异钩藤碱对急性脑梗死大鼠认知障碍的影响及机制研究

Study on the effect and mechanism of isorhyncophylline on cognitive impairment in rats with acute cerebral infarction

目的探讨异钩藤碱对急性脑梗死大鼠认知障碍的影响及相应机制。方法将144只SD大鼠按照随机数字表法随机分为假手术组、急性脑梗死组、异钩藤碱低剂量组、异钩藤碱高剂量组、尼莫地平组、异钩藤碱高剂量+干扰素基因刺激蛋白(STING)激动剂(ADU-S100)组,每组24只。除假手术组外,其他各组大鼠均采用线栓闭塞大脑中动脉法构建急性脑梗死大鼠模型,假手术组仅游离右侧颈外动脉、颈总动脉和颈内动脉,不做插入线栓处理。建模成功后立即给药,异钩藤碱低剂量组和异钩藤碱高剂量组大鼠分别灌胃5 mg/kg、20 mg/kg异钩藤碱,且均需腹腔注射等量的等渗盐水;尼莫地平组大鼠需灌胃30 mg/kg尼莫地平,且还需腹腔注射等量的等渗盐水;异钩藤碱高剂量+ADU-S100组大鼠需灌胃20 mg/kg异钩藤碱且腹腔注射20 mg/kg ADU-S100;假手术组、急性脑梗死组大鼠均需灌胃10 ml/kg与腹腔注射10 ml/kg的等渗盐水。给药1次/d,持续2周。末次给药处理24 h后,采用Zela-Longa法对所有大鼠进行神经功能评分,并进行Morris水迷宫实验,记录大鼠的逃避潜伏期及原平台象限停留次数。采用酶联免疫吸附试验(ELISA)检测大鼠血清中白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α水平;检测各组大鼠脑组织含水量;采用2,3,5-三苯基四唑氯化物(TTC)染色测定各组大鼠脑梗死体积;采用伊文思蓝检测各组大鼠血-脑屏障通透性;采用实时定量聚合酶链反应(qRT-PCR)检测大鼠脑组织中闭锁小带蛋白1(ZO-1)、闭合蛋白(occludin)信使核糖核酸(mRNA)表达;采用免疫印迹检测大鼠脑组织中STING、磷酸化TANK结合激酶1(p-TBK1)、磷酸化干扰素调节因子3(p-IRF3)蛋白表达,并进行组间比较。结果(1)与假手术组相比,急性脑梗死组末次给药处理24 h后神经功能评分[(2.96±0.32)分比0分]、脑组织含水量[(86.9±3.2)%比(71.8±3.1)%]、血清TNF-α[(86.7±3.5)ng/L比(35.6±1.7)ng/L]、IL-6[(167.8±6.1)ng/L比(50.2±2.2)ng/L]、脑梗死体积[(28.6±1.3)mm 3比0 mm 3]、脑组织中伊文思蓝含量[(1.57±0.13)g/L比(0.96±0.08)g/L]及STING[(1.83±0.16)比(0.86±0.08)]、p-TBK1[(0.89±0.07)比(0.41±0.03)]、p-IRF3[(0.67±0.05)比(0.13±0.01)]蛋白表达均升高,脑组织中ZO-1 mRNA[(0.45±0.04)比(1.00±0.00)]、occludin mRNA[(0.23±0.02)比(1.00±0.00)]表达均降低,逃避潜伏期延长[(33.6±1.6)s比(12.3±0.5)s],原平台象限停留次数减少[(5.9±0.2)次比(15.7±0.4)次],组间差异均有统计学意义(均P<0.05)。(2)与急性脑梗死组比较,异钩藤碱低剂量组、异钩藤碱高剂量组、尼莫地平组大鼠末次给药处理24 h后神经功能评分[(2.37±0.21)、(1.14±0.17)、(1.18±0.13)分比(2.96±0.32)分]、脑组织含水量[(81.8±3.0)%、(74.9±3.0)%、(74.3±2.9)%比(86.9±3.2)%]、血清TNF-α[(71.1±1.4)、(43.4±2.0)、(41.5±1.9)ng/L比(86.7±3.5)ng/L]、IL-6[(129.8±5.4)、(81.2±3.8)、(80.0±3.6)ng/L比(167.8±6.1)ng/L]、脑梗死体积[(21.7±1.0)、(10.5±0.5)、(10.7±0.5)mm 3比(28.6±1.3)mm 3]、脑组织中伊文思蓝含量[(1.39±0.12)、(1.16±0.10)、(1.18±0.19)g/L比(1.57±0.13)g/L]及STING[(1.50±0.14)、(1.02±0.11)、(1.01±0.09)比(1.83±0.16)]、p-TBK1[(0.75±0.05)、(0.54±0.04)、(0.52±0.05)比(0.89±0.07)]、p-IRF3[(0.51±0.05)、(0.25±0.02)、(0.27±0.02)比(0.67±0.05)]蛋白表达均降低,脑组织中ZO-1 mRNA[(0.58±0.05)、(0.87±0.07)、(0.89±0.09)比(0.45±0.04)]、occludin mRNA[(0.36±0.03)、(0.71±0.06)、(0.69±0.05)比(0.23±0.02)]表达均升高,逃避潜伏期缩短[(28.6±1.0)、(16.5±0.7)、(16.4±0.7)s比(33.6±1.6)s],原平台象限停留次数增加[(8.2±0.3)、(12.8±0.5)、(12.9±0.5)次比(5.9±0.2)次],组间差异均有统计学意义(均P<0.05)。(3)与异钩藤碱高剂量组比较,异钩藤碱高剂量+ADU-S100组末次给药处理24 h后神经功能评分[(2.12±0.14)分比(1.14±0.17)分]、脑组织含水量[(78.7±3.2)%比(74.9±3.0)%]、血清TNF-α[(59.7±2.1)ng/L比(43.4±2.0)ng/L]、IL-6[(118.9±4.6)ng/L比(81.2±3.8)ng/L]、脑梗死体积[(16.6±0.4)mm 3比(10.5±0.5)mm 3]、脑组织中伊文思蓝含量[(1.36±0.10)g/L比(1.16±0.10)g/L]及STING[(1.37±0.12)比(1.02±0.11)]、p-TBK1[(0.67±0.05)比(0.54±0.04)]、p-IRF3[(0.39±0.03)比(0.25±0.02)]蛋白表达均升高,脑组织中ZO-1 mRNA[(0.63±0.05)比(0.87±0.07)]、occludin mRNA[(0.46±0.05)比(0.71±0.06)]表达均降低,逃避潜伏期延长[(23.4±1.0)s比(16.5±0.7)s],原平台象限停留次数减少[(9.6±0.3)次比(12.8±0.5)次],组间差异均有统计学意义(均P<0.05)。结论异钩藤碱可抑制急性脑梗死大鼠炎症、减少血-脑屏障损伤、降低脑水肿程度,改善认知障碍,其机制可能与抑制STING/TBK1/IRF3通路有关。

