Study On The Protective Mechanism Of Demethyleneberberine On SH-SY5Y Cells Induced By MPP~

Parkinson’s disease（PD）is a common neurodegenerative disease,and its main pathological features are the selective loss of dopaminergic neurons in the substantia nigra and the appearance of Lewy bodies.The main component of Lewy bodies isα-synuclein.The current treatment methods for PD are mainly drug therapy,and the main drugs used are dopamine analogs,dopaminergic receptor agonists,neuroprotective agents,and monoamine oxidase-B（MAO-B）inhibitors.However,most drugs for the treatment of PD have certain toxicity,so finding a drug with high safety and good efficacy has become a top priority for the treatment of PD.According to existing studies,for PD models,although demethyleneberberine（DMB）has a certain protective effect,the mechanism of its protective effect is still unclear.Therefore,in this experiment,the SH-SY5Y cell line was used to establish a PD cell model,and molecular biology and other research methods were used to explore the protective effect of demethyleneberberine in PD and analyze its possible mechanism.In the experiment,the precursor berberine of DMB,which has been proved to have neuroprotective effect,and selegiline,which has also been proved to have neuroprotective effect and is an MAO-B inhibitor with DMB,were used to form a positive control by comparing with DMB.The experimental results are as follows:1.Different concentrations of DMB(0,10^-5,10^-4,10^-3,10^-2,10^-1,1,10,100μM)treated SH-SY5Y cells for 24 h,drugs at different concentrations had no significant effect on cell viability.After 1 m M MPP⁺treatment for 24 h,the cell viability decreased to 37%（P<0.001）.Co-treatment with MPP⁺at 10^-5-100μM DMB significantly increased cell viability.Among them,the survival rate of SH-SY5Y cells in the co-treatment group of 10^-3μM DMB and MPP⁺was the highest（P<0.001）.Therefore,10^-3μM DMB was selected as the optimal concentration for subsequent experiments.2.Compared with the control group,the mitochondrial membrane potential（Δψm）level of cells in the MPP⁺treatment group was significantly reduced,and the level of reactive oxygen species（ROS）was significantly increased.Compared with the MPP⁺treatment group,DMB inhibited the decrease ofΔψm（P<0.001）and the increase of ROS（P<0.001）.There was no significant difference in the effects of the three drug treatments onΔψm and ROS（P>0.05,P>0.05）.3.Compared with the control group,the bcl-2/bax ratio of the MPP⁺treatment group decreased.The decrease of bcl-2/bax ratio was inhibited by DMB treatment（P<0.001）.Among the three drug treatment groups,the effect of selegiline was stronger than that of BBR and DMB（P<0.05,P<0.05）.4.Compared with the control group,the expression of cleaved caspase-3 protein in the MPP⁺treatment group was up-regulated.DMB treatment inhibited the up-regulation of cleaved caspase-3 protein expression（P<0.001）.There was no significant difference in the effects of the three drugs（P>0.05）.5.Compared with the control group,the expression of tyrosine hydroxylase（TH）in the MPP⁺treatment group was significantly decreased（P<0.001）.DMB treatment could significantly inhibit the reduction of TH expression（P<0.001）.There was no significant difference in the effects of the three drugs（P>0.05）.6.Compared with the control group,the expression of phosphorylated Akt protein in the MPP⁺treatment group was significantly reduced（P<0.001）.DMB treatment could inhibit the expression of phosphorylated Akt protein（P<0.001）.Compared with the three drug treatment groups,the effects of selegiline and DMB were stronger than those of BBR（P<0.001,P<0.001）,and the effect of selegiline was also stronger than that of DMB（P<0.001）.There was no significant difference in the expression of total Akt protein among different treatment groups（P>0.05）.7.Compared with the control group,the expression of phosphorylated AMPK protein in the MPP⁺treatment group increased（P<0.05）.The phosphorylation level of AMPK further increased after DMB treatment and was higher than that of MPP⁺treatment group（P<0.001）.Compared among the three drug treatment groups,the effects of selegiline and DMB were stronger than those of BBR（P<0.001,P<0.001）.There was no significant difference in the expression of AMPK total protein among different treatment groups（P>0.05）.8.Compared with the control group,the level of p62 protein in the MPP⁺treatment group increased（P<0.001）.DMB treatment can promote the degradation of p62 protein（P<0.001）.Compared among the three drug treatment groups,the effect of BBR was stronger than that of selegiline（P<0.01）.9.Compared with the control group,the expression level of cleaved caspase-8 in the MPP⁺treatment group was significantly increased（P<0.001）.DMB treatment could inhibit the increased expression of cleaved caspase-8（P<0.001）.Compared among the three drug treatment groups,the effect of selegiline was stronger than that of BBR（P<0.05）.10.Compared with the control group,the cellular MAO-B level of the MPP⁺treatment group was significantly increased.DMB treatment could inhibit the increase of MAO-B level（P<0.01）.Compared among the three drug treatment groups,the effect of selegiline was stronger than that of BBR（P<0.05）.The above results indicated that in MPP⁺-induced SH-SY5Y cells,DMB can maintain mitochondrial membrane potential,reduce ROS level and inhibit the decrease of TH protein expression;it can also inhibit the mitochondria-related caspase-dependent apoptosis pathway.DMB can activate cellular Akt and AMPK signaling pathways,and promote the degradation of p62,thereby maintaining mitochondrial homeostasis and inhibiting the caspase-8 apoptosis pathway.DMB can reduce the level of MAO-B in cells,and MAO-B can not only catalyze the oxidative deamination of dopamine,but the reduction of its level is also conducive to the maintenance of mitochondrial homeostasis.It is suggested that the protective effect of DMB may be realized through various pathways related to mitochondria.