.249 and 2.978 ± 0.285 in the cells cultured with normal culture medium or high glucose culture medium,respectively,with a significant difference between them (t =5.056,P＜0.01).The relative expressions of TNF-α and MCP-1 in the mRNA levels were significantly different among the empty vector group,empty vector+high glucose group,ApoM transfected group and ApoM transfection + high glucose group (F =5.966,P =0.026;F =14.410,P =0.002).Compared with the empty vector+high glucose group,the relative expressions of TNF-α and MCP-1 mRNA were considerably reduced in the ApoM transfection+high glucose group (P=0.017,0.004).Conclusions High glucose environment up-regulates the expression of ApoM,MCP-1 and TNF-α in HRECs.Overexpression of ApoM inhibits the up-regulation of MCP-1 and TNF-α expression induced by high glucose.
ABSTRACT
Background Diabetic retinopathy (DR) is one of the common complications of diabetes.Retinal pigment epithelium (RPE) cells,as important constituent cells of the retina,play important roles in the development and progression of DR.Objective This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells (hRPECs) following high glucose culture.Methods HRPECs were divided into control group,high-glucose group and 3-MA+high glucose group.The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group,and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA (for 1 hour firstly) and 30 mmol/L glucose in the 3-MA+high glucose group.The cells were inoculated into 24-well plate with the content of 1 ×105/well,and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0.5％ fetal bovine serum after achieved attached 80％ confluence.The morphology and uhrastructure of the cells were examined by optics microscope and transmission electronic microscope.The proliferative rate of the cells was assayed by CCK-8 kit.The expression of autophagy-related gene microtubule associated protein light chain 3B (LC3B) in the cells was detected by Western blot and LC3B-Ⅱ/LC3B-Ⅰ value was quantitatively evaluated among the groups.Results The cells grew well with uniform size and regulatory arrangement in the control group.The cells in the high glucose group enlarged and the number of cells evidently increased.In the 3-MA+high glucose group,the cells decreased with a disorder arrangement.Under the transmission electron microscope,the cells were normal with the round-or oval-like nucleus and normal organelles in the control group,and autolysosome could be seen in the cells in the high glucose group.In the 3-MA+high glucose group,some autophagic bodies were found.The proliferative rate of the cells was (100.0±2.0) ％,(116.9-±5.2)％ and (103.7 ±4.7)％ in the control group,high glucose group and 3-MA+high glucose group respectively,showing a significant difference among the groups (F =13.526,P =0.006).The proliferative rate was considerably raised in the high glucose group compared with the control group and 3-MA+high glucose group (both at P＜0.05),but there was no significant difference in the proliferative rate of the cells between the control group and 3-MA+high glucose group (P＞0.05).Compared with the control group,the expressing intensity of LC3B-Ⅰ protein was weakened and that of LC3-Ⅱ protein was enhanced,and the expression intensity of LC3B-Ⅰ and LC3B-Ⅱ proteins in the 3-MA+high glucose group was similar to that in the control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.131 ±0.065,2.504±0.097 and 0.274±0.007 in the control group,high glucose group and 3-MA+high glucose group,respectively,with significant differences among the groups (F =1 694.676,P =0.000),and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in the high glucose group in comparison with the control group and the 3-MA+high glucose group (all at P＜0.05).No significant difference was found in the LC3B-Ⅱ/LC3B-Ⅰ ratio between the control group and 3-MA + high glucose group (P ＞ 0.05).Conclusions High glucose culture of hRPECs can activate autophagy process and promote cell proliferation.3-MA,an autophagy inhibitor,suppresses the high glucoseinduced growth of HRPECs by inhibiting autophagy process.
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Objective To cultivate mice glomerular podocyte with high glucose and high Ang-Ⅱ,observe the morphocytology of podocyte,and investigate protein kinase C (PKC) activity,the expressions of nephrin and CD2-associated protein (CD2AP),and apoptosis of podocyte with injuries cultivations.Methods The conditions immortalization mice podocyte cell lines was cultivated,after passage and differential induce,the cell lines was divided into six groups:normal group,high glucose group,high Ang-Ⅱ] group,the above two mixed group,intervened group of PKC inhibitor,and hypertonic group.After 72 hours,cell apoptosis rate was detected with flow cytometer; PKC activity was detected with enzyme linked immunosorbent assay (ELISA) in 24 hours and 48 hours ; The expressions of nephrin and CD2AP mRNA were detected by real time-polymerase chain reaction (RT-PCR).Results (1)After 24 hours,the PKC of high glucose group and high Ang-Ⅱ group were more activated than normal group [(47.09 ± 1.19) pmol/L,(42.93 ±0.71) pmol/L,P ＜0.05].The activated phenomenon was more obvious in the mixed group.Compared to 24 hours [(58.75 ±0.71) pmol/ L,P ＜ 0.01],PKC was more activated in 48 hours (P ＜ 0.05).(2) Nephrin mRNA was reduced in podocyte exposed to high glucose and high Ang-Ⅱ at least 24 hours (56.87 ± 0.74,48.54 ± 0.86,P ＜ 0.05),and was reduced more in mixed group (11.7 ± 1.54,P ＜ 0.01).(3) CD2AP mRNA was reduced in podocyte exposed to high glucose,high Ang-Ⅱ,and mixed environment at least 48 hours (56.09 ± 1.46,67.68 ±2.58,and 54.08 ±2.74,P ＜0.05).The decrease trend of nephrin and CD2AP mRNA was restrained by PKC inhibitor(90.75 ± 1.33,P ＜0.05).(4) After 48 hours and 72 hours,the apoptosis rates of high glucose group,high Ang-Ⅱ group,and mixed group were risen compared to normal group (P ＜ 0.05).The increase of apoptosis rate can be restrained by PKC inhibitor.Conclusions High glucose,and high Ang-Ⅱ can activate the podocytes PKC activity,inhibit the expression of nephrin and CD2AP mRNAs,and induce podocyte apoptosis.PKC signaling pathway plays an important role in the injury mechanism of Ang-Ⅱ and high glucose to mice podocyte.