| Coordinated motor behaviors refer to the organized harmonious movements of sports organs when conducting certain activities. In the process of body aging, the age-related behavior defects will occur, including hypoesthesia, slow movement, decreased myodynamia and behavior defect of coordinated motor, which are one of the symbols of aging behaviors. The age-related motor behavior defects are closely related to the degenerative change of the nigra-striatal dopaminergic system during the normal aging process. The degenerative change of nigrostriatal dopaminergic system leads to storage, release and metabolic disorder of dopamine(DA) neurotransmitter, causing defects of spontaneous locomotor behaviors, myodynamia and coordinated motor behaviors of aged rats.Many experiments have proven that anabolic androgenic steroids(AAS)can affect the behaviors of organisms. Supplement of testosterone propionate(TP) can enhance anti-anxiety behavior of gonadectomized rats, increase immobile-sniffing, exploratory behavior, motor behavior and grooming behavior of gonadectomized rats and aged rats, recover mating behavior of gonadectomized rats, maintain sexual function of aged rats. After birth, early subcutaneous injection of TP can change rats’ motor behaviors in adolescence.In the central nervous system, androgen has many biological effects, the replacement of TP increases functional activities of dopaminergic neuron of normal male rats’ brain, and changeovers the function reduction of central dopaminergic system caused by castration and improves the function of nigrostriatal dopaminergic system of aged rats. It can also increase the concentration of dopamine in the brain and its metabolites of normal rats and gonadectomized rats.In the nigrostriatal dopaminergic system, vesicular monoamine transporter type 2(VMAT2) is a kind of transmembrane glycoprotein located in Monoamine neurons synaptic vesicle membrane, mainly mediating the storage and release of DA by synaptic vesicles and reducing the neuronal damage from free DA oxidative metabolism in the cytoplasm. The VMAT2 content in the brain can indirectly reflect the function status of dopaminergic neuron in the brain. Studies have shown that VMAT2 dysfunction caused by various reasons may affect the storage and release of DA in the brain, and finally result in retrogression of dopaminergic neuron. Previous studies have shown that VMAT2 expression of nigrostriatal dopaminergic system is reduced during aging. Epidemiological studies have found that the dopaminergic system disorders associated with aging(e.g. Parkinsonism) often happens in elderly men, and their serum testosterone level has decreased significantly. We suppose that the decline of VMAT2 expression is related to the decline of testosterone levels in the process of aging.Therefore, the coordinated motor behaviors of aged male rats after intranasal or subcutaneous administration of TP treatment were observed by the descending a wire mesh pole, tapered beam walking test and adhesive removal test; the level of DA and its metabolites content were observed by HLPC-MS; the expression of VMAT2 were observed by RT-PCR, western blot and immunocytochemistry. To explore the mechanisms of parameters influenced by giving reserpine(VMAT2 inhibitor), and the relationship of improving VMAT2 and androgen receptor in SH-SY5 Y.Part1 Amelioratory effect of testosterone propionate on the impaired coordinated motor behaviors in aged ratsObjective: To study the effects of TP on coordinated motor behaviors in aged rats.Methods: Descending a wire mesh pole, tapered beam walking test and adhesive removal test were used to analyze the effects of chronic intranasal or subcutaneous administration TP on coordinated motor behaviors in rats.Results:1 Descending a wire mesh pole: The time of descending a wire mesh pole was decreased in 24 Mon rats(P<0.01). After TP treatment, the time of descending a wire mesh pole was significantly increased in 24Mon-TP rats(intranasal administration 95.21%, subcutaneous injection 55.02%)(P<0.01) compared to 24 Mon rats.2 Tapered beam walking test: The score of tapered beam walking test was increased in 24 Mon rats(P<0.01). After TP treatment, the score of tapered beam walking test was significantly decreased in 24Mon-TP rats(left hindlimb: intranasal administration 22.56%, subcutaneous injection 11.24%; right hindlimb: intranasal administration 19.33%, subcutaneous injection 13.32%)(P<0.01) compared to 24 Mon rats.3 Adhesive removal test: Latency to remove left and right snout stimulus was increased in 24 Mon rats(P<0.01). After TP treatment, latency to remove left and right snout was significantly decreased in 24Mon-TP rats(left snout: intranasal administration39.29 %, subcutaneous injection 38.29%; right snout: intranasal administration50.11%, subcutaneous injection 40.64%)(P<0.01) compared to 24 Mon rats. Latency to remove left and right forepaw stimulus was increased in 24 Mon rats. After TP treatment, latency to remove left and right forepaw was significantly decreased in 24Mon-TP rats(left forepaw: intranasal administration 48.31%, subcutaneous injection 57.63%; right forepaw: intranasal