摘要
目的探究木犀草素(Lut)对脑卒中大鼠认知障碍和神经细胞凋亡的影响及其机制。方法将健康雄性SD大鼠随机分为Sham组、中动脉闭塞(MCAO)组、Lut组、低氧诱导因子-1α(HIF-1α)拮抗剂(YC-1)组、Lut+HIF-1α激动剂(AG1)组,每组20只。除Sham组外,其余各组大鼠均建立MCAO模型。Sham组大鼠正常喂养,给予等量生理盐水;MCAO组大鼠灌胃给予等量体积含5%二甲基亚砜(DMSO)的生理盐水;Lut组大鼠灌胃给予Lut(40 mg/kg);YC-1组大鼠腹膜内给予YC-1(5 mg/kg);Lut+AG1组大鼠灌胃给予Lut(40 mg/kg),腹膜内给予AG1(10 mg/kg)。再灌注24 h后记录上述各组大鼠神经功能缺损评分;核磁共振成像测试用于确定脑梗死体积;苏木精-伊红(HE)和原位缺口末端标记法(TUNEL)染色用于观察脑组织病理变化和神经细胞凋亡;莫里斯水迷宫测试用于评估大鼠的空间学习和记忆能力;相应的试剂盒测定超氧化物歧化酶(SOD)、丙二醛(MDA)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)表达水平;Western blot检测HIF-1α/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)信号通路相关蛋白表达。结果与Sham组比较,MCAO组大鼠神经功能缺损评分(2.86±0.22)和脑梗死体积(22.34±2.15)%显著升高,并显示出明显的空间学习和记忆缺陷,病变神经细胞(2.16±0.18)%和TUNEL阳性细胞(30.26±3.18)%显著增多,MDA(6.57±0.58)nmol/mg、TNF-α(379.65±29.27)pg/mg、IL-6(158.65±13.25)pg/mg水平显著升高,SOD水平(115.32±11.26)U/mg显著降低,HIF-1α(0.89±0.08)、NLRP3(0.96±0.08)和半胱氨酸蛋白酶-1(caspase-1)(0.75±0.07)蛋白表达显著上调,以上差异均有统计学意义(P均<0.05)。与MCAO组比较,Lut组大鼠神经功能缺损评分(1.60±0.15)和脑梗死体积(8.65±0.87)%显著降低,空间学习和记忆缺陷得到明显改善,病变神经细胞(0.68±0.06)%和TUNEL阳性细胞(8.36±0.78)%显著减少,MDA(3.09±0.28)nmoL/mg、TNF-α(222.36±15.48)pg/mg、IL-6(79.65±6.54)pg/mg水平显著降低,SOD水平(186.36±15.36)U/mg显著升高,HIF-1α(0.37±0.03)、NLRP3(0.49±0.04)和caspase-1(0.32±0.03)蛋白表达显著下调,以上差异均有统计学意义(P均<0.05);YC-1组改变趋势与Lut组相似。与Lut组比较,Lut+AG1组大鼠神经功能缺损评分(2.29±0.18)和脑梗死体积(16.29±1.54)%显著升高,并显示出明显的空间学习和记忆缺陷,病变神经细胞(1.56±0.14)%和TUNEL阳性细胞(19.68±1.87)%显著增多,MDA(4.58±0.42)nmoL/mg、TNF-α(305.69±22.45)pg/mg、IL-6(110.32±10.13)pg/mg水平显著升高,SOD水平(153.68±13.65)U/mg显著降低,HIF-1α(0.63±0.06)、NLRP3(0.72±0.07)和caspase-1(0.58±0.05)蛋白表达显著上调,以上差异均有统计学意义(P均<0.05)。结论Lut可通过抑制HIF-1α/NLRP3信号通路减轻认知障碍和神经细胞凋亡,对MCAO大鼠发挥保护作用。
Objective To explore the influences of luteolin(Lut)on cognitive impairment and neuronal apoptosis in stroke rats and its mechanism.Methods Healthy male SD rats were randomly divided into Sham group,middle cerebral artery occlusion(MCAO)group,Lut group,hypoxia inducible factor-1α(HIF-1α)antagonist(YC-1)group,Lut+HIF-1αagonist(AG1)group,20 mice in each group.Except for the Sham group,the MCAO model was established in the other groups.The rats in the Sham group were fed normally and given the same amount of normal saline;the rats in the MCAO group were given the same volume of normal saline containing 5%dimethyl sulfoxide(DMSO)by gavage;the rats in the Lut group were given Lut(40 mg/kg)by gavage;the rats in YC-1 group were given intraperitoneal YC-1(5 mg/kg);the rats in Lut+AG1 group were given Lut(40 mg/kg)by gavage and AG1(10 mg/kg)intraperitoneally.After 24 hours of reperfusion,the neurological deficit scores of the above groups were recorded;MRI was used to determine the volume of cerebral infarction;hematoxylin and eosin(HE)staining and TdT mediated d UTP nick end labeling(TUNEL)staining were used to observe the pathological changes of brain tissue and neuronal apoptosis;morris water maze test used to assess spatial learning and memory ability in rats;corresponding kits were