牡荆素调控PERK-CHOP内质网应激途径对帕金森病模型小鼠神经功能的改善作用研究

Improvement effect study of Vitexin on neurological function in Parkinson′s disease model mice by regulating PERK-CHOP endoplasmic reticulum stress pathway

目的探讨牡荆素调控蛋白激酶R样内质网激酶(PERK)-C/EBP同源蛋白(CHOP)内质网应激途径对帕金森病(PD)模型小鼠神经功能的改善作用。方法选取C57BL/6J小鼠60只,通过腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)构建PD小鼠模型,将小鼠随机分为激活剂组(50 mg/kg牡荆素+2 mg/kg的PERK激活剂CCT020312)、模型组、阳性对照组(20 mg/kg左旋多巴)、高剂量牡荆素组(50 mg/kg)、低剂量牡荆素组(25 mg/kg),每组12只,再选12只正常C57BL/6J小鼠,灌胃等体积的生理盐水作为对照组,以牡荆素、左旋多巴、CCT020312分组干预后,通过转棒实验、爬杆实验评估小鼠运动功能;HE染色观察脑部黑质区病理形态;免疫组化法检测小鼠脑组织黑质区TH表达;TUNEL染色检测小鼠海马区神经元凋亡情况;酶联免疫吸附试验(ELISA)检测小鼠脑组织中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6、IL-1β水平;免疫印迹检测小鼠脑组织PERK、CHOP、Bcl-2相关X蛋白(Bax)、半胱氨酸蛋白酶3(Caspase-3)蛋白表达。结果与对照组相比,模型组海马神经元凋亡率、小鼠爬杆时间、黑质区病理损伤、脑组织IL-6、IL-1β、TNF-α水平、脑组织CHOP、Bax、Caspase-3、PERK蛋白表达显著升高,差异有统计学意义(P<0.05),下落潜伏期、TH阳性细胞数目显著降低,差异有统计学意义(P<0.05);与模型组比较,低、高剂量牡荆素组和阳性对照组小鼠爬杆时间、黑质区病理损伤、海马神经元凋亡率、脑组织TNF-α、IL-1β、IL-6水平、脑组织Bax、Caspase-3、PERK、CHOP蛋白表达显著降低,差异有统计学意义(P<0.05),下落潜伏期、TH阳性细胞数目显著升高,差异有统计学意义(P<0.05);CCT020312减弱了高剂量牡荆素对PD小鼠海马区神经元凋亡、黑质区病理损伤和炎症的抑制作用。结论牡荆素可改善PD模型小鼠运动功能障碍,发挥神经保护作用,可能是通过抑制PERK-CHOP内质网应激途径实现。

Objective To investigate the improvement effect of Vitexin on the neurological function in Parkinson′s disease(PD)model mice by regulating protein kinase R-like ER kinase(PERK)-C/EBP homologous protein(CHOP)endoplasmic reticulum stress pathway.Methods A total of 60 C57BL/6J mice were selected,and PD mouse models were constructed by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).Mice were randomly grouped into activator group(50 mg/kg Vitexin+2 mg/kg PERK activator CCT020312),model group,positive control group(20 mg/kg Levodopa),high-dose Vitexin group(50 mg/kg),low-dose Vitexin group(25 mg/kg),with 12 animals in each group,another 12 normal C57BL/6J mice were selected,and were given an equal volume of physiological saline by gavage as the control group.After grouping and intervention with Vitexin,Levodopa and CCT020312,the motor function of mice was evaluated by rotarod test and climbing rod test.HE staining was used to observe the pathological morphology of substantia nigra.The expression of TH in the substantia nigra of mouse brain was detected by immunohistochemistry.TUNEL staining was used to detect neuronal apoptosis in mouse hippocampus.The enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-1βin mouse brain tissue.Western blot was used to detect the protein expression of PERK,CHOP,Bcl-2-associated X protein(Bax)and cysteine protease 3(Caspase-3)in mouse brain tissue.Results Compared with the control group,the apoptosis rate of hippocampal neurons,climbing time of mice,pathological damage to the substantia nigra,levels of IL-6,IL-1βand TNF-αin brain tissue,and protein expression of CHOP,Bax,Caspase-3,and PERK in brain tissue in the model group were significantly increased,and the differences were statistically significant(P<0.05),and the fall latency and the number of TH-positive cells were obviously decreased,and the differences were statistically significant(P<0.05).Compared with the model group,the climbing time of mice,pathological damage to the substantia nigra,apoptosis rate of hippocampal neurons,levels of TNF-α,IL-1β,IL-6 in brain tissues,the protein expressions of Bax,Caspase-3,PERK and CHOP in brain tissue were obviously decreased in the low and high dose Vitexin groups and the positive control group,and the differences were statistically significant(P<0.05),and the fall latency and the number of TH-positive cells were obviously increased,and the differences were statistically significant(P<0.05).CCT020312 attenuated the substantia nigra inhibitory effects of high-dose Vitexin on neuronal apoptosis,pathological damage to the substantia nigra and inflammation in the hippocampus of PD mice.Conclusion Vitexin could ameliorate motor dysfunction in PD model mice,and plays a neuroprotective role,which may be achieved by inhibiting the PERK-CHOP endoplasmic reticulum stress pathway.