Asiatic Acid Ameliorates MPTP-induced Parkinson’s Disease-like Motor Symptoms In Mice And Protects Sh-SY5Y Cells Against Rotenone-induced Neurotoxicity

Object: To investigate the effect of asiatic acid on the MPTP-induced Parkinson’s disease-like motor symptoms in mice and on SH-SY5 Y cells damaged by rotenone,then to explore new pharmacological activities of asiatic acid and to reveal its possible neuroprotective mechanisms,which help to broaden the clinical application of asiatic acid.Methods: 45 male C57BL/6 mice were randomly divided into 5 groups as follows: control group,MPTP treated model group,low dose of asiatic acid treated group,high dose of asiatic acid treated group and levodopa treated positive control group(nine mice each group).PD mouse model was induced with 25 mg/kg MPTP by peritoneal injection for 7 days.The mice in control group were injected intraperitoneally with saline.Both the mice in control group and model group were orally administrated with CMC-Na solution for 11 days.The low-dose and high-dose intervention groups were daily administrated with asiatic acid at a dosage of either12.5 mg/kg or 25 mg/kg respectively by intra-gastric gavage for 11 days.The mice in positive control group were treated with Levodopa at a dosage of 75 mg/kg daily by intra-gastric gavage for 11 days.On the twelfth day,the motor function was evaluated by pole test and traction test and then all the animals were sacrificed.The blood samples were taken from the eyeballs and the levels of IL-1β and TNF-α in the serum were measured by ELISA.The expression of TH in substantia nigra was determined by immunostaining and western blot.The m RNA expression levels of i NOS,COX-2,TNF-α and IL-1β were measured by real time PCR.MDA content was evaluated by using MDA kit to assess oxidative stress in midbrain.SH-SY5 Y cells were pretreated with different concentrations of asiatic acid for 24 hours and then they were incubated with 20μmol / L rotenone for 24 hours.Cell vibility was analyzed by MTT assay.The production of ROS and mitochondrial membrane potential were detected by applying DCFH-DA dye and JC-1 dye respectively.The protein expressions of Bax and Bcl-2 in SH-SY5 Y cells were evaluated by western blot.Results: The mice treated with asiatic acid showed better performance in behavior tests than the mice in model group,as the time of the mice treated with asiatic acid spent on climbing down the pole was reduced and the suspension time was prolonged.MPTP injection caused obviously reduction of TH expression and loss of TH positive cells in the substantia nigra.Asiatic acid effectively rescued the dopaminergic neurons in the substantia nigra,as the expression of TH was upregulated and the number of TH positive cells increased.The m RNA levels of IL-1β,TNF-α,i NOS and COX-2 in midbrain were markedly suppressed by asiatic acid.Meanwhile,we also found that asiatic acid reduced the levels of IL-1β and TNF-α in the serum.Furthermore,decreased content of the MDA in the midbrain tissue was detected in asiatic acid-treated mice.In vitro experiments showed that asiatic acid effectively improved the cell viability in a concentration-dependent manner.Asiatic acid inhibited the production of ROS and improved the mitochondrial membrane potential to alleviate oxidative stress and regulate the mitochondrial function.In addition,asiatic acid can upregulate Bcl-2 expression and suppress the expression of Bax,which suggested that asiatic acid protected SH-SY5 Y cells against rotenone-induced apoptosis.Conclusion: Asiatic acid attenuated MPTP-induced motor dysfunction and dopaminergic neuronal deficits in PD mice.Asiatic acid supplement effectively suppressed inflammatory responses both in the central neuron system and in the peripheral immune system and alleviated oxidative stress in middle brain.In vitro experiments demonstrated that asiatic acid could protect cellular damage of SH-SY5 Y induced by rotenone.Asiatic acid inhibited the production of ROS and enhanced mitochondrial membrane potential to alleviate oxidative stress and to improve the mitochondrial function,which reduced SH-SY5 Y apoptosis.These results suggested that the neuroprotective mechanisms of asiatic acid may attribute to its anti-oxidative,anti-inflammatory and mitochondria protection activities.Taken together,the present study demonstrated that asiatic acid was expected to be a novel therapeutic candidate for PD.