Neurorotective Effect of Chaihu Jia Longgu Mulitang on Parkinson's Disease with Depression Model Rats and Its Mechanism Based on AMPK/mTOR Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo observe the protective effect of Chaihu Jia Longgu Mulitang （CLMT） on dopaminergic neurons in Parkinson's disease with depression （PDD） model rats， and to explore the mechanism based on adenosine monophosphate-activated protein kinase/mammalian target of rapamycin （AMPK/mTOR） signaling pathway. MethodAmong the 80 male SD rats， 10 were randomly selected as normal group and the rest were treated with long-term low-dose subcutaneous injection of rotenone in the neck and back combined with chronic unpredictable mild stress （CUMS） to establish PDD rat model. The successfully modeled PDD rats were randomly divided into model group， western medicine group （madopar 0.032 g·kg-1+fluoxetine hydrochloride 0.002 g·kg-1）， CLMT low-dose， medium-dose and high-dose groups （5， 10 and 20 g·kg-1）， 10 rats in each group. Normal group and model group were administrated with the same amount of normal saline by gavage for 4 consecutive weeks. Behavioral changes of rats in each group were evaluated by open field test and pole climbing test. The content of dopamine （DA） and 5-hydroxytryptamine （5-HT） in cerebrospinal fluid was determined by high performance liquid chromatography （HPCL）. The pathological changes of dopaminergic neurons in substantia nigra of rats were observed by hematoxylin-eosin （HE） staining. The positive expression of tyrosine hydroxylase （TH） and expression of α-synuclein in substantia nigra were detected by immunohistochemistry （IHC） and immunofluorescence （IF）， repsectively. The protein expression of microtubule-associated protein 1 light chain 3 （LC3）， adenosine monophosphate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， mammalian target of rapamycin （mTOR） and phosphorylated mTOR （p-mTOR） was detected by Western blot. ResultCompared with the conditions in normal group， the total horizontal distance and the activity time in the central region in open field test and the content of DA and 5-HT in cerebrospinal fluid were decreased （P<0.05， P<0.01）， and the time of pole climbing was shortened （P<0.01）， with increased score （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group increased the total horizontal distance and activity time in the central region and the content of DA and 5-HT （P<0.05， P<0.01）， and extended the time of climbing pole （P<0.05）， with decreased score （P<0.05， P<0.01）. Compared with those in normal group， the number of dopaminergic neurons in the substantia nigra was reduced， with narrowed and loosely arranged cell body. The fluorescence expression of α-synuclein was enhanced （P<0.01）， and the positive expression of TH was decreased （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group showed elevated number of dopaminergic neurons in the substantia nigra， with enlarged cell body， and decreased fluorescence expression of α-synuclein， and enhanced the positive expression of TH （P<0.05， P<0.01）. Compared with normal group， model group had lowered expression of LC3Ⅱ/Ⅰ， p-AMPK/AMPK in striatum （P<0.05， P<0.01） and increased expression of p-mTOR/mTOR （P<0.01）. Compared with those in model group， LC3Ⅱ/Ⅰ and p-AMPK/AMPK expression were increased （P<0.05， P<0.01） and p-mTOR /mTOR expression was decreased （P<0.01） in CLMT high-dose group and western medicine group. ConclusionCLMT exerts a neuroprotective effect by inhibiting rotenone neurotoxicity. It enhances the level of DA， and thus improves the depression condition in rats with Parkinson's disease. The underlying mechanism may be related to the regulation of AMPK/mTOR signaling pathway， activation of autophagy， and promotion of degrading α-synuclein.