Objective To investigate the effect of isorhyncophylline on cognitive impairment in rats with acute cerebral infarction and its corresponding mechanism.Methods SD rats were separated into acute cerebral infarction group,sham operation group,low-dose isorhyncophylline group,high-dose isorhyncophylline group,nimodipine group,and high-dose isorhyncophylline+ADU-S100 group,with 24 rates for each group.Except the sham operation group,the rats in other groups were treated with filament model to construct the model of acute cerebral infarction.In the sham operation group,only the right external carotid artery,common carotid artery and internal carotid artery were exposed,and the filament was not inserted.After successful modeling,the medication was administered once a day for 2 weeks.After the modeling was successful,the rats in the low dose group and the high dose group were given 5 mg/kg and 20 mg/kg respectively,and the same amount of isotonic saline was injected intraperitoneally.Nimodipine group rats were given 30 mg/kg nimodipine by intragastric administration,and the same amount of isotonic saline was also injected intraperitoneally.The rats in high dose isorhyncophylline+ADU-S100 group were given 20 mg/kg isorhyncophylline by intragastric administration and 20 mg/kg ADU-S100 intraperitoneally.Rats in sham operation group and acute cerebral infarction group were injected with 10 ml/kg isotonic saline by intragastric administration and intraperitoneal injection.The medication was administered once a day for 2 weeks.The Zela-Longa neurological function score was evaluated in all the rats 24 h after the final medication,and then Morris water maze test was conducted and their escape latency and the number of stays in the original platform quadrant were recorded.The levels of interleukin 6(IL-6)and tumor necrosis factor(TNF)αin serum were detected by enzyme-linked immunosorbent assay(ELISA).The water content of brain tissue was detected in each group.The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Evans blue was applied to detect blood-brain barrier permeability.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of ZO-1 and occludin messenger RNA(mRNA)in brain tissue of each group.The expression of STING,phosphorylated tank-bound kinase 1(p-TBK1)and phosphorylated interferon regulatory factor 3(p-IRF3)in rat brain tissues was detected by western blot and compared between groups.Results Compared with sham operation group,the neurological function score([2.96±0.32]vs.0),brain tissue water content([86.9±3.2]%vs.[71.8±3.1]%),serum TNF-α([86.7±3.5]ng/L vs.[35.6±1.7]ng/L)and IL-6([167.8±6.1]ng/L vs.[50.2±2.2]ng/L)levels,cerebral infarction volume([28.6±1.3]mm 3 vs.0 mm 3),evans blue content([1.57±0.13]g/L vs.[0.96±0.08]g/L),STING([1.83±0.16]vs.[0.86±0.08]),p-TBK1([0.89±0.07]vs.