| Male aging is typically accompanied by reduced serum levels oftestosterone in human and rodent, which are associated with declines inreproductive function, muscle strength, bone density and other physiologicalparameters. The Leydig cells produce testosterone in response to hormonalstimulation by the pituitary gonadotropin luteinizing hormone (LH). LH levelsdo not decrease significantly with age though serum levels of testosterone dodecline, suggesting that although age-related functional changes may (andprobably do) occur within the hypothalamic–pituitary axis, a primary gonadallesion principally contributes to age-related testosterone decline. The numberof Leydig cells per testis seems unchanged compared to young controls. Thesefindings suggest that functional changes to the Leydig cells, rather than theirloss, accounts for the observed reduction in serum testosterone levels.In recent years, oxidative stress has been recognized and attracts thepublic attention more and more. The free radical theory of aging posits thatcells are in a chronic state of oxidative stress as a consequence of imbalancebetween pro-oxidants and the antioxidant defense system, leading to reactiveoxygen species (ROS) accumulation and a variety of macromolecularoxidative modifications, including lipid peroxidation of membranes, enzymeinactivation, protein oxidation, and DNA damage. At the same time, the agingbody also has formed a set of oxidative stress response system to deal with thefree radical. Antioxidant enzymes (AOEs) play an important role in theprotection of cells from reactive oxygen species. Nuclear factor erythroid2-related factor (Nrf2) is one of the critical transcription factors involved inthe regulation of anti-oxidant enzymes through its recruitment to theantioxidant response element (ARE) in their gene promoters. It is the mostimportant endogenous antioxidant stress pathway so far. In addition to oxidative stress, chronic inflammatory events are known to participate in thedecline in physiological functions of aging organs. Nuclear factor kappa beta(NF-κB) is a redox-sensitive transcription factor that is involved in theexpression of genes encoding pro-inflammatory molecules (such as TNF-α,IL-1β, IL-2, iNOS and COX2). NF-κB activation and chronic inflammationseem to be a generalized phenomenon during aging.This study aimed to investigate the roles of the antioxidant stresspathways of Nrf2and chronic inflammation pathway of NF-κB in age-relatedtestosterone reduction and the mechanism underlying this process in testiculartissue and Leydig cells of mice through physiological, morphological andmolecular biology techniques. Our study consisted of three parts:(1) Toinvestigate the changes of transcriptional factor Nrf2and NF-κ b signalingpathway in testicular tissue with aging.(2) To find the evidences thatage-related changes in ROSâ†'p38MAPKâ†'COX2, NF-κBâ†'COX2and Nrf2signaling pathways contribute to the decreased testosterone production by theLeydig cells from old mice.(3) To determine the effects of moderate exerciseover different phases on age-related physiological dysfunction in testes ofmice.Part â… Oxidative stress and inflammatory response increase with agingin the SAMs mice testis: the role of transcriptional factor Nrf2and NF-κBsignaling pathwaysObjective: The decline in blood testosterone concentration during thecourse of male aging results in decreases in many physiological functions.However, the mechanism responsible for this decline is not clear. Previousstudies have shown that testosterone reduction of aged rodent was associatedwith oxidative damage and pro-inflammation increases. In our study，weexplored the changes of redox state and inflammatory response with aging inthe testes of varying ages (2,4,8,10months old) of senescence-acceleratedmouse8(SAMP8, P8) with the control mice (SAMR1，R1), and detected therole of transcriptional factor Nrf2and NF-κB signaling pathway. Methods:1Two,4,8, and10-month-old male P8and R1mice were provided byFirst Hospital of Hebei Medical University (N=6mice per age).2Serum testosterone concentrations from individual mice were assayedby radioimmunoassays (RIA). The sensitivity and intra-assay and inter-assaycoefficients of variation of the RIA were13g/tube,8.9%and13.6%,respectively.3Immunohistochemical and immunofluorescence staining were used toexamine the changes of COX2, Nrf2and NF-κB.4Biochemical analyses were used to examine the oxidation levels ofMDA and protein carbonyls and the activity of SOD, CAT and GPX.5Luminex multi-analyte cytokine profiling technology was used todetect the levels of pro-inflammatory TNF-α, IL-1β.6RT-PCR and Western blotting were used to detect the expression ofStAR, Nrf2, NF-κB, COX2, P450scc, P-P38and P38.7Data analysis: All data were analyzed