applied to measure the expressions of superoxide dismutase(SOD),malondialdehyde(MDA),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6);Western blot was applied to evaluate the expressions of HIF-1α/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway-related proteins.Results Compared with the Sham group,the neurological deficit score(2.86±0.22)and the cerebral infarction volume(22.34±2.15)%of the rats in the MCAO group were obviously increased,and showed obvious spatial learning and memory deficits;diseased nerve cells(2.16±0.18)%and TUNEL positive cells(30.26±3.18)%were obviously increased;the levels of MDA(6.57±0.58)nmol/mg,TNF-α(379.65±29.27)pg/mg,IL-6(158.65±13.25)pg/mg were obviously increased,the level of SOD(115.32±11.26)U/mg was decreased;the protein expressions of HIF-1α(0.89±0.08),NLRP3(0.96±0.08)and cysteinyl aspartate specific proteinase-1(caspase-1)(0.75±0.07)were obviously upregulated,and the above differences were all statistically significant(P<0.05).Compared with the MCAO group,the neurological deficit score(1.60±0.15)and the cerebral infarction volume(8.65±0.87)%in the Lut group were obviously reduced,and the spatial learning and memory deficits were obviously improved;the lesion nerve cells(0.68±0.06)%and TUNEL positive cells(8.36±0.78)%were obviously less;the levels of MDA(3.09±0.28)nmoL/mg,TNF-α(222.36±15.48)pg/mg,IL-6(79.65±6.54)pg/mg were obviously decreased,the level of SOD(186.36±15.36)U/mg was increased;the protein expressions of HIF-1α(0.37±0.03),NLRP3(0.49±0.04)and caspase-1(0.32±0.03)were obviously down-regulated,and the above differences were all statistically significant(P<0.05);the changing trend of YC-1 group was similar to that of Lut group.Compared with the Lut group,the neurological deficit score(2.29±0.18)and the cerebral infarction volume(16.29±1.54)%of the rats in the Lut+AG1 group were obviously increased,and showed obvious spatial learning and memory deficits;diseased nerve cells(1.56±0.14)%and TUNEL positive cells(19.68±1.87)%were obviously increased,the levels of MDA(4.58±0.42)nmoL/mg,TNF-α(305.69±22.45)pg/mg,IL-6(110.32±10.13)pg/mg were obviously increased,the level of SOD(153.68±13.65)U/mg was obviously decreased;the protein expressions of HIF-1α(0.63±0.06),NLRP3(0.72±0.07)and caspase-1(0.58±0.05)were obviously up-regulated,and the above differences were all statistically significant(P<0.05).Conclusion Lut could alleviate cognitive impairment and neuronal apoptosis by inhibiting HIF-1α/NLRP3 signaling pathway,and exert a protective effect on MCAO rats.
作者
郭彦芳
董秀芳
王瑜龙
董欣
徐贝贝
程瑶
GUO Yan-fang;DONG Xiu-fang;WANG Yu-long;DONG Xin;XU Bei-bei;CHENG Yao(Department of Neurology,Xingtai First Hospital,Xingtai,Hebei 054000;Department of Encephalopathy,Tai'an Hospital of Traditional Chinese Medicine,Tai'an,Shandong 271000,China)
出处
《热带医学杂志》
CAS
2022年第9期1196-1202,1325,共8页
Journal of Tropical Medicine
基金
山东省中医药科技项目(2021M063)
