Figure 2
Combination of MH with HUK, MK-801 or Ngb prevented neuronal apoptosis against OGD/R injury.
Upper panel: Population analysis or cells with Annexin V/PI Staining. The percentages of apoptosis cells (Q2+Q4) in primary cortical neurons after OGD/R treated by MH combined with HUK, were less than MH or HUK alone. The primary cortical neurons were cultured in 6-well plates. In DIV6, cells were subjected to OGD for 3 hrs, then treated with HUK, HT, or both of them and reoxygenation were simultaneously made. 48 hrs later, apoptosis was measured with the Annexin V/PI Staining Assay Kit (Sigma, St. Louis, MO) and detected by FACS. As showed on upper panel, necrosis cells stained by propidium iodide (PI) appeared in the first quadrant, late apoptosis cells stained by annexin V-FITC and PI were in the second quadrant, alive cells in the third quadrant and the early apoptosis cells stained by annexin V-FITC presented in the fourth quadrant. Lower panel: Combinations of each of three Neuroprotectants and MH Prevented OGD/R Neurons from Apoptosis. Processing of primary cortical neurons and analysis of apoptosis by flowcytomery were performed as above. This figure illustrated bar graphs of apoptosis analysis results. The bars represented population percentage by means ± s.d. (n = 4). *, P<0.05 compared with the other groups and **, P<0.05, compared with one or two groups. Three reagents, HUK, MK-801 and Ngb, combined with MH prevented much more greatly against the apoptosis of primary cerebral neurons after OGD/R, compared with single agent (P< 0.05). (a) The co-treatment of BDNF and MH reduced the total apoptosis percentage, was less than MH (t = 2.561, P = 0.043) but not BDNF (t = −1.867, P = 0.111). (b) The co-treatment of GBC and MH reduced the total apoptosis percentage, was less than MH (t = 2.804, P = 0.031) but more than GBC (t = −4.594, P = 0.004). (c) The co-treatment of HUK and MH reduced the total apoptosis percentage, was less than HUK (t = 11.504, P<0.001) or MH (t = 11.360, P<0.001). (d) The co-treatment of MK-801 and MH reduced the total apoptosis percentage, was less than MK-801 (t = 4.031, P = 0.007) or MH (t = 9.196, P<0.001). (e) The co-treatment of Ngb and MH reduced the total apoptosis percentage, was less than Ngb (t = 9.504, P<0.001) or MH (t = 13.595, P<0.001). Abbrevation: Q: quadrant; FACS: Fluorescence Activated Cell Sorter.
