褪黑素对脑缺血再灌注大鼠神经干细胞增殖的影响及机制研究

The effect of melatonin on the proliferation of neural stem cells in rats with cerebral ischemiareperfusion and its mechanism

目的探讨褪黑素对缺血再灌注(IR)大鼠脑内神经干细胞(NSCs)增殖的影响及其相关机制。方法将72只大鼠采用随机数字表法随机分为正常对照组(n=12)、模型组(n=30)及褪黑素组(n=30),根据IR后的时间分别将模型组与褪黑素组大鼠分为6h、24h、72h和7d 4个亚组。通过HE染色法观察褪黑素对IR大鼠损伤侧室管膜下区(svz)细胞损伤的影响,采用增殖细胞核抗原(PCNA)/巢蛋白(Nestin)免疫荧光双标法观察褪黑素对损伤侧SVZ内源性NSCs增殖的影响,通过免疫组织化学法和蛋白质印迹法检测褪黑素对损伤侧SVZ Toll样受体4(TLR4)及核转录因子(NF)一KBp65蛋白表达的影响。通过一般线性回归分析褪黑素组与模型组PCNA^+Nestin^+DAN^+细胞数差值与TLR4^+、NF.KB p65^+细胞数差值之间的相关性。结果HE染色显示模型组大鼠损伤侧SVZ细胞排列紊乱,形态不规则;褪黑素组大鼠损伤侧SVZ细胞排列较整齐,形态较规则。免疫荧光双标染色显示模型组(498.47±26.44/mm^2)与褪黑素组(623.10±39.70/mm^2)大鼠各时间点损伤侧SVZ PCNA^+Nestin^+DAPI^+细胞数逐渐增加,IR后7d达较高水平,均显著高于正常对照组(203.91±2.23/mm^2)(F=35.193、170.344、277.536、285.947,均P<0.01),褪黑素组大鼠PCNA^+Nestin^+DAPI^+细胞数均显著高于模型组(F=102.561、91.244、168.502、38.013,均P<0.01)。免疫组织化学染色显示模型组(740.02±31.63/mm^2;710.01±26.59/mm^2)和褪黑素组(555.57±25.28/mm^2;528.85+30.60/mm^2)大鼠各时间点损伤侧SVZTLR4^+与NF—KB p65^+细胞数逐渐增多,IR后7d达较高水平,且均显著多于正常对照组(F=21.413、263.059、873.691、1037.098,均P<0.01;F=26.374、372.940、854.826、929.018,均P<0.01),褪黑素组TLR4^+与NF—KB p65’细胞数均较模型组显著下降(F=7.641、25.135、66.094、103.753;F=18.612、69.597、113.113、119.814;均P<O.05)。蛋白质印迹结果显示建模后7d,模型组和褪黑素组大鼠TLR4(0.87±O.08,0.68±0.06)与NF-KB p65(0.72±0.05,0.58±0.05)蛋白相对表达量显著高于正常对照组(0.35±0.04,0.3l±0.03;F=107.43,F=132.51,均P<0.01),褪黑素组TLR4与NF—KB p65蛋白表达量均较模型组显著下降(均P<0.01)。一般线性回归显示建模后各时间点褪黑素组与模型组的PCNA^+Nestin^+DAPI^+细胞数差值与TLR4^+、NF—KB p65^+细胞数差值均呈负相关(r^2=0.838,r^2=0.813,均P<0.01)。结论褪黑素可抑制TLR4、NF—KB p65蛋白的表达,促进IR大鼠脑内源性NSCs的增殖。

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 (rt=30)and melatonin group (n=30)aecording to the random number table.The rats in the model group and melatonin group were divided into four subgroups:6h,24h,72h and 7d 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 immunofluorescenec staining;the effects of melatonin ontoll-like receptor 4(TLR4)and nuclear factor (NF)-KB p65protein were examined by immunohistochemistry staining and Western blotting analysis.The correlation between the proliferating NSCs and TLR4 protein or the NF-KB 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/mm^2)and melatonin groups (623.10+39.70/mm^2)increased gradually,and reached a higher level after IR for 7days,which were significantly higher than the normal control group (203.91±32.23/mm^2)(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-KB p65+cells in the SVZ of the model (740.02±31.63/mm^2;710.01±26.59/mm^2)and melatonin groups (555.57±25.28/mm^2;528.85±30.60/mm^2)increased gradually,and reached a higher level 7d after IR,which were significantly higher than the normal control group (107.97+12.84/mm^2;109.80±13.89/mm^2)(F=21.413,263.059,873.691,1037.098,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-KB 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-KB 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-KB 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-KB p65^+ cells in the melatonin group (P=0.838,r^2=0.813,both P<0.01).Conclusion Melatonin can inhibit the expression of TLR4 and NF-KB p65 protein,thus promote the proliferation of endogenous NSCs in cerebral ischemia reperfusion rats.