“抗帕颗粒”对帕金森病小鼠结肠ICC细胞及线粒体自噬相关蛋白Beclin-l/LC3B表达的影响

Anti-Parkinson's disease granules on colon ICC cells of Parkinson's disease mice and the influence of mitochondrial autophagy related protein Beclin-l/LC3B expression

目的探讨中药“抗帕颗粒”通过调控Cajal间质细胞(ICC)及线粒体自噬改善帕金森病(PD)肠道动力障碍的作用机制。方法PD小鼠模型制作采用MPTP腹腔注射法,随机法分组:空白组(A组)、PD对照组(B组)和PD干预组(“抗帕颗粒”)(C组),每组10只。A、B组予生理盐水灌胃(1ml·d^(-1)),C组“抗帕颗粒”灌胃(40mg·kg^(-1)·d^(-1))。4个月后每组中随机取5只小鼠结肠组织被用于免疫组化检测,每组余下5只小鼠结肠壁组织被用于提取蛋白Western blotting法检测,检测内容c-kit、Beclin l、LC3B的表达;透射电子显微镜观测结肠壁ICC细胞结构。结果(1)免疫组化结果显示:与A组相比,B组、C组C-kit、Beclin1、LC3B表达水平均显著升高(^(a)P<0.01),与B组相比,C组C-kit、LC3B表达水平显著降低(^(c)P<0.01),而Beclin1的表达水平无显著变化;(2)Western blot结果显示,与A组相比,B组、C组Beclin1、LC3B、C-kit表达水平均显著升高(^(a)P<0.01);与B组相比,C组的Beclin1、LC3B、C-kit表达水平均显著降低(^(c)P<0.01);(3)透射电子显微镜观察显示,PD模型小鼠的结肠ICC中出现自噬囊泡,“抗帕颗粒”干预后PD模型小鼠自噬囊泡减少。结论“抗帕颗粒”通过抑制肠道ICC过度自噬,保护细胞结构;并且抑制了由ICC过度自噬导致的细胞程序性死亡而出现的ICC数量反应性增加;其主要作用是改善了ICC结构的异常。这可能是“抗帕颗粒”改善PD小鼠肠道传输功能的机制之一。

Objective To discuss the possible machining of anti-Parkinson's disease(PD)granules in improving intestinal motility disorders in PD by regulating Cajal interstitial cells(ICC)and mitochondrial autophagy.Methods MPTP was used to build PD model,the experimental micewere randomly divided into blank control group(group A),PD control group(group B)and PD intervention group(group C),with 10 mice in each group.The concentration of anti-PD granules in group C was 800mg·mL^(-1) and the dose was 40mg·kg^(-1)·d^(-1).Groups A and B were given 1ml·d^(-1) normal saline by gavage.After 4 months of cage feeding,colon tissues of 5 mice in each group were randomly selected for immunohistochemical detection,and the colon wall tissues of the remaining 5 mice in each group were used for protein extraction Western blotting detection,and the detection contents were as follows:c-kit,Beclin l,LC3B expression;The structure of ICC cells in colon wall was observed by transmission electron microscope.Results①In terms of immunohistochemistry,the C-Kit,Beclin 1 and LC3B levels ofgroup B and C were significantly increased(^(a)P<0.01)than group A;and thelevels of C-KIT and LC3B in group C were remarkably decreased(^(b)P<0.01)than group B,but the Beclin 1 had no significant change between group B and C;②Western blot results:to group A,the expression levels of Beclin1,LC3B and C-Kit in groups B and C were significantly increased(^(c)P<0.01);to group B,Beclin1,LC3B and C-KIT levels in group C were notably decreased(^(d)P<0.01);③Transmission electron microscopy showed that autophagy vesicles appeared in the colon ICC of PD model mice,and the autophagy vesicles of PD model mice were reduced after the intervention of anti-PD granules.Conclusion Anti-PD granules can protect the cellular structure of intestinal ICC by inhibiting excessive autophagy.The increase in ICC number reactivity caused by programmed cell death caused by ICC autophagy was inhibited.Its main function is to improve the anomaly of ICC structure.This may be one of the mechanisms by which Anti-PD granules improve the intestinal transport function of PD mice.