Study On The Effects Of COXâ…¦a On Partial Androgen Deficiency Syndrome Of The Aging Male Rats And Its Mechansim

Due to the decrease in androgen levels gradually with their aging, middle-aged males appear fatigue, weakness, depression, insomnia, mood swings, irritability, decreased libido, erectile dysfunction, weight loss and osteoporosis and a series of clinical syndrome, which are known as partial androgen deficiency syndrome of the aging male (PADAM). PADAM is associated with many diseases, such as cardiovascular disease, diabetes and so on. The higher incidence of various age-related diseases seriously affects older men's health and their quality of life. Concerning about the age-related diseases, improving the quality of life of older persons and extending their life span have become the important medical issues of the world's attention.COXⅦa (Cytochrome c Oxidase SubunitⅦa) is nuclear encoded subunit of complex IV of mitochondrial respiratory chain, involved in the complex composition and plays an important role in electron transfer of mitochondria respiratory chain oxidation. Studies indicate that COXⅦa in the testis of the aging male was significantly increased, suggesting that it may be involved in androgen dysfunction and partial androgen deficiency syndrome of the aging male, but the exact mechanism remains unclear, and there are rare domestic and foreign reports. PADAM model was established by injection of D-galactose. Some techniques of modern molecular biology such as gene transfection and RNA interference were used to study the effects and mechanism of COXⅦa on PADAM rats in vivo and in vitro, aiming to provide the theoretical basis for exploring the mechanism of PADAM and gene targeting therapy. To study the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA and acute regulatory protein expression in rat testes of different periodsand the correlation between them and testosterone concentration.1. Male SD rats aged 1 week,3 weeks,5 weeks,3 months and 24 months were studied, each group 6 animals(1 week group was 12).2. The serum testosterone was determined by chemiluminescence immunoassay (CLIA), variation coefficient of inter-group and intra-group was 2.2% to 2.4% and 3.4% to 4.7% respectively.3. RT-PCR was used to examine the changes of COXⅦa,3β-HSD,17β-HSD, P450scc and StAR mRNA of the testicular tissues among the above groups.4. Western blotting and immunohistochemistry were employed to detect the difference of COXⅦa,3β-HSD,17β-HSD, and P450scc and StAR protein expression of the testicular tissues among the above groups.1. The testosterone level in the rat serum decreased gradually from 3 months to 24 months,5 weeks,3 weeks and 1 week group. The difference between 3 months,24 months group and the other groups was statistically significant respectively (p<0.05).2. COXⅦa mRNA level in 24 month group was the highest, and that in 3 month group was the lowest, and the differences between 3 months,24 months group and the other groups were statistically significant(p<0.05); 3β-HSD,17β-HSD and P450scc mRNA levels in 3 months group and 5 weeks group were significantly higher than them in other groups(p<0.05); StARmRNA level in 3 months group was the highest,5 weeks and 24 months group were followed,1 week and 3 weeks group were the lowest, the difference was statistically significant(p<0.05).3. COXⅦa protein expressed in plasmid and nucleus of Leydig cells and the nucleus of spermatogonia. It was the highest in 24 month group and the lowest in 3 months group.3β-HSD protein was expressed in plasmid of Leydig cells, 17β-HSD protein in plasmid of Leydig cells and nucleus of spermatogonia, P450scc protein in plasmid, nucleus and membrane of Leydig cells, and plasmid of spermatid, StAR protein in the plasmid and membrane of Leydig cells and plasmid of spermatogonia.3β-HSD,17β-HSD, P450scc and StAR protein was significantly higher in 3 months group and 5 weeks group than them in other groups(p<0.05).4. There was negative correlation between COXⅦa protein and serum testosterone concentration,3p-HSD,17β-HSD, P450scc and StAR protein. The differences were statistically significant except StAR (p<0.05,p<0.01).1. The expression of COXⅦa had significant change in different periods and it was higher in old rats than in young rats, and was negatively related to serum testosterone concentration, suggesting that COXⅦa may be associated with testosterone synthesis and involved in the occurrence of PAD AM.2. The expression of 3β-HSD,17β-HSD, P450scc and StAR had significant change in rat testes of different periods, namely,3 months and 5 weeks group was significant higher than 1 week,3 weeks and 24 months group.3. There was negative correlation between COXⅦa and 3β-HSD,17β-HSD, P450scc, indicating that COXⅦa regulated testosterone synthesis through steroidogenesis enzymes but not StAR.1. To explore the impact of COXⅦa overexpression to testosterone synthesis by construction pEGFP-N1-COXⅦa recombinant plasmid and transient transfection primary cultured rat Leydig cells extracted from 3 months.2. To explore the impact of COXⅦa suppression to testosterone synthesis by application of RNA interference inhibition of COXⅦa in Leydig cells. 