| The teenager`s binge drinking and the aging are current two major concerns. Preventing the brain damage caused by alcohol and delaying brain aging are the focus of present studies. The proportion of binge drinking is higher in the male than in the female at teenage. The brain damage by alcohol is severer in women than that in men, which suggests that the testosterone might be closely related to the brain damage. The brain is one of the major target organs of ethanol and oxidative stress is involved in the change of brain structure and function caused by alcohol abuse. Ageing is one of the greatest risk factors for neurodegenerative disorders. Aged men have a greater prevalence of PD than aged women and testosterone deficiency in men increases the incidence of AD. Ageing is associated with a progressive disruption of the redox balance leading to recurrent damage resulting from mitochondrial oxidative stress. With advancing age, the level of testosterone in aged male is significantly reduced. The decrease of androgen levels in old age is likely to be related to mitochondrial dysfunction during the aging process.Mitochondria are compartmentalized with two lipid membranes: the inner mitochondrial membrane and the outer mitochondrial membrane. The inner mitochondrial membrane covers the mitochondrial matrix, which contains tricarboxylic acid and betaoxidation. Between the inner and outer membrane of the mitochondria is a intermembrane cavity. It contains high concentration of protons, which maintains the mitochondrial membrane potential. The electron transport chain including four mitochondrial complexes(I, II, III, IV) is located in the inner mitochondrial membrane. It works for electron transfer and ATP generation. Different from other organelles, mitochondria contain their own DNA(Mitochondrial DNA,mt DNA). Mt DNA encodes 7 subunits(ND1, 2, 3, 4, 4L, 5, and 6) of the 46 subunits constituting complex I, one of 11 subunits of complex III, 3 of 13 subunits of complex IV, and 2 of 17 subunits of complex V. Mitochondria are essential for the protection of mammalian cells. Normal neuronal activities are critically dependent on mitochondrial function. Abnormalities in mitochondrial structure and function may lead to age-related neurodegenerative diseases. Mitochondria are both the primary sources of reactive oxygen species(ROS) and the primary targets of ROS damage. The mitochondrial dysfunction may lead to the increased ROS, unbalance of neuronal redox and increased oxidative stress damage.Therefore, the involvement of testosterone propionate in the mitochondrial oxidative stress in ethanol-treated male rats and the amelioratory effects of testosterone propionate on the activity of mitochondrial electron transport chain in aged male rats were investigated in the present paper in order to analyze the role of androgen in mitochondrial dysfunction.Part1 Involvement of testosterone propionate in the mitochondrial oxidative stress in ethanol-treated ratsObjective: To observe whether the altered oxidative stress parameters in ethanol-treated gonadectomized rats could be improved by testosterone propionate replacement.Methods:(1) All the rats were divided into Intact, Sham, GDX and GDX-TP(0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 2.5 and 3mg/kg) groups. GDX was performed on rats by removing testis and Sham was done by procedures similar to GDX without removing testis.(2) The rats were divided into Intact, Sham, GDX, GDX-TP, GDX-eth, GDX-TP-eth and Intact-eth groups. Ethanol was given to the rats in GDX-eth, GDX-TP-eth and Intact-eth groups by intraperitoneal injection of ethonal(3g/kg), one injection per day for two consecutive days, followed by two days with no injections. A total of 20 injections was performed.Morris water maze and open field test, as well as biochemical detection were adopted to analysis the behavioral changes and oxidative stress parameters respectively.Results:1 Study on TP dose screening(1) Open field test: The amount of exploratory behaviors and motor behaviors in GDX rats was lower than that in the Sham rats(P<0.01). Supplement of TP at the dosage from 0.5mg/kg to 3 mg/kg increased the amount of exploratory and motor behaviors in GDX rats. The amount of above behaviors in GDX-TP rats at 0.75, 1.0 and 1.25 mg/kg was restored to that in the Sham rats.(2) Morris water maze testThe orientation navigation experiment: Analyses of escape latencies within each day revealed a significant effect of TP on days3, 4 and 5 in Sham. GDX and GDX-TP groups(P<0.01). The escape latency in the GDX rats was longer than that in the Sham rats on test days 3, 4 and 5(P<0.01). Supplement of TP significantly reduced the escape latency of GDX rats on test days 3, 4 and 5 at the dosage of 0.75, 1.0 and 1.25 mg/kg, restored the escape latency of GDX rats to the level of the Sham rats.The probe trails: The percent time in target quadrant and the number of probe platform crossings were less in the GDX rats than in the Sham rats(P<0.01). TP supplement at dosage of 0.75, 1.0 and 1.25 mg/kg restored the percent time in target quadrant and the number of probe platform crossings of GDX rats to the level of Sham rats.