Pharmacodynamic Evaluation And Mechanism Of Bushen Yizhi Formula In Treating Parkinson’s Disease

ObjectiveParkinson’s disease(PD)is the second severest neurodegenerative disease associated with aging following Alzheimer’s disease(AD).Hence,it is of great significance to elucidate the pathophysiological mechanism of PD and to find effective drugs to alleviate the course of PD.Bushen Yizhi formula(BSYZ)is a compound of traditional Chinese medicine for tonifying Zang of kidney and benefiting wisdom.The clinical research has proved that it has the effect of anti-dementia and improving the score of nerve function.Neuroinflammatory theory plays an important role in the pathological process of PD.Inhibition of central inflammation may be an effective strategy to alleviate PD process.In recent years,the study of inflammatory corpuscles of nod like receptor heat domain associated protein 3(nod-like receptor pyrin domain containing 3,NLRP3)has attracted extensive attention of researchers in central nervous degenerative diseases such as ad and PD.It has been proved that inhibiting the activation of NLRP3 inflammatory corpuscles can effectively inhibit the central inflammation and relieve the limb motor disorders and dopamine neuritis neurodegenerative changes in PD model animals.In this study,we aimed to investigate the therapeutic effect of Bushen Yizhi recipe on limb motor disorder induced by MPTP in PD animal model.Furthermore,the possible mechanism of BSYZ was verified on the model of BV2 cells activation induced by LPS and ATP in vitro,which provides theoretical support for the treatment of PD by BSYZ.MethodsThe acute MPTP model of mice was established by intraperitoneal injection of MPTP(MPTP 18 mg/kg,i.p.,4 times/day at 2h intervals)in C57BL/6 mice.One week before model establishment,BSYZ(1.46,2.92,5.84 g/kg/d)was given orally in different doses to the treatment group,while the positive control group was treated with piroxicam orally.On the 1st,3rd and 5th days after the model was established,the mice were transferred to a stick experiment,and on the 6th day,the Y-maze test was used to detect the motor coordination and balance ability of the mice.Mice were continued to be treated by gavage while the behavioral experiment is under way.On the second day after the end of behavior test,the animals were killed,the whole brain was taken out,the midbrain and striatum of mice were isolated,and the pathological and biochemical indexes were detected at the later stage.Nissl’s staining was used to observe the basic morphology of dopaminergic neurons in the substantia nigra pars compacta.Immunofluorescence staining was used to observe the expression of TH-positive cells in substantia nigra pars compacta and the activation of microglia and astrocytes.Western blotting was used to detect the expression of TH protein,NLRP3 inflammasome related proteins(NLRP3,Caspase-1,Pro-Caspase-1,ASC,pro-IL-1β,Il-1β)and inflammatory nuclear transcription factor NF-κB s level.In vitro,BV2 microglia induced by LPS and ATP were used to mimic the activation of microglia and to induce the activation of NLRP3 inflammatory bodies.The morphology of microglia induced by LPS and ATP was observed under microscope.The positive control group was treated with NLRP3 inhibitor MCC950.Western blotting assay was used to detect the levels of NLRP3 inflammatory corpuscle associated protein(NLRP3 caspase-1)and inflammatory nuclear transcription factor(NF-κB)in cells.The nuclear metastasis of NF-κB was observed by immunofluorescence labeling.Reults1.BSYZ formula could significantly improve the dysfunction of MPTP model mice and alleviate the damage of dopaminergic neurons induced by MPTP;2.BSYZ formula could significantly inhibit the excessive activation of microglia and astrocytes induced by MPTP;3.BSYZ formula could significantly inhibit the activation of NLRP3 inflammatory corpuscles induced by LPS and ATP and the nuclear metastasis of NF-κB in BV2 microglia cells.ConclusionBSYZ formula could ameliorate limb motor disorder,alleviates dopaminergic neuron injury and reduces central inflammatory response in PD model mice.It may play a key role by inhibiting activation of inflammatory bodies of NLRP3,nuclear transfer of NF-κB and activation of microglia.