Study On The Mechanism Of Buyin Qianzheng Regulating Calcium Homeostasis And Inhibiting The Loss Of DA Neurons In PD Mice

BackgroundParkinson's disease(PD)is the second highest neurodegenerative disease in the world,especially among middle aged and elderly people.The number of patients in China is about half of that in the world,and with the increase of the aging population in China,the incidence rate is increasing year by year.The typical pathological change of PD is degeneration and death of dopamine(DA)neurons in the substantia nigra of the middle brain,resulting in decreased DA content in striatum,accmpanied with motor symptoms such as motor retardation,myotonia and quiescence tremor as well as non-motor symptoms such as constipation.dysphagia,sleep disorder and depression.The pathogenesis of PD is complicated.In recent years,the research on the relationship between apoptosis and Ca2+has been increasing,and it is generally believed that the imbalance of calcium homeostasis leads to the apoptosis of DA neurons and then leads to PD,which is one of the main mechanisms.Currently,there is no effective cure for PD.Drugs like Dopamine replacement drugs,monoamine oxidase inhibitors,catecholol oxymethyl transferase inhibitors,anticholinergic drugs have certain effects for improving clinical symptoms of patients,but the curative effects are reduced and side effects are obvious while long-term using of these drags.Traditional Chinese medicine which is based on the overall concept and the theoretical system of syndrome differentiation and treatment plays an important role in the treatment of PD with traditional Chinese medicines,which can significantly improve the motor symptoms and non-motor symptoms of patients with lasting and stable curative effects and few adverse reactions.Research reveals that the proportion of PD with liver and kidney Yin deficiency in clinical practice ranks the first among all syndromes,and the other syndrome types are derived from it.The method of nourishing liver and kidney is effective in the treatment of PD.Buyin Qianzheng Formula is the combination of Dabu Yinwan and Qianzheng San and composed of commonly used drugs,such as rhizoma anemarrhenae,prepared rehmannia root,turtle board,stiffened silkworm and scorpion.It can nourish the liver and kidney to cure the root cause of the disease,calm the wind and clear the collaterals to treat the symptoms of the disease.In our previous research.PD model mice mediated by the neurotoxin 1-methy l-4-phenyl-1,2,3.6tetrahydropyridine(MPTP)were treated with Buyin Qianzheng Formula,showing a significant protective effect on the number and morphology of DA neurons in the substantia nigra.On this basis,from the perspective of Ca2+,this study further explores the mechanism and regulation of Buyin Qianzheng Formula on the apoptosis of DA neurons in PD.ObjectiveFrom the perspective of regulating Ca2+ homeostasis by Buyin Qianzheng Formula.this study revealed the correlation between the protective effect of this formula on the functions of mitochondria and endoplasmic reticulum in the brain of PD mice and the regulation of Ca2+concentration in cytoplasm,mitochondria and endoplasmic reticulum and related factors during the pathogenesis of PD.In this study,we investigated the link of Buyin Qianzheng Formula in inhibiting the apoptosis of DA neurons in mitochondrial and endoplasmic reticulum pathways and maintaining Ca2+ homeostasis,and further clarified the molecular mechanism on Buyin Qianzheng Formula to protect DA neurons,providing experimental basis for the clinical application of Buyin Qianzheng Formula in the treatment of PD.MethodsExperiment 1:The inhibitory effect of Buyin Qianzheng Formula on the apoptosis of neurons in the brain of PD mice.The experiment was divided into 3 groups,?control group:no treatment;?model group:intraperitoneal injection of MPTP;?traditional Chinese medicine group:in addition to intraperitoneal injection of MPTP,intragastric administration of Buyin Qianzheng Formula decoction.The behavior of mice was detected by the pole test and hanging experiment.The changes of TH positive neurons in the substantia nigra were observed by immunofluorescence method.The content of DA and its metabolites in forebrain was determined by HPLC.Tunel method