胡椒碱对帕金森痴呆模型小鼠小肠部位的自噬作用机制研究

Autophagic activity of piperine on small intestine in dementia model mice with Parkinson′s disease

该文旨在研究胡椒碱对帕金森痴呆(Parkinson′s disease with dementia,PDD)模型小鼠小肠部位的作用机制。将96只无特定病原体级别的雄性C57BL/6小鼠随机分为8组:正常组,模型组,自噬抑制剂组(6-氨基-3-甲基嘌呤,3MA,30 mg·kg^-1),自噬激活剂组(雷帕霉素,1 mg·kg^-1),胡椒碱低、中、高剂量组(10,20,40 mg·kg^-1)和美多巴组(112.5 mg·kg^-1)。除正常组外,其他各组小鼠均皮下注射利血平(0.1 mg·kg^-1),每48 h给药1次,持续40 d。此外,在给药第20天时,除正常组外,其余各组小鼠均再进行双侧颈总动脉阻断术,最终制备为PDD模型。同时,各组均给予相对应的药物治疗,每天1次,持续40 d。末次给药结束后,采用自主活动试验和热板试验观察小鼠的行为学变化;采用免疫组化法检测小肠组织中α-突触核蛋白(α-synuclein,α-syn)和酪氨酸羟化酶(tyrosinehydroxylase,TH)表达;免疫荧光法检测小肠组织中beclin-1、微管相关蛋白轻链3B(microtubule-associated protein 1 light chain 3B,LC3B)和p62表达;苏木精-伊红染色法观察各组小肠组织的病理形态;酶联免疫吸附法检测小鼠组织β淀粉样蛋白前体蛋白(β-amyloid precursor protein,APP)、磷酸化tau蛋白(phospho tauprotein,p-tau)、乙酰胆碱转移酶(choline acetyltransferase,ChAT)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;实时荧光定量聚合酶链式反应法检测小肠组织α-syn,TH,beclin-1,微管相关蛋白轻链3(microtubule-associated protein 1 light chain 3,LC3)和p62的mRNA及mmu-miR-99a-5p表达。结果发现,与模型组比较,3MA组、胡椒碱各剂量组和美多巴组的活动次数、ChAT、TH和p62表达显著增加(P<0.05),3MA组、胡椒碱各剂量组和美多巴组的首次舔足时间缩短,APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3B和mmu-miR-99a-5p表达显著减少(P<0.05),雷帕霉素组的活动次数、ChAT、TH和p62表达显著减少(P<0.05),雷帕霉素组的APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3B和mmu-miR-99a-5p表达显著增加(P<0.05);与3MA组比较,胡椒碱低、中剂量组和雷帕霉素组的活动次数、ChAT、TH和p62表达显著减少(P<0.05),首次舔足时间显著延长,APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3B和mmu-miR-99a-5p表达显著增加(P<0.05);与美多巴组比较,胡椒碱低剂量组和雷帕霉素组的活动次数、ChAT、TH和p62表达显著减少(P<0.05),首次舔足时间显著延长,APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3B和mmu-miR-99a-5p表达显著增加(P<0.05)。此外,与正常组相比,模型组和雷帕霉素组的小鼠肠上皮细胞大量脱落,肠黏膜受损严重,小肠绒毛出现水肿及脱落现象;给药治疗干预后,3MA组、胡椒碱各剂量组和美多巴组的肠上皮细胞轻度受损,绒毛少量脱落。综上所述,胡椒碱对帕金森痴呆模型小鼠小肠部位有保护作用,表现为减少mmu-miR-99a-5p、促炎因子和自噬因子的表达,其减缓PDD病理症状的机制可能与抑制自噬有关。

This article is to investigate the effect of piperine on the small intestine of mice with Parkinson′s disease with dementia(PDD).Ninety-six C57 BL/6 mice of SPF grade were randomly divided into 8 groups(male,12 in each group):normal group,model group,autophagy inhibitor group(6-amino-3-methylpurine,3 MA,30 mg·kg^-1),autophagy activator group(rapamycin,1 mg·kg^-1),low,medium,and high dose piperine groups(10,20,40 mg·kg^-1),and medopar group(112.5 mg·kg^-1).Except for the normal group,mice in each group were injected subcutaneously with reserpine(0.1 mg·kg^-1)once every 48 hours for 40 days.In addition,on the 20 th day of administration,except for the normal group,the mice in the other groups were subjected to bilateral common carotid artery occlusion to finally prepare PDD models.At the same time,each group was given the corresponding drug treatment once a day for 40 days.After the last administration,the behavioral changes of mice were observed by autonomic activity experiment and hot plate experiment.The expression levels ofα-synuclein(α-syn)and tyrosine hydroxylase(TH)in the small intestine were detected by immunohistochemistry.The expression levels of beclin-1,microtubule-associated protein 1 light chain 3 B(LC3 B)and p62 in the small intestine were detected by immunofluorescence assay.Hematoxylin-eosin staining was used to observe the pathological morphology of small intestine tissues in each group.Enzyme-linked immunosorbent assay was adopted for detection ofβ-amyloid precursor protein(APP),p-tau,acetylcholine transferase(ChAT),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in small intestine.Real-time fluorescent quantitative polymerase chain reaction was used to detect the expression ofα-syn,TH,beclin-1,microtubule-associated protein 1 light chain 3(LC3),and p62 mRNA and mmu-miR-99 a-5 p in the small intestine.The results of this study showed that,as compared with the model group,the number of activities,the expression levels of ChAT,TH,and p62 were significantly increased in the 3 MA group,the various piperine dose groups,and the medopar group(P<0.05),and their first foot licking time was shortened;APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3 B and mmu-miR-99 a-5 p expression levels were significantly reduced(P<0.05).However,as compared with the model group,the number of activities,ChAT,TH,and p62 expression levels in the rapamycin group were significantly reduced(P<0.05),and the APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3 B and mmu-miR-99 a-5 p expression levels were significantly increased(P<0.05).As compared with the 3 MA group,the number of activities,ChAT,TH,and p62 expression levels were significantly reduced in the low and medium dose piperine groups and rapamycin group(P<0.05);howe-ver,their first foot licking time was significantly prolonged,APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3 B and mmu-miR-99 a-5 p expression levels were increased significantly(P<0.05).As compared with the medopar group,the number of activities,ChAT,TH,and p62 expression levels were significantly reduced in low dose piperine group and rapamycin group(P<0.05),but their first foot licking time was significantly extended,and APP,p-tau,IL-6,TNF-α,α-syn,beclin-1,LC3 B and mmu-miR-99 a-5 p expression levels were significantly increased(P<0.05).In addition,as compared with the normal group,the small intestinal epithelial cells of the model group and the rapamycin group were shed off a lot,with severe damages of intestinal mucosa as well as edema and shedding of the small intestine villi.After administration of the therapeutic interventions,the small intestinal epithelial cells of the 3 MA group,each dose group of piperine,and the medopa group were slightly damaged and the villi were slightly shed off.In summary,piperine has a protective effect on the small intestine of PDD model mice,showing reduced expression of mmu-miR-99 a-5 p,pro-inflammatory factors and autophagy factors,and the mechanism of slowing PDD pathological symptoms may be related to the inhibition of autophagy.