1. Improved Wang Xiaoyun's differential adhesion speed method was used in the isolation of Leydig cells; 3β-hydroxysteroid dehydrogenase (3β-HsD) staining and immunohistochemical staining were employed in identifying the cell purity,0.4% solution of trypan blue in checking cell viability.2.pEGPF-N1-COXⅦa plasmid construction, tranfection and function examination2.1 Construct pEGPF-N1-COXⅦa plasmid:COXⅦa DNA was amplified by RT-PCR from 24 month rat testis and was digested with Bgl II and EcoR I, then was linked to the pGM-T vector by T4 ligase. E. coli including pGM-T-COXⅦa plasmid and E. coli including p-EGPF-N1 plasmid were propagated in a large scale. After extracted, two kinds of plasmid were digested with Bgl II and EcoR I. Then pEGPF-N1 and COXⅦa gene was linked together by T4 linkase.2.2 Transfection:After the sequence of the recombinant plasmid was proved correct, the recombinant plasmid was transferred into the primary cultured Lydia cells.2.3 Identification to positive cells:Cells with green fluorescence under the fluorescent microscope were positive. Co focal laser scanning microscopy was used to observe in the expression and localization of pEGFP-Nl-COXⅦa within the cell which were incubated with MitoTracker (?) Red CM-H2Xros together after 20 hours transfected with pEGFP-N1-COXⅦa.2.4 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively, and immunocytochemistry for protein localization.3. RNA Interference3.1 Transfer:SiRNA Oligo with the most powerful inhibitory effect was screened out to be tranferred into PADAM rat Leydig cells with Lipofectamine 2000. Blank control group, Lipofectamine control, negative control, RNA interference group, positive control and fluorescent labeled negative control group were formed.3.2 Transfer efficiency:Fluorescent microscope was used to observe fluorescent labeled negative control cells to determine transfer efficiency.3.3 Function examination:Cell BlueTM Kit was used to determine cell viability, CLIA was used to examine testosterone concentration, RT-PCR and western blotting were used to detect COXⅦa mRNA and protein expression respectively.1. The purity of isolated Leydig cells was more than 95% in accordance with the test requirements.2. pEGPF-N1-COXⅦa plasmid sequence was confirmed correct by using the sequencing apparatus.3. Both the viability and testosterone level of Leydig cells overexpressing COXⅦa was significantly decreased than them of blank control, lipofectamine and empty plasmid group (p<0.05).4. COXⅦa protein was detected in Leydig cell plasmid and nucleus. COXⅦa mRNA and protein of Leydig cells overexpressing COXⅦa was significantly higher than that of blank control, lipofectamine and empty plasmid group (p<0.05).5. SiRNA suppressed COXⅦa mRNA and protein expression in the Leydig cells, which were significantly lower than that in blank control, Lipofectamine and negative control group(p<0.05).6. Both the viability and testosterone level of Leydig cells with SiRNA was significantly higher than that of blank control, Lipofectamine and negative control group (p<0.05).COXⅦa protein can suppress cell proliferation and testosterone secretion. To study the impact of COXVIIa to testosterone biosynthesis in rats and the relation between it and steroidogenesis enzymes and acute regulatory protein in vivo and to explore the new therapy by using RNA interference and Polygonum Multiflorum Trunb.1. The impacts of COXⅦa overexpression to testosterone synthesis, steroidogenesis enzymes and acute regulatory protein in young rats:1.1 Animal groups:3 months SD male rats were randomly divided into pEGFP-N1-COXⅦa group (M group), empty plasmid group (P group), lipofectamine (L group), glucose control (G group) and blank control group (Bc group), each group 8 animals.1.2 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting was used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively.2. The impacts to the Testosterone synthesis and steroidogesis enzyme and protein treated with RNA interference and Polygonum Multiflorum Trunb in PADAM model rats.2.1 Establishment of PADAM model:clean 8 weeks male SD rats were treated with D-galactose 300mg/kg/d intraperitoneally, for continuous 50d.2.2 Animal groups:normal control group (Bc group), PADAM model group (M group), RNA interference group (Si group), testosterone injection group (T group), Polygonum Multiflorum Trunb treated group (H group) and model control group (Mc group), each group 8 animals.2.3 CLIA was used to determine the concentration of total testosterone in serum. Biochemical methods were used to examine SOD, T-AOC viability and MDA level; RT-PCR and western blotting were used to detect COXⅦa, 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression respectively. 