CAJ | 학술논문

目的：观察大鼠惊厥持续状态（SC）后脑组织海马中Toll样受体4（TLR4）、核因子-κB（NF-κB）和半胱氨酸门冬氨酸特异性蛋白酶3（Caspase-3）的表达及神经细胞凋亡的变化，并探讨白藜芦醇（Res）对三者的影响。方法106只SD大鼠随机分为对照组（ A）、SE组（ B）和白藜芦醇干预组（ C），其中B组再随机分为B1～B4组（分别于惊厥后4h、24h、48h和72h处死），B组和C组采用氯化锂-匹罗卡品法制作大鼠SC模型，C组在大鼠惊厥终止后30min，给予30 mg/kg白藜芦醇腹腔注射，每天1次，连用3d，于惊厥后72 h处死。免疫组织化学法检测海马TLR4、NF-κB/p65蛋白的表达变化；RT-PCR技术检测海马TLR4、Caspase-3 mRNA表达的动态改变；TUNEL法检测神经细胞凋亡数的变化。结果免疫组织化学显示，B组各时间点TLR4蛋白的表达均明显高于A组（ P＜0.05），并随时间延长明显增高；C组TLR4蛋白表达较B4组显著降低（ P＜0.01）。 B组可见NF-кB/p65蛋白在胞核内有不同程度表达，与A组比较差异有统计学意义（ P＜0.05）。 C组与B4组比较，NF-κB/p65蛋白表达水平明显降低（P＜0.01）。 RT-PCR显示，B组各时间点TLR4、Caspase-3 mRNA的表达均较A组高（P＜0.05），且随时间延长逐步升高，惊厥后72h达高峰；C组海马TLR4、Caspase-3mRNA的表达明显低于B4组（P＜0.05）。 B组在惊厥24h后海马CA1区TUNEL阳性细胞数已显著高于A组（P＜0.01），72h达高峰，而C组TUNEL阳性细胞数较B4组显著下降（P＜0.01）。结论 SC后大鼠海马TLR4、NF-κB/p65和Caspase-3的表达增强。白藜芦醇可下调匹罗卡品致惊厥持续状态大鼠海马TLR4、NF-κB/p65和Caspase-3的表达，并使神经细胞凋亡减少；提示白藜芦醇对SC引起的脑损伤可能有保护作用。
목적：관찰대서량궐지속상태（SC）후뇌조직해마중Toll양수체4（TLR4）、핵인자-κB（NF-κB）화반광안산문동안산특이성단백매3（Caspase-3）적표체급신경세포조망적변화，병탐토백려호순（Res）대삼자적영향。방법106지SD대서수궤분위대조조（ A）、SE조（ B）화백려호순간예조（ C），기중B조재수궤분위B1～B4조（분별우량궐후4h、24h、48h화72h처사），B조화C조채용록화리-필라잡품법제작대서SC모형，C조재대서량궐종지후30min，급여30 mg/kg백려호순복강주사，매천1차，련용3d，우량궐후72 h처사。면역조직화학법검측해마TLR4、NF-κB/p65단백적표체변화；RT-PCR기술검측해마TLR4、Caspase-3 mRNA표체적동태개변；TUNEL법검측신경세포조망수적변화。결과면역조직화학현시，B조각시간점TLR4단백적표체균명현고우A조（ P＜0.05），병수시간연장명현증고；C조TLR4단백표체교B4조현저강저（ P＜0.01）。 B조가견NF-кB/p65단백재포핵내유불동정도표체，여A조비교차이유통계학의의（ P＜0.05）。 C조여B4조비교，NF-κB/p65단백표체수평명현강저（P＜0.01）。 RT-PCR현시，B조각시간점TLR4、Caspase-3 mRNA적표체균교A조고（P＜0.05），차수시간연장축보승고，량궐후72h체고봉；C조해마TLR4、Caspase-3mRNA적표체명현저우B4조（P＜0.05）。 B조재량궐24h후해마CA1구TUNEL양성세포수이현저고우A조（P＜0.01），72h체고봉，이C조TUNEL양성세포수교B4조현저하강（P＜0.01）。결론 SC후대서해마TLR4、NF-κB/p65화Caspase-3적표체증강。백려호순가하조필라잡품치량궐지속상태대서해마TLR4、NF-κB/p65화Caspase-3적표체，병사신경세포조망감소；제시백려호순대SC인기적뇌손상가능유보호작용。
Objective To study the expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-кB) and Caspase-3 and the change of neuron apoptosis of the hippocampus in the status convulsion rat , and to explore the effect of resveratrol on them.Methods A total of 106 male Sprague-Dawley(SD)rats were randomly divided into the control (A), convulsion (B) and resveratrol treatment (C) groups.Group B was further randomly divided into four subset groups (B1-B4) which were executed at 4h, 24h, 48h, 72h after convulsion discontinued .Continuous epilepticus was induced by injecting lithium chloride and pilocarpine , and group C was daily injected with 30mg/kg resveratrol 30minutes after convulsion stopped for 3 days.TLR4 and NF-κB/p65 proteins were detected by immunohistochemistry ( IHC ); the expressions of TLR4 and Caspase-3 mRNA were detected by RT-PCR.The neuron apoptosis was observed by TUNEL . Results The IHC staining of TLR4 protein in group B was significantly higher than in group A (P<0.05), and that in group C was much lower than in B 4 group ( P<0.01 ) .The expression of NF-кB/p65 showed that hippocampal neurons had positive expression in cell nuclei in group B compared with group A (P<0.05), and the expression of NF-кB/p65 protein in group C was much lower than that in group B 4 (P<0.01).The mRNA expressions of TLR4 and Caspase-3 in the rat hippocampus of group B were significantly elevated than that in group A ( P <0.05 ) , and the tendency was increased gradually , reaching the peak at 72 hours after seizure , and that in group C was much lower than that in B 4 group (P<0.01).The TUNEL positive cells in hippocampus CAl of B group were more than that of group A after the SC 24hours (P<0.01),reached the highest level at the 72nd hour, and that in group C the TUNEL positive cells were lower than that in B4 group (P<0.01).Conclusion The expression of TLR4, NF-кB and Caspase-3 increased after SC.Resveratrol can down regulate the expression of TLR4, NF-кB and Caspase-3 in the hippocampus with pilocarpine-induced seizures, reduced the number of neuronal apoptosis .These results suggest that resveratrol may have a protective effect against the hippocampus damage caused by status convulsion .