| Objective:1.We aim to explore the effect of Retenone and MPP+,the common inducers of Parkinson’s disease,on dopaminergic(DA)neurons injury,and the mRNA level of ferroptosis-related pathways,in order to investigate the role of ferroptosis-related pathways in DA neuron injury.2.We aim to explore the protective effect of JTW on DA neuron damage and its possible molecular mechanism through in vivo and in vitro studies,in order to provide pharmacological and molecular basis for the treatment of JTW in PD.Methods:In vitro study:(1)Retenone and MPP+were used to induce the damage of MN9D dopaminergic neurons,in order to construct an in vitro DA neuron damage model.We first selected the appropriate modeling concentration through the MTT experiment,and then observed the effect of Rotenone and MPP+ on the mRNA expression of the ferroptosis pathway by PCR assays.Next,JTW were applied and the effects of JTW on Rotentone and MPP+-induced MN9D cells were evaluated through living/death cells staining,ROS and JC-1 experiments.The protective mechanism of JTW on DA neuron cell injury was explored by PCR experiments.(2)An in vitro model of PD dopaminergic neuron damage was constructed,and Erastin(iron death inducer)was used.Induced neuronal cell line(PC12)damage,constructed an in vitro neuronal iron death cell model,and after selecting a suitable modeling concentration by MTT experiment,based on this,observed different modeling methods for iron death pathway-related genes by PCR.The impact of expression.Next,based on the previous work,drug intervention was conducted,and the protective effects of JTW on two injured cell models were evaluated by MTT experiments,live-dead cell staining,ROS,and JC-1 experiments.Cellular immunofluorescence staining experiments and PCR verification experiments were conducted to study the protective mechanism of JTW on neuronal cell injury model.2.In vivo study:Forty-eight C57/BL6 male mice were randomly divided into control group(Contorl),MPTP group(MPTP),JTW low-dose group(JTW-L)and JTW high-dose group(JTW-H),12 mice in each group.Except the Control group,the remaining three groups were induced by MPTP intraperitoneal injection.In the JTW group,the corresponding dose of JTW was administered by intragastric administration 7 days before modeling,and the MPTP group and Control group were given equal volumes of saline.After modeling,each group was continued to drug administration for 1 week.After that,we prepared frozen sections of brain tissue to label DA neurons in the brain with specific antibodies,and observed the damage of DA neurons in the brain of mice.Then,we took midbrain tissue homogenate and extract RNA to observe genes related to ferroptosis pathway changes in mRNA levels.Results:1.Results of in vitro experiments:(1)Experimental results of Rotenone and MPP+ induced MN9D cells.The results of MTT experiments showed that after induction of Rotenone and MPP+,the survival rate of MN9D cells in the model group was significantly decreased than that in the Control group.PCR results showed that the GSH synthesis-related genes including GPX4,GSS,GCLC mRNA levels were increased after Retenone and MPP+intervention,meanwhile,SLC7A11 and SLC3A2 mRNA in Xc-system and key genes of NFE2L2 mRNA and HOMX1 levels in anti-oxidant pathway were also increased,while TFRC and SLC3A2 mRNA levels which is related to iron transport were decreased.ALOX12 and ACSL4 which is related to fatty acid metabolism were up-regulated after Rotenone and MPP+ administration.The above differences were statistically significant(P<0.05 or P<0.01).(2)The protective effect of JTW on Rotenone and MPP+-induced MN9D cells.Results of staining of living/dead cells,ROS and JC-1 experiments showed that compared with the Control group,the number of dead cells in the Model group increased significantly after Rotenone and MPP stimulation,and at the same time,the release of ROS in the cells increased significantly.After Rotenone stimulation,the cell mitochondrial membrane The potential level decreased,however,there was no significant change in the mitochondrial membrane potential of the MPPstimulated group.JTW can significantly reduce the number of MN9D cell death cells after Rotenone and MPP stimulation.It also can reduce the release of ROS in cells,JTW could increase the level of mitochondrial membrane potential induced by Rotentone,however,it has no effect on MPP+induced mitochondrial membrane potential of MN9D cells.The PCR results suggest that compared with the model group,Rotenone and MPP+-induced MN9D cells in the JTW intervention group showed a trend of decreasing DPP4,NCOA4,GPX4,GCLC,GSS and GLS2 mRNA expression.The expression of SLC11A2 mRNA in MN9D cells stimulated by Rotenone induced by Rotenone in the JTW intervention group was up-regulated,and the expression of TFRC mRNA was up-regulated in the model group.JTW did not change the expression of SLC11A2 in MN9D cells stimulated by MPP+ significantly,but down-regulated the expression of TFRC mRNA.There are statistical differences in the above differences(P<0.05 or P<0.01).(3)Erastin-induced PC12 cell experiment with JTW intervention.Compared with the model group,the cell viability was significantly increased.The results of live-dead cell staining showed that the number of dead cells was significantly reduced.The results of ROS experiments showed that the release of ROS was significantly reduced.JC-1 The experimental results showed that the mitochondrial membrane potential level increased significantly.2.In vivo experiments:(1)Comparison results of brain tissue dopaminergic neuron damage:Compared with mice in Control group,the number of TH-positive neuron cells in the SNpc area of the MPTP group was significantly reduced.Compared with the MPTP group,the loss of DA neurons in the JTW treatment group was significantly improved.The differences were statistically significant(P<0.05 and P<0.01).(2)Results of PCR quantitative comparison of genes related to iron death pathway in mouse midbrain tissue:Compared with Control group,mice in MPTP group had increased expression of SLC7A11,ALC3A2,TFRC,SLC11A2,and Hmoxl,NFE2L2 mRNA were not obvious Changes(P<0.05 or P<0.01).Compared with MPTP group,the expression of SLC7A11,ALC3A2,TFRC,SLC11A2,GPX4 and NFE2L2 mRNA in the JTW group were up-regulated,and the difference was statistically significant(P<0.01).However,compared with the MPTP group,the expression of Hmoxl did not change significantly after JTW administration.Conclusion:1.Retinone and MPP+ stimulation can induce changes in gene levels of ferroptosis pathways in DA neurons and induce DA neuron damage.2.JTW can alleviate Retinone,MPP+and MPTP-induced dopaminergic neuron damage both in vivo and in vitro,and its mechanism may be related to its regulation of iron death signaling pathway.3.JTW could inhibit Erastin-induced ferroptosis in vitro. |