1. To compare with P group, L group, G group and Bc group, the expression of the several indicators in M group was as follows:1.1 Serum testosterone concentration was decreased (p<0.05).1.2 The SOD and T-AOC activity was decreased, MDA level was increased (p<0.05).1.3 COXⅦa mRNA and protein expression increased respectively (p<0.05).1.4 3β-HSD,17p-HSD, P450scc and StAR mRNA and protein expression was decreased respectively (p<0.05).2. Comparison between Si group, T group and M group and Bc group after RNA interference in PADAM model rats.2.1 Serum testosterone concentration of Si group and T group was significantly higher than that of M group, while lower than that of Bc group (p<0.05).2.2 The SOD, T-AOC viability of Si group and T group was higher than that of M group, while lower than that of Bc group, MDA content in the contrary (p<0.05).2.3 COXⅦa mRNA and protein expression of Si group and T group decreased compared with M group, still higher than that of Bc group (p<0.05).2.4 3β-HSD,17β-HSD, P450scc and StAR mRNA and protein expression significantly increased compared with M group, but still lower than that of Bc group (p<0.05).1. COXⅦa overexpression in young rats could decrease testosterone synthesis and expression of steroidogenesis enzymes and acute regulatory protein.2. RNA interference in PADAM model rats could lead to partial restoration of testosterone synthesis of rats, increased expression of steroidogenesis enzymes and acute regulatory protein.3. COXⅦa may be play a role in the aging process and related to steroidogenesis enzymes and the acute-regulated protein.4. RNA interference targeting COXⅦa gene and Polygonum Multiflorum Trunb can delay senescence of rats.To explore whether the pathway of ERK and JNK participates in the process that COXⅦa regulates testosterone synthesis of Leydig cells.Leydig cells extracted from young rats and RADAM model rats were transfected with pEGPF-N1-COXⅦa recombinant plasmid and SiRNA respectively, then the experiments were as followed:1. Flow Cytometry was used to detect cell apoptosis rate.2. PCR was used to detect 3β-HSD,17β-HSD, P450scc and StAR mRNA.3. Immunocytochemistry was used to detect 3β-HSD,17β-HSD, P450scc, StAR, Bax and Bcl-2 protein expression.4. Western blotting was used to detect 3β-HSD,17β-HSD, P450scc, StAR and Bax, Bcl-2, ERK1/2, p-ERK1/2, JNK, p-JNK, P38, p-P38 protein expression.1. To compare with control group, empty vector group and Lipofectamine group, the expression of the several indicators in pEGPF-N1-COXⅦa group was as follows: 1.1 The apoptosis rate increased significantly (p<0.05).1.2 The level of 3β-HSD,17β-HSD, P450scc mRNA decreased significantly (p<0.05), while there was no significant difference of StAR mRNA.1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression decreased significantly (p<0.05).1.4 Bcl-2 expression decreased, while Bax expression increased, Bcl-2/Bax decreased (p<0.05).1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK increased significantly (p<0.05); p-P38 no positive bands. 2. To compare with the negative control group, Lipofectamine control group and blank control group, the expression of the several indicators in the interference group with was as follows: 1.1 The apoptosis rate decreased significantly (p<0.05). 1.2 The level of 3β-HSD,17P-HSD, P450scc mRNA increased significantly (p<0.05), while there was no significant difference of StAR mRNA. 1.3 The expression of 3β-HSD,17β-HSD, P450scc and StAR protein expression increased significantly (p<0.05). 1.4 Bcl-2 expression increased, while Bax expression decreased, Bcl-2/Bax increased (p<0.05). 1.5 ERK1/2, JNK and P38 showed no significant difference; p-ERK1/2 and p-JNK significantly reduced (p<0.05); p-P38 no positive bands.1. In the Leydig cells overexpressing COXⅦa, p-ERK1/2 and p-JNK expression and the apoptosis rate increased, while testosterone concentration, the ratio of Bcl-2/Bax, and 3β-HSD,17β-HSD, P450scc and StAR protein decreased, suggesting that p-ERK1/2 and p-JNK could suppress steroid synthesis and promote cell apoptosis.2. The results of RNA interference were in accordance with those of Leydig cells overexpressing COXⅦa.3. COXⅦa protein exerting its function may be through the route of ERK1/2 and JNK.