Protective Effects Of TanshinoneⅡa On Dopaminergic Neurons In Mouse Model Of Parkinson’s Disease And Related Mechanisms

Objectives To investigate the effects of Tanshinone ⅡA (Tan ⅡA) on the expressionof tyrosine hydroxylase (TH), CD11b, p47-phox and inducible nitric oxide synthase (iNOS)in the substantia nigra (SN) of the mouse model of Parkinson’s disease (PD) induced by1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP), and to explore the protective effectsof Tan ⅡA on dopaminergic neurons and its possible mechanism.Methods One hundred and twenty healthy male C57BL/6N mice were randomly dividedinto six groups, each20.(1)3d model group: Mice received4intraperitoneal injections ofMPTP (20mg/kg) dissolved in saline at2hours intervals. After12hours of the lastinjection of MPTP, mice were treated with dimethyl sulfoxide (DMSO,5mg/ml) once aday for3days. Mice were sacrificed and brains were removed at3day after the lastinjection of MPTP.(2)7d model group: Mice received4intraperitoneal injections ofMPTP (20mg/kg) dissolved in saline at2hours intervals. After12hours of the lastinjection of MPTP, mice were treated with dimethyl sulfoxide (DMSO,5mg/ml) once aday for7days. Mice were sacrificed and brains were removed at7day after the lastinjection of MPTP.(3)3d Tan ⅡA intervention group: In addition to giving the sametreatment of MPTP as that of3d model group, mice were intraperitoneally injected withTan ⅡA (25mg/kg body weight) dissolved in DMSO (5mg/ml) at12hours after the lastinjection of MPTP, and once a day for3days. Mice were sacrificed and brains wereremoved at3day after the last injection of MPTP.(4)7d Tan ⅡA intervention group: Inaddition to giving the same treatment of MPTP as that of7d model group, mice wereintraperitoneally injected with Tan ⅡA (25mg/kg body weight) dissolved in DMSO(5mg/ml) at12hours after the last injection of MPTP, and once a day for7days. Micewere sacrificed and brains were removed at7day after the last injection of MPTP.(5)3dblank control group: Mice received4intraperitoneal injections of saline which was asmuch as that of model and intervention groups at2hours intervals. After12hours of thelast injection of saline, mice were treated with DMSO which was as much as that of modeland intervention groups once a day for3days. Mice were sacrificed and brains wereremoved at3day after the last injection of saline.(6)7d blank control group: Micereceived4intraperitoneal injections of saline which was as much as that of model andintervention groups at2hours intervals. After12hours of the last injection of saline, micewere treated with DMSO which was as much as that of model and intervention groupsonce a day for7days. Mice were sacrificed and brains were removed at7day after the lastinjection of saline. We used the immunohistochemical method to detect the number of TH,CD11b, p47-phox and iNOS immunopositive cells in the SN of mice of each group, anddetect the TH immunopositive fibers density in the striatum of mice of each group. We used the western blot to detect the protein expression of TH, CD11b, p47-phox and iNOSin the SN of mice of each group. The number of positive cells of immunohistochemistryand the special protein brand of Western blot were analysed semi-quantitatively withComputer Image Analysis System. All data were processed with statistical analysissoftware of SPSS13.0.Results Compared with the control group of mice, model mice showed typical symptomsof PD. The number of CD11b (12.33±1.00vs3.11±0.78), p47-phox (101.45±48.12vs30.40±6.06) and iNOS (88.17±27.45vs47.46±4.60) immunoreactive cells wassignificantly increased in the SN of mice at the3d after the last injection of MPTP(P<0.05), as well as that of protein levels. The number of TH (82.60±35.45vs173.59±5.60)immunoreacive neurons in the SN of mice was almostly reduced as much as50percent atthe7d after the last injection of MPTP (P<0.05), as well as that of protein level. Tan ⅡAtreatment ameliorated the PD symptoms of mice that had been treated with MPTP.Compared with the model group of mice, the number of CD11b (4.89±1.16vs12.33±1.00),p47-phox (76.61±25.56vs101.45±48.12) and iNOS (69.96±16.28vs88.17±27.45)immunoreactive cells in the SN of mice was obviously decreased after the treatment of TanⅡA at3d after the last injection of MPTP (P<0.05), as well as that of protein levels.Compared with the control group, the number of TH (115.09±23.78vs173.59±5.60)immunoreactive neurons in the SN of mice treated with Tan ⅡA was only reduced25percent at the7d after the last injection of MPTP, as well as that of protein level.Conclusions Tanshinone ⅡA could mitigate the loss of dopaminergic neurons in the PDmouse model induced by MPTP. The mechanism of neuprotective effect may be related tothe inhibition of microglial activation and NADPH oxidase, iNOS expression.