[Interleukin-6 and tumor necrosis factor-alpha levels of endothelial cells in different hypoxia modes: in vitro experiment].

Objective: To investigate the damage of different patterns of intermittent hypoxia (IH) and continuous hypoxia (CH) on endothelial cells.

Methods: Human umbilical vein endothelial cells of the line ECV304 were cultured in a program-controlled gas delivery system newly developed and divided into 8 groups to undergo different IH/reoxygenation (ROX) cycles so as to simulate the patterns of hypoxic episode seen in recurrent apnea and chronic obstructive pulmonary disease: intermittent normoxia (IN) group (exposed to 21% O2 15 s/21% O2 225 s for 60 cycles), IH group (exposed to 1.5% O2 15 s/21% O2 225 s for 30 or 60 cycles), IH hypercapnia group (exposed to 1.5% O2 and 20% CO2 15 s/21% O2 and 5% CO2 225 s, for 60 cycles), continuous hypoxia (CH group, exposed to 1.5% or 10% O2 for 15, 30 or 60 min), CH hypercapnia group (exposed to 10% O2 and 10% CO2 for 15, 30 or 60 min), CH added to IH group (exposed to 1.5% O2 15 s/10% O2 225 s for 60 cycles), different intermittent hypoxia extent group (exposed to 1.5% or 10% O2 15 s/21% O2 225 s for 60 cycles), different intermittent hypoxia frequency group (exposed to 1.5% O2 15 s/21% O2 225 s 315 s, 495 s or 105 s for 60 cycles), and different intermittent hypoxia duration group (exposed to 1.5% O2 15 s or 30 s/21% O2 225 s for 60 cycles). Then ELISA was conducted to examine the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFalpha) Bicinchoninic acid method was used to standardize the cell total protein level.

Results: The levels of IL-6 and TNFalpha levels in the IH group were (770.40 +/- 21.60) and (126.93 +/- 2.58) pg.ml(-1).100 mg protein(-1) respectively, both significantly higher than those in the IN group [(374.06 +/- 38.10) and (31.96 +/- 13.64) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002], but significantly lower than those in the IH hypercapnia group [(829.27 +/- 7.16) and (78.77 +/- 4.00) pg.ml(-1).100 mg protein(-1) respectively, U = 0.000, P = 0.002]. The IL-6 levels of the CH hypercapnia 15, 30, and 60 min subgroups were all significantly higher than those of the corresponding CH subgroup (U = 0.000, P = 0.002). The IL-6 and TNFalpha levels of the CH added to IH group were (536.74 +/- 14.97) and (51.10 +/- 6.80) pg.ml(-1).100 mg protein(-1) respectively, both were significantly higher than those of the IN group, but significantly lower than those of the IH group (chi(2) = 23.4, P < 0.05). The levels of IL-6 and TNFalpha increased along with the increase of the IH degree (chi(2) = 23.4, P < 0.05). The level changes of IL-6 and TNFalpha of the groups with different intermittent hypoxia frequency and with different intermittent hypoxia duration were complicated.

Conclusion: IH and CH significantly damage the endothelial cells dose-dependently, especially combined with hypercapnia. In IH/ROX, the inflammatory damage comes from ROX phase but not IH phase. Hypoxia duration and hypoxia frequency are also important parameters in the activation of inflammation.