The Protective Effect And The Mechanism Of Coenzyme Q₁₀ On Rotenone-induced Dopaminergic Neurons Injuring

Propose:To observe the toxic effects of different nanomolar concentrations of rotenone to mesencephalic dopaminergic cell cultures on the 6th DIV for various hours, and study the manner of the toxic effects; To investigate the neuroprotective effects and the mechanism of CoQ₁₀ on rotenone-induced dopaminergic neurons injuring.Method:1. Primarily cultured mesencephala dopaminergic neurons which from the mesencephala of embryonic 14 d mouse were treated with nanomolar concentrations of rotenone and (or) CoQ₁₀ on the 6th in vitro as followed: control group: without rotenone; group rotenone A, B and C: respectively treated with 5, 15 and 30 nmol·L^-1 rotenone for 24 h; group rotenone D, B, and E: treated with 15 nmol·L^-1 rotenone respectively for 12 , 24 and 48 h; group CoQ₁₀(group X,Y,Z): pretreat the mesencephalic dopaminergic neurons with CoQ₁₀(10,50 and 100μmol·L^-1) for 12 h, then addition of 15 nmol·L^-1 rotenone to cultures for 24 h.2. The neurons were observed under the inverted phase contrast microscope and assessed by immunostaining using tyrosine hydroxylase (TH) antibodies and PI fluorescence staining. The viability of neurons was reflected by the transudatory amount of lactate dehydrogenase (LDH) measured with LDH assay kit. By measuring the intracellular Rhodamine 123 fluorescene density, mitochondrial potential (△ψm) was evaluated.Results:1. Primarily cultured mesencephala dopaminergic neurons which from the mesencephala of embryonic 14 d mouse on the 6th in vitro were assessed by immunostaining using TH antibodies. The results shows there are a number of TH⁺ neurons.2. Addition of 5 nmol·L^-1 rotenone to cultures for 24 h (Group A) decreased the survival of TH⁺ neurons, and the total length and number of neurites of dopaminergic neurons were extensively decreased,while the number of PI⁺ cells was obviously increased. Moreover, the release of LDH (U·mL^-1) increased from 329.41±7.784 to 18.62±13.54 (P<0.05), and the△ψm(%) decreased from 100±0 to 82.69±0.54 (P <0.05). While after addition of 15 and 30 nmol·L^-1 rotenone for 24 h (Group B and C), the number of TY⁺ cells was progressively decreased and the total length and number of neurites of dopaminergic neurons was extensively decreased, and the number of PI⁺ cells was advanced increased. The release of LDH (U·mL^-1) respectively increased to 434.27±3.75 and 447.62±6.53 (P <0.05), and the△ψm (%) respectively decreased to 80.91±1.49 and 70.54±7.59 (P <0.05). The release of LDH (U·mL^-1) between group A and group C was statistically different (P <0.05).3.Addition of 15 nmol·L^-1 rotenone to cultures for 12 h (Group D) decreased the survival of TH⁺ neurons , and the total length and number of neurites of dopaminergic neurons was extensively decreased, while the number of PI⁺ cells was obviously increased; the release of LDH (U·mL^-1) increased from 329.41±7.784 to 420.93±6.45 (P <0.05), and the△ψm (%) decreased from 100±0 to 82.36±0.44 (P <0.05), while a longer treatment for 24 h or 48 h (Group B and E) progressively decreased the number of TH⁺ cells and increased the number of PI⁺ cells. The total length and number of neurites of dopaminergic neurons was extensively decreased after exposure to 15 nmol·L^-1 rotenone for 48 h. The release of LDH (U·mL^-1) respectively increased to 434.27±3.75 and 452.21±11.44 (P <0.05), and the△ψm (%) respectively decreased to 80.91±1.49 and 62.66±2.50 (P <0.05). The release of LDH (U·mL^-1) between group D and group E was statistically different (P <0.05).4. Compared to group rotenone B, neurons of Group X did not show marked difference. However, the number and total length and number of neurites of dopaminergic neurons of Group Y and Z were extensively increased compared to group rotenone B and the number of PI⁺ cells obviously decreased. The release of LDH (U·mL^-1) respectively decreased to 410.24±11.29 (P<0.05) and 414.57±2.94 (P <0.05), and the△ψm (%) respectively increased from 80.91±1.49 to 86.83±1.34 (P <0.05) and 91.13±5.01 (P<0.05).Conclusion:1. The rotenone had the dramatically neurotoxic effect on dopaminergic neurons; the cell viability and the△ψm were dramatically decreased. The neurotoxic effect of rotenone destroyed dopaminergic cells in a dose- and time-dependent manner.2. The neurotoxic effect of rotenone on dopaminergic cells possibly resulted from mitochondrial damage.3. CoQ₁₀ can effectively protect rotenone-induced dopaminergic neurons injuring, represented in significantly protecting the cell membrane and preventing from mitochondrial depolarization in a dose-dependent manner.4. The protective effect of CoQ₁₀ on rotenone-induced dopaminergic neurons possibly by protecting the mitochondrial function.