| Background:Many Researches showed that autophagy is strictly regulated in neurons,and the dysfunction of autophagy is tightly related to the degeneration of neurons.Autophagy plays a key role in clearing the misfolded proteins and easily aggregated proteins related to neurodegenerative diseases,especially in the occurrence and development of Parkinson's disease(PD).ATP and its analogues affect the response of immune cells to inflammation by activating different purine receptors.P2X7R(P2X7R)has a strong pro-inflammatory factor release effect.In the central nervous system,abnormal expression or function of P2X7 R may cause DA mage to neurons and glial cells.Brilliant blue G(BBG)is a powerful unselective antagonist of P2X7 R.Previous studies have shown that BBG can increase dopamine neurons in PD model,but the specific mechanism is not described.Objective: 1.To explore the role of BBG in MPTP mouse model.2.To study the changes of autophagy in MPTP mice.3.To explore whether BBG plays a role in MPTP injury model through autophagy.Methods: 1.Animal selection and grouping: C57/BL6 J male mice(23-26 g,8-10 weeks old)with moderate SPF weight and mouse age were randomly divided into control group(control-ns group),model group(MPTP group),intervention group(MPTP + BBG group)and intervention control group(BBG group).2.Model preparation: MPTP group was intraperitoneally injected(ip)with MPTP solution for 5 consecutive days,and control group was intraperitoneally injected with normal saline in the same proportion with MPTP solution at the same time every day.In MPTP + BBG group,BBG was injected intraperitoneally one day before and one day after MPTP.In BBG group,only equal proportion of BBG solution was injected.3.Behavioral experiments: 3 days after the last injection of BBG solution,open field experiment,climbing pole experiment and tail suspension experiment were carried out to detect the movement and anxiety state of mice in each group.4.The mice were perfused and fixed,and then the mouse brain was frozen and sectioned,double immunofluorescence staining of p62 and LC3-?,Immunohistochemistry to evaluate of cell survival and autophagy activation.5.Decapitate the brain,extract the protein,and detect the expression of TH,?-synuclein(?-syn),Cleaved Caspase 3,m TOR,p-m TOR,AMPK,p-AMPK,p62,LC3-? and other proteins by Western blot.the amount.Results: 1.BBG as the intervention control group,its results were not statistically significant compared with the control group(P<0.05).2.Compared with MPTP group,MPTP + BBG group significantly increased the activity time and entry times in the central area of the open field(P<0.05);in the pole Pole test,MPTP + BBG group decreased the turning time compared with MPTP group(P<0.05).The score of MPTP + BBG group was higher than that of MPTP group(P<0.05).3.Western blot showed that compared with MPTP group,MPTP + BBG group significantly increased TH expression in substantia nigra and striatum(P<0.05),and decreased ?-syn expression(P<0.05).4.Immunohistochemistry showed that compared with control group,MPTP reduced the in striatum and substantia nigra(P<0.05),while BBG improved the DA mage by MPTP(P<0.05).5.Compared with MPTP group,the expression of p-m TOR and p62 protein in BBG group decreased(P<0.05),but the expression of p-AMPK,LC3-? increased(P<0.05).Fluorescence colocalization showed that the number of TH cells in the control group was more than that in the MPTP group,and that in the MPTP group,there was significantly reduced.The fluorescence intensity of p62 was stronger in MPTP(P<0.05),but decreased after BBG intervention(P<0.05).Conclusions:1.BBG reduced the anxiety state produced by MPTP in mice.2.BBG reduced dopaminergic neuron DA mage produced by MPTP in mice.3.The autophagy pathway was inhibited in MPTP mice,while BBG enhanced autophagy in striatum and substantia nigra of MPTP mice,which played a protective role in MPTP injury model. |
PDF Full Text Request