ABSTRACT
Objective To investigate the effects of melatonin on the proliferation of neural stem cells (NSCs) in cerebral ischemia reperfusion (IR) rats, and to explore the possible mechanisms. Methods Seventy-two rats were randomly divided into the normal control group (n=12), model group (n=30) and melatonin group (n=30) according to the random number table. The rats in the model group and melatonin group were divided into four subgroups: 6 h, 24 h, 72 h and 7 d subgroups according to the time after IR. The morphological changes of the subventricular zone (SVZ) were examined by HE staining;the effects of melatonin on NSCs proliferation were examined by immunofluorescence staining;the effects of melatonin on toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB p65 protein were examined by immunohistochemistry staining and Western blotting analysis. The correlation between the proliferating NSCs and TLR4 protein or the NF-κB p65 protein was analyzed by linear regression analysis. Results HE staining showed that the cells in the SVZ of rats in the model group were in disorder and irregular in shape. In the melatonin group, the cells in the SVZ of the injured side were relatively well arranged. Immunofluorescence staining showed that the number of proliferating cell nuclear antigen (PCNA)+Nestin+4',6-diamidino-2-phenylindole (DAPI)+cells in the SVZ of the model (498.47 ± 26.44/mm2) and melatonin groups (623.10 ± 39.70/mm2) increased gradually, and reached a higher level after IR for 7 days, which were significantly higher than the normal control group (203.91 ± 32.23/mm2) (F=35.193, 170.344, 277.536, 285.947, all P＜0.01). The number of PCNA+Nestin+DAPI+cells in the melatonin group rats at each time points was significantly higher than that in the model group (F=102.561, 91.244, 168.502, 38.013, all P＜0.01). Immunohistochemistry staining showed that the numbers of TLR4+and NF-κB p65+cells in the SVZ of the model (740.02±31.63/mm2;710.01± 26.59/mm2) and melatonin groups (555.57 ± 25.28/mm2;528.85 ± 30.60/mm2) increased gradually, and reached a higher level 7 d after IR, which were significantly higher than the normal control group (107.97±12.84/mm2;109.80±13.89/mm2) (F=21.413, 263.059, 873.691, 1 037.098, all P＜0.01;F=26.374, 372.940, 854.826, 929.018, all P＜0.01). There were less TLR4+(F=7.641, 25.135, 66.094, 103.753, all P＜0.05) and NF-κB p65+cells (F=18.612, 69.597, 113.113, 119.814, all P＜0.01) in the melatonin group as compared with those in the model group at each time points. Western blotting analysis showed that the expression of TLR4 (0.87±0.08;0.68±0.06) and NF-κB p65 (0.72±0.05;0.58±0.05) protein was higher in the model and melatonin groups as compared with the normal control group (0.35±0.04, 0.31±0.03;F=107.43, F=132.51, both P＜0.01). The expression of the TLR4 and NF-κB p65 protein was lower in the melatonin group as compared with that in the model group (P＜0.01). Linear regression analysis showed that the differences of PCNA+Nestin+DAPI+cells were all negatively correlated with that of the TLR4+cells and NF-κB p65+cells in the melatonin group (r2=0.838, r2=0.813, both P＜0.01). Conclusion Melatonin can inhibit the expression of TLR4 and NF-κB p65 protein, thus promote the proliferation of endogenous NSCs in cerebral ischemia reperfusion rats.