(3) Oxidative stress parametersAntioxidant parameters:The activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN and in HIP were significantly reduced in the GDX rats compared to the Sham rats(P<0.01). Supplement of TP increased the activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN and in HIP of GDX rats with the peak value at the dosage of 1.0 mg/kg, restored to the level of Sham rats.Lipid peroxide parameters: The level of MDA and LPO in SN and in HIP were significantly increased in GDX rats compared to the Sham rats(P<0.01).Supplement of TP decreased the level of MDA and LPO in SN and in HIP of GDX rats with the inverted peak value at the dosage of 1.0 mg/kg, restored to the level of Sham rats.2 The influence of testosterone propionate upon the behavioral and oxidative stress parameters in gonadectomized rats treated by ethanol(1) Open field test: The amount of immobile-sniffing events, exploratory behaviors,the duration of movement in the central area and the latency of grooming in the Intact-eth rats were less than in the Intact rats(P<0.01). The amount of motor behaviors, the number of grooming events and the duration of grooming in the Intact-eth rats were greater than in the Intact rats(P<0.01). The amount of immobile-sniffing events, exploratory behaviors, motor behaviors, grooming events and the duration of grooming in the GDX rats were lower than in the Sham rats(P<0.01), and the amount of the above behaviors in the GDX rats were restored to the level of the Sham rats after subcutaneous administration of 1.0mg/kg TP.The number of immobile-sniffing events, the duration of movement in the central area and the latency of grooming were less in the GDX-TP-eth rats than in the GDX-eth rats(P<0.01). The amount of motor behaviors, grooming events and the duration of grooming were more in the GDX-TP-eth rats than in the GDX-eth rats(P<0.01).(2)Oxidative stress parametersAntioxidant parameters:The activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN were significantly reduced in the Intact-eth rats compared to the Intact rats(P<0.05). The activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN were significantly reduced in the GDX rats compared to the Sham rats(P<0.01);TP supplement at dosage of 1.0 mg/kg restored the activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN of GDX rats to the level of Sham rats. The activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN were significantly increased in the GDX-TP-eth rats compared to the GDX-eth rats(P<0.05).Lipid peroxide parameters: The level of MDA and LPO in SN were significantly increased in Intact-eth rats compared to the Intact rats(P<0.01). The level of MDA and LPO in SN were significantly increased in GDX rats compared to the Sham rats(P<0.01); TP supplement at dosage of 1.0 mg/kg restored the level of MDA and LPO in SN of GDX rats to the level of Sham rats.The level of MDA and LPO in SN were significantly reduced in GDX-TP-eth rats compared to the GDX-eth rats(P<0.01).Conclusions:1 Testosterone propionate supplement at 0.75, 1 or 1.25 mg/kg can restore the relevant behavioral and oxidative stress parameters in gonadectomized rats.2 Testosterone propionate ameliorates oxidative stress damage in ethanol treated rats.Part2 Amelioratory effects of testosterone propionate on the activity of complex I in the substantia nigra of aged male ratsObjective: To analyse whether the activity of mitochondrial electron transfer chain was altered in aged male rats and whether testosterone propionate supplement ameliorated the activity of electron transfer chain and the expression of mt DNA encoding subunits.Methods: Male SD rats were divided into adult group(6Mon), aged group(24Mon) and 24-month-old group(24Mon-TP). The changes of mitochondrial membrane potential in rat substantia nigra and hippocampus were observed by flow cytometry. Mitochondrial oxidative stress parameters in rat substantia nigra and hippocampus were detected by biochemical methods. The enzymatic activity of mitochondrial electron transport chain was analyzed by UV spectrophotometry. Real time fluorescent quantitative PCR(q PCR) and Western blot method were used to analyze the expression of mt DNA-encoded complex I subunit in substantia nigra of rats.Results:1 Mitochondrial membrain potential: The value of Rh123 fluorescence intensity in SN and in HIP of the 24 Mon rats were significantly less compared to the 6Mon rats(P<0.01). TP supplement increased the value of Rh123 fluorescence intensity in the SN(P<0.01) and HIP(P<0.05) of 24 Mon rats.The value of Rh123 fluorescence intensity both in SN and in Hip of 24Mon-TP rats failed to differ significantly from that of 6Mon rats(P<0.01).2 Oxidative stress parameters: The activity of Mn-SOD and GSH-PX, as well as the level of GSH in SN and in HIP were significantly reduced in the 24 Mon rats compared to the 6Mon rats(P<0.01).The level of MDA and LPO in SN and in HIP were significantly increased in the 24 Mon rats compared to the 6Mon rats(P<0.01).Supplement of TP increased the activity of Mn-SOD and GSH-PX, as well as the level of GSH and decreased the level of MDA and LPO in SN and in HIP of 24 Mon rats; the activity of Mn-SOD and GSH-PX, as well as the level of GSH, MDA and LPO in SN and in HIP of 24Mon-TP rats failed to differ significantly from that of 6Mon rats(P<0.01).3 Mitochondrial complex activities:No significant differences were found in SN on