administration 49.81%, subcutaneous injection 61.34%)(P<0.01) compared to 24 Mon rats.Conclusions:1 The function of locomotion was significantly lower in aged rats than in adult rats.2 Chronic treatment of TP ameliorated the function of locomotion of aged rats.3 Intranasal route improved the impaired coordinated motor behaviors better than subcutaneous injection.Part2 The dopaminergic activity enhanced in nigrostriatal dopaminergic neruons of aged rats by testosterone propionate administrationObjective: To study the influence of TP on the level of DA and its metabolites in caudate-putamen of aged rats.Methods: The HLPC-MS was used to analyse the effects of chronic intranasal or subcutaneous administration TP on the level of DA and its metabolites in caudate-putamen of rats.Results: The HLPC-MS: The level of DA and its metabolites in caudate-putamen were decreased, and the ratio of DA metabolites/DA were increased in 24 Mon rats(P<0.01). After TP treatment, the level of DA and its metabolites were significantly increased in 24Mon-TP rats(DA: intranasal administration 131.46%, subcutaneous injection 53.87%; DOPAC: intranasal administration 38.24%, subcutaneous injection 27.82%; HVA: intranasal administration 33.25%, subcutaneous injection 15.97%)(P<0.01) compared to 24 Mon rats. After TP treatment, the ratio of DA metabolites/DA were significantly decreased in 24Mon-TP rats(DOPAC+HVA)/DA: intranasal administration 35.79%, subcutaneous injection 18.61%; DOPAC/DA: intranasal administration 35.24%, subcutaneous injection 16.48%; HVA/DA: intranasal administration 37.26%, subcutaneous injection24.07%)(P<0.01) compared to 24 Mon rats. The level of DA and its metabolites in caudate-putamen of intranasal administration TP rats restored to the level of 6Mon rats(P>0.05).Conclusions:1 The level of DA and its metabolites in caudate-putamen were significantly decreased in aged rats compared to adult rats.2 Chronic treatment of TP ameliorated the dopamine and its metabolites in caudate-putamen of aged rats. It suggests TP could improve the function of nigrostriatal dopaminergic neurons.3 Intranasal route improved the impaired dopamine and its metabolites in caudate-putamen better than subcutaneous injection.Part3 Testosterone propionate increased the expression of VMAT2 in nigrostriatal dopaminergic neurons of aged ratsObjective: To explore the influence of chronic administration of TP on the expression of VMAT2 in nigrostriatal dopaminergic neurons of aged rats.Methods: Immunocytochemistry, Western blot and RT-PCR detection of chronic intranasal or subcutaneous administration TP on the expression of VMAT2 in nigrostriatal dopaminergic neurons of rats.Results:1 The RT-PCR: The RQ of VMAT2 m RNA in SN was decreased in 24Mon(P<0.01). After TP treatment, the RQ of VMAT2 m RNA in SN were significantly increased in 24Mon-TP(intranasal administration 77.14%, subcutaneous injection 35.69%)(P<0.01). The RQ of VMAT2 m RNA in SN of intranasal administration TP rats restored to the level of 6Mon rats(P>0.05).2 The Western blot: The expression of VMAT2 in SN were decreased in 24Mon(P<0.01). After TP treatment, the expression of VMAT2 in SN were significantly increased in 24Mon-TP(Glyco-VMAT2: intranasal administration 65.71%, subcutaneous injection 9.31%; Partially glyco-VMAT2: intranasal administration 25.84%, subcutaneous injection 20.15%; Native-VMAT2: intranasal administration72.43%, subcutaneous injection 77.57%)(P<0.01). The Partially glyco-VMAT2 in SN of intranasal administration TP rats restored to the level of 6Mon rats(P>0.05).3 The Western blot: The expression of VMAT2 in CPu were decreased in 24Mon(P<0.01). After TP treatment, the expression of VMAT2 in CPu were significantly increased in 24Mon-TP(Glyco-VMAT2: intranasal administration 57.29%, subcutaneous injection 49.22%; Partially glyco-VMAT2: intranasal administration 34.40%, subcutaneous injection 9.14%; Native-VMAT2: intranasal administration 25.24%, subcutaneous injection 19.23%)(P<0.01). The Partially glyco-VMAT2 in CPu of intranasal administration TP rats restored to the level of 6Mon rats(P>0.05).4 The immunocytochemistry: The number of VMAT2-ir cells in SN were decreased in 24Mon(P<0.01). After TP treatment, the number of VMAT2-ir cells in SN were significantly increased in 24Mon-TP(intranasal administration 27.97%, subcutaneous injection 22.88%)(P<0.01).5 The immunocytochemistry: The AOD of VMAT2-ir cells in SN were decreased in 24Mon(P<0.01). After TP treatment, the AOD of VMAT2-ir cells in SN were significantly increased in 24Mon-TP(intranasal administration 6.90%, subcutaneous injection 8.75%)(P<0.01).6 The immunocytochemistry: The AOD of VMAT2-ir terminals in CPu were decreased in 24Mon(P<0.01). After TP treatment, the AOD of VMAT2-ir terminals in CPu were significantly increased in 24Mon-TP(intranasal administration 20.18%, subcutaneous injection 6.95%)(P<0.01). The AOD of VMAT2-ir terminals in CPu of intranasal administration TP rats restored to the level of 6Mon rats(P>0.05).Conclusions:1 The number of VMAT2-ir cells in SN were decreased in aged rats compared to adult rats. Chronic treatment of TP increased the number of VMAT2-ir cells in SN