[0.41±0.03]),and p-IRF3([0.67±0.05]vs.[0.13±0.01])protein expression in brain tissue were increased,expression of ZO-1([0.45±0.04]vs.[1.00±0.00])and occludin mRNA([0.23±0.02]vs.[1.00±0.00])in brain tissue were decreased,the escape latency was prolonged([33.6±1.6]s vs.[12.3±0.5]s),and the number of stays in the original platform quadrant([5.9±0.2]times vs.[15.7±0.4]times)was decreased in the acute cerebral infarction group 24 h after last medication administration(all P<0.05).(2)Compared with acute cerebral infarction group,the neurological function score([2.37±0.21],[1.14±0.17],[1.18±0.13]vs.[2.96±0.32]),brain tissue water content([81.8±3.0]%,[74.9±3.0]%,[74.3±2.9]%vs.[86.9±3.2]%),serum TNF-α([71.1±1.4]ng/L,[43.4±2.0]ng/L,[41.5±1.9]ng/L vs.[86.7±3.5]ng/L)and IL-6([129.8±5.4]ng/L,[81.2±3.8]ng/L,[80.0±3.6]ng/L vs.[167.8±6.1]ng/L)levels,cerebral infarction volume([21.7±1.0]mm 3,[10.5±0.5]mm 3,[10.7±0.5]mm 3 vs.[28.6±1.3]mm 3),evans blue content([1.39±0.12]g/L,[1.16±0.10]g/L,[1.18±0.19]g/L vs.[1.57±0.13]g/L),STING([1.50±0.14],[1.02±0.11],[1.01±0.09]vs.[1.83±0.16]),p-TBK1([0.75±0.05],[0.54±0.04],[0.52±0.05]vs.[0.89±0.07]),and p-IRF3([0.51±0.05],[0.25±0.02],[0.27±0.02]vs.[0.67±0.05])protein expression in brain tissue were decreased,expression of ZO-1([0.58±0.05],[0.87±0.07],[0.89±0.09]vs.[0.45±0.04])and occludin mRNA([0.36±0.03],[0.71±0.06],[0.69±0.05]vs.[0.23±0.02])in brain tissue were increased,the escape latency were shortened([28.6±1.0]s,[16.5±0.7]s,[16.4±0.7]s vs.[33.6±1.6]s),and the number of stays in the original platform quadrant([8.2±0.3]times,[12.8±0.5]times,[12.9±0.5]times vs.[5.9±0.2]times)were increased in the low-dose isorhyncophylline group,high-dose isorhyncophylline group,and nimodipine group(all P<0.05).(3)Compared with high-dose isorhyncophylline group,the neurological function score([2.12±0.14]vs.[1.14±0.17]),brain tissue water content([78.7±3.2]%vs.[74.9±3.0]%),serum TNF-α([59.7±2.1]ng/L vs.[43.4±2.0]ng/L)and IL-6([118.9±4.6]ng/L vs.[81.2±3.8]ng/L)levels,cerebral infarction volume([16.6±0.4]mm 3 vs.[10.5±0.5]mm 3),evans blue content([1.36±0.10]g/L vs.[1.16±0.10]g/L),and STING([1.37±0.12]vs.[1.02±0.11]),p-TBK1([0.67±0.05]vs.[0.54±0.04]),and p-IRF3([0.39±0.03]vs.[0.25±0.02])protein expression in brain tissue were increased,expression of ZO-1([0.63±0.05]vs.[0.87±0.07])and occludin mRNA([0.46±0.05]vs.[0.71±0.06])in brain tissue were decreased,the escape latency was prolonged([23.4±1.0]s vs.[16.5±0.7]s),and the number of stays in the original platform quadrant([9.6±0.3]times vs.[12.8±0.5]times)was decreased in the isorhyncophylline high-dose+ADU-S100 group(all P<0.05).Conclusion Isorhyncophylline can inhibit inflammation,reduce blood-brain barrier damage,reduce cerebral edema,and improve cognitive impairment in rats with acute cerebral infarction,and the mechanism of which may be related to the inhibition of STING/TBK1/IRF3 pathway.