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Objective: To explore the mechanism and role of Cavelion-1 in injury of oxidative stress induced by intermittent high glucose in human umbilical vein endothelial cells.Methods:HUVECs were exposed to DMEM culture media containing 5.5 mM (Normal group, NG), 20 Mm (High glucose group, HG) glucose, 20/5.5 mM (Intermittent glucose group, IG) and 14.5 mM mannitol (Mannitol group, MG) for 2, 7 or 14 days respectively. The changes of cell morphological were observed.The intracellular maleic dialdehyde (MDA) was assayed by thioabrbituric acid method and the antioxidant ability of superoxide dismutase (SOD) was detected by xanthine oxidase technique.The mRNA of Cav-1, PKCαand PKCβ2 were determined by Real-time PCR and the protein were determined by Western blot. Experimental data were statistically analyzed by SPSS16.0.Results:1. After 2, 7 or 14days, NG and MG cells were observed with clear outline, fusiform. Cytoplasmic and nucleus were visible. HG cells were observed volume increased, cobblestone-like, cytoplasmic increased and small black scattered granular of secrete metabolites. IG cells were observed without clear outline, volume increased, disorganized, ill-defined with cytoplasm and nucleus, small black scattered granular of secrete metabolites. The changes became more serious in time-dependent manner.2. After 2 days, the content of MDA in HG and IG were higher than that in NG (P<0.05), there was no difference between IG and HG (P>0.05). After 7 or 14days, the content of MDA in IG and HG was significantly higher than that in NG (P<0.01), while compared with HG, it was higher in IG (P<0.05). The content of MDA was increased in time-dependent manner, when the HUVECs were cultured with different conditions. The level of MDA was no difference in MG at anytime (P>0.05).3. After 2 days, the activity of SOD in HG and IG were lower than that in NG (P<0.05), there was no difference between IG and HG (P>0.05). After 7 or 14days, the activity of SOD in IG and HG was significantly lower than that in NG (P<0.01), while compared with HG, it was lower in IG (P<0.05). The activity of SOD was decreased in time-dependent manner, when the HUVECs were cultured with different conditions. The activity of SOD was no difference in MG at anytime (P>0.05).4. After 2 days, the expression of Cav-1 mRNA and protein in HG and IG was higher than that in NG (P<0.05), while there was no difference between HG and IG (P>0.05). After 7 or 14days, the expression of Cav-1 mRNA and protein in IG and HG was significantly higher than that in NG (P<0.01), while compared with HG, it was higher in IG (P<0.05). The expression of Cav-1 mRNA and protein increased in time-dependent manner, when the HUVECs were cultured with different conditions. The expression of Cav-1 mRNA and protein was no difference in MG at anytime (P>0.05).5. Treated HUVECs under different conditions after 2, 7 or 14days,there was no difference in the expression of PKCαmRNA and protein among HG, NG, IN and MG (P<0.05).6. After 2 days, the expression of PKCβ2 mRNA and protein in HG and IG was higher than that in NG (P<0.05), while there was no difference between HG and IG (P>0.05). After 7 or 14days, the expression of PKCβ2 mRNA and protein in IG and HG was significantly higher than that in NG (P<0.01), while compared with HG, it was higher in IG (P<0.05). The expression of PKCβ2 mRNA and protein increased in time-dependent manner, when the HUVECs were cultured with different conditions. The expression of PKCβ2 mRNA and protein was no difference in MG at anytime (P>0.05)Conclusions:1. Intermittent high glucose induces oxidative stress through PKCβ2 pathway. Furthermore, intermittent high glucose is more deleterious compared with constant high glucose.Thus, this result demonstates intermittent high glucose makes more significant glucose toxicity. 2. Constant and intermittent high glucose upregulate the expression of Cav-1. It is indicated that Cav-1 may play an important role during high glucose-induced endothelial cell injury.3. It has been showed that the expression of Cav-1 and PKCβ2 has the same developing trend.There is two possibilities. One is that intermittent high glucose induced the injury of oxidative stress through PKCβ2-Cav-1 pathway, another is that enhance of Cav-1 is adopted regulation to protect the cells from the injury caused by PKCβ2.4. Whether constant and intermittent high glucose, the injury of oxidative stress and the expression of PKCβ2, Cav-1 are time-dependent manner. It is suggesting that an early removing of the high glucose toxicity is important.
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