in aged rats.2 Chronic treatment of TP ameliorated the VMAT2 m RNA and VMAT2 protein in nigrostriatal dopaminergic neurons.3 It suggests TP could ameliorate the expression of VMAT2 in nigrostriatal dopaminergic neurons in aged rats.4 Intranasal route improved the impaired nigrostriatal dopaminergic neurons in aged rats better than subcutaneous injection.Part4 Vesicular monoamine transporter type 2 is involved in the impaired coordinated motor behaviors improved and dopaminergic activity enhanced by testosterone propionateObjective: To investigate the TP improve the function of nigrostriatal dopaminergic neurons in aged rats whether or not involving VMAT2.Methods: Reserpine was injected previously. The descending a wire mesh pole, tapered beam walking test and adhesive removal test were used to analyse the effects of chronic intranasal or subcutaneous administration TP on coordinated motor behaviors in rats. The HLPC-MS was used to analyse the effects of chronic intranasal or subcutaneous administration TP on the level of DA and its metabolites in caudate-putamen of aged rats. The immunocytochemistry, Western blot and RT-PCR detection of chronic intranasal or subcutaneous administration TP on the expression of VMAT2 in nigrostriatal dopaminergic neurons of aged rats.Results:1 24Mon-R group and 24Mon-R.TP group of rats couldn’t do the descending a wire mesh pole and tapered beam walking test.2 Adhesive removal test: Latency to remove left snout, right snout left forepaw and right forepaw was significantly increased in 24Mon-R group compared to 24 Mon group(P<0.01). Latency to remove left snout, right snout left forepaw and right forepaw was significantly increased in 24Mon-R.TP group compared to 24Mon-R group(P<0.01).3 The HLPC-MS: The level of DA and its metabolites in caudate-putamen were decreased, and the ratio of DA metabolites/DA were increased in 24Mon-R group compared to 24 Mon group(P<0.01). The level of DA and its metabolites were no difference in 24Mon-R.TP group compared to 24Mon-R group(P>0.05). The ratio of DA metabolites/DA were significantly increased in 24Mon-R.TP group compared to 24Mon-R group(P<0.01).4 The RT-PCR: The RQ of VMAT2 m RNA in SN was no difference in 24 Mon group and 24 Mon group(P>0.05). The RQ of VMAT2 m RNA in SN was significantly increased in 24Mon-R.TP group compared to 24Mon-R group and 24 Mon group(P<0.01).5 The Western blot: The expression of VMAT2 in SN and CPu were no difference in 24 Mon group and 24 Mon group(P>0.05). The expression of VMAT2 in SN and CPu were significantly increased in 24Mon-R.TP group compared to 24Mon-R group and 24 Mon group(P<0.01).Conclusions:1 Reserpine inhibit the function of VMAT2, decrease the content of DA and its metabolites, increase the ratio of DA metabolites/DA and impair the locomotion.2 It suggests TP could improve the function of nigrostriatal dopaminergic neurons through improving VMAT2.Part5 Increased VMAT2 in SH-SY5 Y cells by testosterone is associated with androgen receptorObjective: To investigate T increasing the expression of VMAT2 and neuroprotective effect concerning to AR.Methods: SH-SY5 Y cells were differentiated with RA/TPA. After T and/or H2O2 separately treated, MTT and LDH were used to examine cell activity; immunofluorescence and western blot were used to examine the expression of VMAT2. Then flutamide(AR antagonist) was added, to investigate T increasing the expression of VMAT2 and neuroprotective effect concerning to AR.Results:1 RA / TPA differentiated SH-SY5 Y cells, rounded cell bodies appeared smaller, growth projections and other morphological changes. Neu N test results show that RA/TPA differentiated cells expressing Neu N was significantly higher than undifferentiated cells(P<0.01).2 MTT and LDH assay showed: Con, T and T+F activity among the three groups of cells did not change significantly(P>0.05), H, T+H and T+F+H activity compared three groups of cells Con, T and T+F three groups lower(P <0.01), H and T+F+H cell activity was lower than T+H group(P<0.01).3 Immunofluorescence: There is no AOD values significant difference between Con and T+F group(P>0.05), no significant difference H, T and T+F+H(P>0.05), H, T, and T+F+H three groups were significantly higher than Con and T+F group(P<0.01), T+H group was significantly higher than the other five groups(P<0.01).4 Western blotting: The relative ratio of Glyco-VMAT2 and GAPDH immunoblot bands was no significant difference H, T and T+F+H groups(P>0.05), H, T, and T+F+H groups were significantly higher than Con and T+F group(P<0.01), T+H group was significantly higher than the other five groups(P<0.01). Partially glyco-VMAT2 and Native-VMAT2 and GAPDH immunoblot bands relative absorbance ratio, Con, H, T, T+F+H and T+F groups were not statistically significant(P>0.05), T+H group was significantly higher than the other five groups(P<0.01).Conclusions:1 It suggests T have neuroprotective effects against H2O2 damage SH-SY5 Y cells through AR.2It suggests T could improve the expression of VMAT2 in SH-SY5 Y cells through AR.3 Oxidative stress can increase VMAT2 expression through non-AR pathway. |
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