Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy

WANG Xiaopeng; YANG Huiju; SUN Mingming; LIU Jing; WU Bensheng; YUE Yinzi; QIN Yuanyuan; CHEN Yinghui; TIAN Lie; LI Yan; WANG Yahui; YAN Shuai

doi:10.13422/j.cnki.syfjx.20230340

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Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy

更新时间：2023-06-02

- Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 13, Pages: 45-53(2023)
- 作者机构：
  
  1.南京中医药大学附属苏州市中医医院，江苏苏州 215009
  2.河南中医药大学第三附属医院，郑州 450000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20230340
  CLC： R2-0;R22;R33;R289;R318.14
- Published：05 July 2023，
  
  Published Online：21 February 2023，
  
  Received：13 December 2022，
- 稿件说明：
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王晓鹏,杨会举,孙明明等.枳实-白术调节PINK1/Parkin信号通路介导的线粒体自噬改善慢传输型便秘大鼠结肠动力障碍[J].中国实验方剂学杂志,2023,29(13):45-53.

WANG Xiaopeng,YANG Huiju,SUN Mingming,et al.Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):45-53.
王晓鹏,杨会举,孙明明等.枳实-白术调节PINK1/Parkin信号通路介导的线粒体自噬改善慢传输型便秘大鼠结肠动力障碍[J].中国实验方剂学杂志,2023,29(13):45-53. DOI： 10.13422/j.cnki.syfjx.20230340.

WANG Xiaopeng,YANG Huiju,SUN Mingming,et al.Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(13):45-53. DOI： 10.13422/j.cnki.syfjx.20230340.

摘要

目的

基于线粒体自噬及磷酸酶及张力蛋白同源物诱导的蛋白激酶1（PINK1）/帕金蛋白（Parkin）通路观察枳实、白术及其配伍对慢传输型便秘大鼠结肠动力障碍的改善作用，为临床精准用药提供理论参考。

方法

将56只雄性SD大鼠按体质量随机分成正常组、模型组、自然恢复组、枳实组、白术组、枳实-白术组和莫沙必利组，每组各8只。除正常组外，采用洛哌丁胺连续14 d灌胃（3 mg·kg

-1

·d

-1

）构建慢传输型便秘大鼠模型。造模成功后，除模型组继续洛哌丁胺诱导外，正常组和自然恢复组采用0.9%生理盐水灌胃，枳实组（1.35 g·kg

-1

·d

-1

）、白术组（2.7 g·kg

-1

·d

-1

）、枳实-白术组（4.05 g·kg

-1

·d

-1

）和莫沙必利组（1.56 mg·kg

-1

·d

-1

）大鼠分别给予相应的药物灌胃，连续7 d。观察药物对大鼠粪便数量、粪便含水率及小肠推进率的影响；苏木素-伊红（HE）染色法和阿尔新兰-过碘酸雪夫染色（AB-PAS）观察结肠病理变化；紫外分光光度计测定结肠组织呼吸链复合体活性；透射电子显微镜观察结肠组织超微结构；实时荧光定量聚合酶链式反应（Real-time PCR）检测PINK1、Parkin和p62 mRNA表达；蛋白免疫印迹法（Western blot）检测微管相关蛋白1轻链3（LC3）、线粒体自噬相关蛋白PINK1和Parkin的表达水平。

结果

与正常组比较，模型组和自然恢复组大鼠粪便数量、含水率及小肠推进率均明显下降（

0.05，

0.01），结肠组织线粒体呼吸链复合体Ⅱ、Ⅲ和Ⅳ活性明显降低（

0.05，

0.01），PINK1、Parkin mRNA表达，PINK1、Parkin和LC3蛋白表达显著上调（

0.01），p62 mRNA表达明显下降（

0.05）；与模型组和自然恢复组比较，枳实-白术组大鼠粪便数量、含水率、小肠推进率及线粒体呼吸链复合体Ⅱ、Ⅲ和Ⅳ活性均明显提高（

0.05，

0.01）；透射电镜结果显示，枳实-白术组可减轻结肠组织线粒体肿胀程度，并能明显降低PINK1、Parkin mRNA表达，降低PINK1、Parkin和LC3蛋白表达（

0.05，

0.01），明显升高p62 mRNA表达（

0.05）。

结论

枳实、白术配伍后可显著改善洛哌丁胺诱导的大鼠慢传输型便秘，其机制可能与阻断PINK1/Parkin信号通路抑制结肠组织中Cajal间质细胞线粒体过度自噬有关。

Abstract

Objective

To observe the effects of Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and their combination on slow transit constipation via PTEN-induced putative kinase 1 （PINK1）/Parkin pathway-mediated mitophagy.

Method

Fifty-six male SD rats were randomly assigned into normal group， model group， natural recovery group， Aurantii Fructus Immaturus group， Atractylodis Macrocephalae Rhizoma group， Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group， and mosapride group， with 8 rats in each group. Slow transit constipation model was established by gavage with loperamide （3 mg·kg

-1

·d

-1

） for 14 days in other groups except the normal group. After successful modeling， except that the model group was continuously induced by loperamide， the normal group and the natural recovery group were administrated with 0.9% normal saline by gavage， and the rats in the Aurantii Fructus Immaturus （1.35 g·kg

-1

·d

-1

） group， the Atractylodis Macrocephalae Rhizoma （2.7 g·kg

-1

·d

-1

） group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma （4.05 g·kg

-1

·d

-1

） group， and the mosapride （1.56 mg·kg

-1

·d

-1

） group were administrated with corresponding drugs by gavage for 7 days. The amount of feces， fecal water content， and intestinal propulsion rate of rats were determined. The pathological changes of the colon were evaluated by hematoxylin-eosin （HE） staining and Alcian blue-periodic acid-Schiff （AB-PAS） staining. The activity of respiratory chain complex and the ultrastructure of the colon tissue were determined by ultraviolet spectrophotometry and observed by transmission electron microscopy， respectively. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of PINK1， Parkin， and p62， and Western blot to determine the protein levels of microtubule-associated protein 1 light chain 3 （LC3）， PINK1， and Parkin.

Result

Compared with the normal group， the model group and the natural recovery group showed decreases in the amount of feces， fecal water content， intestinal propulsion rate （

0.05，

0.01）， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ in the colon tissue （

0.05，

0.01）. Further， the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 were up-regulated （

0.01） and the mRNA level of p62 was down-regulated in the model group （

0.05） and the natural recovery group. Compared with the model group and the natural recovery group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group showed increased amount of feces， fecal water content， intestinal propulsion rate， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ （

0.05，

0.01）. Moreover， the combination meliorated the degree of mitochondrial swelling in the colon tissue， down-regulated the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 （

0.05，

0.01）， and up-regulated the mRNA level of p62 （

0.05）.

Conclusion

Aurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma， and their combination may remedy the colonic motility disorders in rats with slow transit constipation by blocking PINK1/Parkin signaling pathway to inhibit the excessive mitophagy in interstitial cells of Cajal in the colon tissue.

关键词

枳实白术慢传输型便秘自噬磷酸酶及张力蛋白同源物诱导的蛋白激酶1/帕金蛋白信号通路

Keywords

Aurantii Fructus ImmaturusAtractylodis Macrocephalae Rhizomaslow transit constipationautophagyPTEN-induced putative kinase 1/Parkin signaling pathway

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