异泽兰黄素对脑出血继发性脑损伤的神经保护作用及相关机制研究

Neuroprotective effect and related mechanisms of of eupatilin on secondary brain injury after intracerebral hemorrhage

目的 探讨异泽兰黄素(EP)对脑出血(ICH)继发性脑损伤的神经保护作用及相关机制。方法 体内动物实验:将无特定病原体雄性小鼠按随机数字表法分为假手术组(0.9%氯化钠溶液)、模型组(0.9%氯化钠溶液)、EP低剂量组(5 mg/kg EP)、EP中剂量组(10 mg/kg EP)、EP高剂量组(20 mg/kg EP),每组18只;除假手术组外,其余4组均采用Ⅳ型胶原酶诱导ICH法构建小鼠ICH模型。检测并比较5组小鼠给药7 d后改良神经功能缺失评分(m NSS)、水迷宫实验结果(逃避潜伏期、平台停留时间、穿越平台次数)、血脑屏障通透性、脑含水量、脑组织病理学改变、氧化应激指标[丙二醛(MDA)、超氧物歧化酶(SOD)、过氧化氢酶(CAT)]、细胞凋亡数、炎症因子指标[肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素1β(IL-1β)]以及凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)、裂解的胱天蛋白酶-3(Cleaved caspase-3)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(Akt)、糖原合成酶-3β(GSK-3β)、磷酸化PI3K(p-PI3K)、磷酸化Akt(p-Akt)、磷酸化GSK-3β(p-GSK-3β)]、水通道蛋白4(AQP4)蛋白表达水平。体外细胞实验:将培养后的人神经母细胞瘤细胞系SH-SY5Y细胞分别设空白组、模型组、EP低剂量组(12.5μmol/L)、EP中剂量组(25μmol/L)、EP高剂量组(50μmol/L),每组设6复孔;除空白组不作任何处理外,其余4组均加入冈田酸构建ICH神经细胞损伤模型。检测并比较4组细胞存活率、细胞凋亡率以及凋亡相关蛋白表达水平。结果 体内动物实验结果显示,与假手术组比较,模型组小鼠脑组织结构破坏明显,神经细胞坏死且大量炎性细胞浸润,m NSS评分、逃避潜伏期、血脑屏障通透性、脑含水量以及脑组织MDA、细胞凋亡数和Cleaved caspase-3、AQP4蛋白表达水平均明显升高(均P<0.05),平台停留时间、穿越平台次数、体重以及脑组织SOD、CAT、Bcl-2/Bax、p-PI3K/PI3K、p-AMPK/AMPK、p-GSK-3β/GSK-3β均明显降低(均P<0.05)。与模型组比较,EP低、中、高剂量组脑组织结构以及上述指标均得到明显改善(均P<0.05),且呈剂量依赖性(均P<0.05)。体外细胞实验结果显示,与空白组比较,模型组细胞存活率、Bcl-2/Bax、p-PI3K/PI3K、p-AMPK/AMPK、p-GSK-3β/GSK-3β均明显降低(均P<0.05),凋亡率明显升高(P<0.05);与模型组比较,EP低、中、高剂量组上述指标均得到明显改善(均P<0.05),且呈剂量依赖性(均P<0.05)。结论 EP能减轻小鼠ICH后神经细胞凋亡和炎症反应,进而保护神经功能,且呈剂量依赖性;其作用机制可能与EP激活PI3K/Akt/GSK-3β信号通路进而对ICH后神经细胞损伤起保护作用有关。

Objective To explore the neuroprotective effect of eupatilin(EP) on secondary brain injury after intracerebral hemorrhage(ICH) and the underlying mechanisms. Methods In vivo animal experiments, male mice were randomly divided into five groups using the random number table method. The sham surgery group(0.9% sodium chloride solution), the model group(0.9% sodium chloride solution), the EP low-dose group(5 mg/kg EP), the EP medium dose group(10 mg/kg EP) and the EP high-dose group(20 mg/kg EP), 18 mice in each group. Except for the sham surgery group, the other 4 groups underwent ICH model construction using type Ⅳ collagenase. After 7 days of medication, we evaluated and compared the modified neurologic severity score(m NSS), water maze test results(escape latency, platform residence time, number of platform crossings), blood-brain barrier permeability, brain water content,pathological changes in brain tissue, oxidative stress indicators [malondialdehyde(MDA), superoxide dismutase(SOD),catalase(CAT)], cell apoptosis, inflammatory factor indicators [tumor necrosis factor alpha(TNF-α), interleukin-6(IL-6),interleukin-1 β(IL-1β)] and apoptosis related proteins [B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax),Cleaved caspase-3, phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), glycogen synthetase-3(GSK-3),phosphorylated PI3K(p-PI3K), phosphorylated Akt(p-Akt), phosphorylated GSK-3β(p-GSK-3β)] and aquaporin 4(AQP4) in 5 groups. In vitro cell experiments, the cultured SH-SY5Y cells were divided into the control group, the model group, the EP low-dose group(12.5 μmol/L), the EP medium dose group(25 μmol/L) and the EP high-dose group(50μmol/L), 6 wells in each group. Except for the control group, the other 4 groups were added with okada acid to construct an ICH neural cell injury model. The cell survival rate, apoptosis rate and expression levels of apoptosis related proteins in 4 groups were detected. Results The results of vivo experiment showed that compared with the sham surgery group, the brain tissue structure of the model group mice was significantly damaged, with nerve cell necrosis and a large number of inflammatory cell infiltration. The m NSS, escape latency, blood-brain barrier permeability, brain water content, MDA, apoptosis, AQP4 in the model group were increased(all P<0.05), the platform residence time, number of platform crossings, body weight, SOD, CAT, Bcl-2/Bax, p-PI3K/PI3K, p-AMPK/AMPK,p-GSK-3β/GSK-3β in brain tissue in the model group were decreased(all P<0.05). Compared with the model group,the brain tissue structure and above indicators of in the EP low, middle and high dose groups were significantly improved(all P<0.05), and showed a dose-dependent manner(all P<0.05). The results of in vitro experiment showed that compared with the control group, the cell survival rate, Bcl-2/Bax, p-PI3K/PI3K, p-AMPK/AMPK, p-GSK-3β/GSK-3β in the model group were significantly reduced(all P<0.05), apoptosis rate was significantly increased(P<0.05).Compared with the model group, the above indicators in the EP low, medium, and high dose groups were significantly improved(all P<0.05), and showed a dose-dependent manner(all P<0.05). Conclusion EP can alleviate neuronal apoptosis and inflammatory response after ICH in muce, thereby protecting neural function in a dose-dependent manner. The mechanism may involve EP activation of the PI3K/Akt/GSK-3β signaling pathway, which plays a protective role against neuronal damage after ICH.