| Parkinson’s disease（PD）is characterized by a progressive loss of dopaminergic neurons in the substantia nigra（SN）and the formation of Lewy bodies which are predominantly composed of a-synuclein.Currently,the treatment of PD is mainly focused on pharmacological therapy.However,the side effects of many drugs have not been well resolved.Berberine is a kind of isoquinoline alkaloid isolated from Coptis chinensis,Phellodendri,which has a wide range of pharmacological activities.It has been found that berberine has a neuroprotective effect in the progress of PD.However,the mechanisms underlying its potential protective effects have not been fully investigated.In this study,SH-SY5Y cell line and C57BL/6 mice were selected to establish PD models in vivo and in vitro.Molecular biology,behavior tests,immunohistochemistry and other methods were used to explore the protective effect of berberine on the pathogenesis of PD.The results are as follows:1.The cell was treatment with different concentrations of berberine.The cell viability was unchanged when treated with berberine up to 1μM.However,a significant decrease of cell viability was observed when cells were treated with above 10μM berberine.The cell viability reduced to 65%after 20μM rotenone treatment for 24 h compared with the control（P<0.001）.Treatment with 10-3-1μM berberine significantly increase the cell viability compared to rotenone treatment group,in which,10-1μM berberine has the strongest protective effect compared with rotenone treatment group（P<0.001）.Therefore,10-1μM berberine was chosen as the optimal concentration for further experiments.2.Treatment with rotenone induced a significant decrease in mitochondrial transmembrane potential（Δψm）and an increase in reactive oxide species（ROS）content compared to the control（P<0.001）.Berberine markedly reversedΔψm（P<0.001）and inhibited ROS generation compared with rotenone treatment group（P<0.05）.3.The ratio of Bcl-2/Bax was significantly decreased and the caspase-3 protein expression was significantly up-regulated in the rotenone-treated SH-SY5Y cells compared with the control（P<0.001）.Treatment with berberine significantly increased the ratio of Bcl-2/Bax and inhibited the expression of caspase-3（P<0.001）.4.Compared with the control,rotenone treatment inhibited the expression of pAkt（P<0.001）.Berberine pretreatment prevented the decrease of Akt phosphorylation induced by rotenone.There was no significant difference in the expression of Akt among different treatment groups（P>0.05）.5.Pole test showed that the time of free crawling from the top of the pole to the bottom was longer than that of the control group in MPTP-treated mice（P<0.001）.The time in the berberine-pretreated mice was less than that of MPTP-treated mice（P<0.01）.Rotarod test showed that the time on the rod of the MPTP-treated mice reduced significantly compared with the control（P<0.001）.Treatment with berberine significantly attenuated MPTP-induced motor impairment compared with MPTP treatment group（P<0.01）.6.Compared with the control,the number of TH positive cells observably decreased in the SN of MPTP-treated mice（P<0.001）.This effect was partly restored by berberine treatment compared with MPTP treatment group（P<0.001）.7.Compared with the control,the TH protein expression was significantly decreased and theα-synuclein expression was significantly increased in the SN of MPTP-treated mice（P<0.001）.Compared with MPTP treatment group,treatment with berberine significantly increased the expression level of TH protein（P<0.01）and decreased the expression level ofα-synuclein（P<0.001）.8.Compared with the control group,the expression of microtubule-associated protein light chain 3-II（LC3-II）was up-regulated（P<0.01）,and the expression of p62 protein was up-regulated in SN of MPTP-treated mice（P<0.001）.The expression of LC3-II protein was significantly higher than that of MPTP-treated mice（P<0.001）,and the expression level of p62 protein was significantly lower than that of MPTP treated mice in SN of berberine-pretreated mice（P<0.01）.9.Compared with the control group,the adenosine 5-monophosphate（AMP）-activated protein kinase phosphorylated protein（pAMPK）was up-regulated in the SN of MPTP-treated mice（P<0.05）.The pAMPK protein was significantly higher than that of MPTP treated mice in the SN of berberine-pretreated mice（P<0.001）.There was no significant difference in total protein expression of AMPK among different treatment groups（P>0.05）.10.Pretreatment with 3-methyl-adenine（3-MA）,an autophagy inhibitor,antagonized the inhibitory effect of berberine on the decrease of cell survival induced by MPP+（P<0.05）.Pretreatment with compound C,an inhibitor of AMPK,could also antagonize the inhibitory effect of berberine on the decrease of cell survival induced by MPP+（P<0.001）.3-MA or compound c treatment alone had no effect on cell survival（P>0.05）.11.Compared with berberine and MPTP co-treatment group,3-MA pretreatment could inhibit the increase ofΔψm（P<0.001）and the decrease of ROS level（P<0.05）.Compound C pretreatment could also antagonize the effect of berberine on the recovery ofΔψm（P<0.01）and decrease of ROS level（P<0.001）.12.Compared with berberine and MPTP co-treatment group,3-MA pretreatment could inhibit the expression of LC3-II protein（P<0.001）,but had no effect on the expression of pAMPK（P>0.05）.Compound C pretreatment could inhibit the expression of LC3-II protein（P<0.001）and p AMPK protein（P<0.001）.13.Compared with berberine and MPTP co-treatment group,3-MA pretreatment increased the protein expression ofα-synuclein（P<0.001）.Compound C pretreatment also increased the protein expression ofα-synuclein（P<0.05）.These results suggest that berberine protects rotenone-treated SH-SY5Y cells by anti-oxidation and activation of Akt signaling pathway.Treatment with berberine significantly ameliorated the degeneration of dopaminergic neurons in the SN and improved the motor impairment in the MPTP-treated mice.Berberine also significantly decreasedα-synuclein accumulation and enhanced the LC3-II-associated autophagy in the SN of MPTP-treated mice.Furthermore,the AMPK was activated by berberine.Berberine’s actions were abolished by pretreated with the autophagy inhibitor,3-MA or AMPK inhibitor,compound C in the MPP+-treated SH-SY5Y cells.These results suggested that the protective effects of berberine on the toxicity of MPTP could be attributed to the berberine-enhanced autophagy via AMPK dependent pathway. |
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