the activity of complex II, complex III and complex IV among the 6Mon, 24 Mon and 24Mon-TP groups. No significant differences were found in HIP on the activity of complex I, complex II, complex III and complex IV among the 6Mon, 24 Mon and 24Mon-TP. The activity of complex I in SN was significantly reduced by 28.94% in the 24 Mon rats compared to the 6Mon rats(P<0.01). The activity of complex I was significantly increased by 19.89% in the 24Mon-TP rats compared to the 24 Mon rats(P<0.01).The activity of complex I in 24Mon-TP rats failed to differ significantly from that in the 6Mon rats(P<0.01).4 Mt DNA-encoded complex I gene expression in SN: No significant differences were found in SN on the m RNA expression of ND2, ND3, ND4 L, ND5 and ND6 among the 6Mon, 24 Mon and 24Mon-TP groups. The ND1 and ND4 m RNA were decreased in the 24 Mon rats compared with that in the 6Mon rats by 27.85%, 29.27%(P<0.01). The expression of ND1 and ND4 m RNA were increased by 21.05%, 22.41% in the 24Mon-TP rats than in the 24 Mon rats(P<0.01). The expression of ND1 and ND4 m RNA in the 24Mon-TP rats failed to differ significantly from that in the 6Mon rats(P<0.01).5 Mt DNA-encoded ND1 and ND4 protein expression in SN: The ND1 and ND4 protein were reduced in SN of 24 Mon rats compared to 6Mon rats by 40.68%,33.33%(P<0.01). Elevated ND1 and ND4 protein were observed in SN of 24Mon-TP rats compared with that of 24 Mon rats by 34.29%, 25%(P<0.01).The expression of ND1 and ND4 m RNA in the 24Mon-TP rats failed to differ significantly from that in the 6Mon rats(P<0.01).6 Serum testosterone level: The levels of serum testosterone is lower in the 24 Mon rats than in the 6Mon rats(P<0.01). TP supplement at dosage of 1.0, mg/kg restored the levels of serum testosterone of 24 Mon rats to the level of 6Mon rats.Conclusions:1 Mitochondrial dysfunction was found in the substantia nigra and hippocampus of aged rats and TP supplement ameliorated the mitochondrial dysfunction.2 The mitochondrial electron transport chain showed the region-specific change of the enzyme activity in aged male rats. The decreased activity of complex I in substantia nigra was significantly improved by TP treatment.3 Testosterone propionate ameliorated the reduced expression of ND1 and ND4 in the substantia nigra of aged male rat.Part3 Androgen is involved in regulating the activity of complex I in substantia nigraObjective: To observe whether androgen is involved in regulating the mitochondrial electron transfer chain enzyme activities of rat brain.Methods: Adult male SD rats were divided into Sham, GDX and GDX-TP group. The changes of mitochondrial membrane potential in rat substantia nigra and hippocampus were measured by flow cytometry. The enzymatic activity of mitochondrial electron transport chain was analyzed by UV spectrophotometry.Real time fluorescent quantitative PCR(q PCR) and Western blot method were used to analyze the expression of mt DNA-encoded complex I subunit in substantia nigra of rats.Results:1 Mitochondrial membrain potential: The value of Rh123 fluorescence intensity in SN and in HIP of the GDX rats were significantly less compared to the Sham rats(P<0.01). Supplement of TP increased the value of Rh123 fluorescence intensity of GDX rats(P<0.01). TP supplement at dosage of 1.0 mg/kg restored the value of Rh123 fluorescence intensity both in SN and in Hip of GDX rats to the level of Sham rats.2 Mitochondrial complex activities: No significant differences were found in SN on the activity of complexâ…¡, complex III and complex IV among the Sham, GDX and GDX-TP groups. No significant differences were found in HIP on the activity of complex I, complexâ…¡, complex III and complex IV among the Sham, GDX and GDX-TP groups. The activity of complex I in SN were significantly reduced in the GDX rats compared to the Sham rats by 24.32%(P<0.01). Supplement of 1.0mg/kg TP increased the activity of complex I in GDX rats by 38.82%(P<0.01).The activity of complex I in GDX-TP rats at 1.0mg/kg was restored to that in the Sham rats.3 Mt DNA-encoded complex I gene expression in SN: No significant differences were found in SN on the m RNA expression of ND2, ND3, ND4 L, ND5 and ND6 among the Sham, GDX and GDX-TP groups. The ND1 and ND4 m RNA were decreased in GDX rats compared with that in Sham rats by 8.97%,14.11%(P<0.01). Supplement of 1.0mg/kg TP increased the expression of ND1 and ND4 m RNA in GDX rats by 9.86%, 15.07%(P<0.01). The expression of ND1 and ND4 m RNA in GDX-TP rats at 1.0mg/kg were restored to that in the Sham rats.4 Mt DNA-encoded ND1 and ND4 protein expression in SN: The ND1 and ND4 protein were reduced in SN of GDX rats compared to Sham rats by 26.23%, 26.47%(P<0.01). Elevated ND1 and ND4 protein were observed in SN of GDX-TP rats compared with that of GDX rats by 33.33%, 30%(P<0.01).There was no significant difference of ND1 and ND4 protein in SN between GDX-TP rats and Sham rats.Conclusions:1 Gonadectomy decreased the activity of complex I and reduced the expression of ND1 and ND4 m RNA in substantia nigra of adult male rats.2 Replacement of TP reversed the activity of complex I and the expression of ND1 and ND4 in male gonadectomized rats.3 Expression of ND1 and ND4 in substantia nigra is androgen dependent. |
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