was used to detect the apoptosis of DA neurons in the substantia nigra.Experiment 2:The effect of Buyin Qianzheng Formula on the function of mitochondria and endoplasmic reticulum in the brain of PD mice.The experiment groups are the same as before.The content of brain ATP was determined by luciferase method.The content of brain ROS was detected by fluorescence staining.The changes of Cyt c positive cells in the substantia nigra were observed by immunofluorescence method.The expression levels of Cyt c,caspase3 and GRP78 proteins in the brain were detected by Western Blot.Experiment 3:The effect of Buyin Qianzheng Formula on Ca2+and related channel proteins in the brain of PD mice.The experiment groups are the same as before.Ca2+ specific fluorescent probes were used to detect the concentration of cytoplasmic free Ca2+,endoplasmic reticulum Ca2+ and mitochondrial Ca2+ in the brain.Western Blot was used to detect the expression levels of VDAC1 protein on the mitochondrial outer membrane and MCU protein on the mitochondrial inner membrane.ResultsExperiment 1:1.Behavior:MPTP increased the climbing time and decreased the hanging score of C57BL/6J mice.Buyin Qianzheng Formula reduced MPTP-induced climbing time and increased MPTP-induced hanging score.2.Neurons and DA content in the substantia nigra:MPTP caused the number of TH positive neurons in the substantia nigra to decrease,the apoptosis rate of neurons in the substantia nigra to increase and the content of DA and its metabolites in the forebrain to decrease.These changes caused by MPTP were improved by Buyin Qianzheng Formula,compared with the model group,mice in the traditional Chinese medicine group had significantly increased number of TH positive neurons in the substantia nigra,decreased apoptosis rate of neurons in the substantia nigra,increased DA content in the forebrain but there was not significant difference in the content of DA metabolites in the forebrain.Experiment 2:1.The function of Mitochondria and endoplasmic reticulum:MPTP decreased ATP content and increased ROS content in the brain of C57BL/6J mice.Buyin Qianzheng Formula increased ATP content and decreased ROS content in the brain of MPTP-treated mice.MPTP increased GRP78 protein level in the brain of C57BL/6J mice.Buyin Qianzheng Formula reduced MPTP-induced overexpression of GRP78 protein.2.Apoptosis mediated by mitochondria pathway and endoplasmic reticulum pathway:MPTP led to increased expression of Cyt c and Caspase-3 proteins in brain of C57BL/6J mice,and enhanced fluorescence of Cyt c in substantia nigra.Buyin Qianzheng Formula reduced the overexpression of Cyt c and Caspase-3 proteins in the brain of PD mice mediated by MPTP as well as the fluorescence intensity of Cyt c in the substantia nigra.Experiment 3:1.Ca2+ concentration in the brain:MPTP caused Ca2+ concentration in the endoplasmic reticulum to decrease,cytoplasmic and mitochondrial Ca2+ to increase in the brain of C57BL/6J mice.Buyin Qianzheng Formula antagonized these changes caused by MPTP,and caused increase of Ca2+ in the endoplasmic reticulum as well as decrease of cytoplasmic and mitochondrial Ca2+in the brain of mice.2.Related Ca2+ channel proteins:MPTP up-regulated the expression of VDAC1 and MCU proteins in C57BL/6J mice.The overexpression of VDAC1 and MCU proteins caused by MPTP reduced by Buyin Qianzheng Formula.Conclusions1.MPTP causes excessive release of Ca2+ from the endoplasmic reticulum to mitochondria,leading to Ca2+ depletion of the endoplasmic reticulum and mitochondrial Ca2+ overload.These changes result in dysfunction of the endoplasmic reticulum and mitochondria,which leads to increased apoptosis of DA neurons,decreased number of DA neurons and decreased content of DA and its metabolites,finally leading to the occurrence of PD.2.Buyin Qianzheng Formula may reduce the expression of VDAC1 and MCU proteins in the brain of PD mice,and then inhibit excessive release of Ca2+ from the endoplasmic reticulum to mitochondria which reduces mitochondrial Ca2+overload and maintains intracellular Ca2+homeostasis,so as to protect the endoplasmic reticulum and mitochondrial function and reduce the expression of apoptosis factors such as Cyt c and Caspase-3,thus inhibiting the apoptosis of DA neurons and protecting DA neurons.