using SPSS16, and results arepresented as the mean value±SD. Significant differences for age (2,4,8,10months) and strain (P8, R1) were determined using a two-way ANOVA. Ina second analysis, a one-way ANOVA was run on the function of age (2,4,8,10months) among the P8, R1mice. Statistical differences between strain (P8,R1) were evaluated by Student t-test. A p-value of less than0.05wasconsidered to be significant.Results:1Age-dependent changes in testosterone: The interaction of strain andage was significant (p=0.000). Plasma testosterone across P8mice2,4,8,and10months of age decreased with increasing age. The mean testosteronelevel of4-month-old P8mice was significantly higher than the means of miceeither8or10months of age at p <0.01. The~44%decrease in testosteronelevel for the8-month-old compared to4-month-old P8mice was detected,whereas testosterone levels for4-and8-month-old RI mice were notsignificantly different. 2Age-dependent changes in the expression of StAR and P450scc: Theresult of StAR expression showed that the interaction of strain and age wassignificant. However, the result of P450scc expression showed that theinteraction of strain and age did not have a statistical effect. The expression ofStAR and P450scc across P8mice2,4,8, and10months of age yielded asignificant effect, with the expression of StAR and P450scc decreasing withincreasing age. The expression of StAR for8-month-old P8mice hadsignificantly lower values than4-month-old P8mice (p <0.01) whereas for4-and8-month-old RI mice were not significantly different. The levels of StARwere significantly higher at2,4months, and significantly lower at8and10months in P8than that of R1(p <0.05). This was consistent with changes intestosterone. The expression of P450scc did not have significantly differentbetween4-and8-month-old P8mice, but the levels of P450scc of10-month-old were significantly lower than2,4,8-month-old mice in bothstrains (p <0.01). The levels of P450scc were significantly lower at10monthsin P8than that of R1.3Age-related alterations in oxidative damage and anti-oxidative enzymesactivities：The result of protein carbonyl showed that the interaction of strainand age was significant, however, the result of MDA showed that theinteraction of strain and age did not have a statistical effect. The results ofSOD, GPX and CAT showed that the interaction of strain and age weresignificant. The protein carbonyl and MDA contents were increased in P8mice at4,8months of age respectively, and gradually elevated with the agingprocess (p <0.05). The levels of SOD and CAT, which are known as enzymesresisting oxidative damage, were decreased at8months of age, however, thelevel of GPX was decreased earlier at4months of age (p <0.05). Theseresults indicate that the decrease in the level of SOD, CAT as well as GPX,may be involved in the increased oxidative stress of P8mice at a younger age,preceding the manifestation of definite deficits in testosterone production inP8mice. MDA, protein carbonyls content at the age of8,10months wasincreased in SAMP8mice compared with R1mice. The levels of anti-oxidative enzymes, SOD, GPX and CAT at the age of4,8,10month inP8mice were less active than that in R1mice. From these results, Comparedwith R1mice, P8mice showed earlier biochemical changes considered to beassociated with the accelerated aging process even8months after birth.4Age-related declined in transcription factor Nrf2: The interaction ofstrain and age was significant. The level of Nrf2in the nuclear fraction oftestes increased in an age-dependent manner in R1, which implies that moreNrf2is translocated into the nucleus in response to increasing oxidative stresswith age. However, the level of Nrf2in the nuclear fraction of testes increasedat4month old compared with2months old (p <0.05), and significantlydecreased at8months old in P8(p <0.05). The level of Nrf2in the nuclearfraction in8appeared to be lower at2months old and became significantlylower at8months old than that in age-matched R1mice. The intensity of Nrf2immunofluorescence staining in the nucleus of the testes in P8was muchweaker than that in R1mice at8months old. Thus, this result supports thealteration of Nrf2protein level in the nucleus of the testes of P8mice.5Age-related alterations in pro-inflammation: The interaction of strainand age was not significant for IL-1β and TNF-α. In both strains of SAMs at8,10months old, increases in the levels of IL-1β and TNF-α were observed(P<0.001). The significant differences in the levels of TNF-α, IL-1β betweenthe strains were detected at4,8,10months in SAMP8(P<0.001). In a word,pro-inflammatory cytokines TNF-α, IL-1β increased with aging. Aged cellswere in a chronic inflammatory state.6Age-related increase in the expression of COX2: The result showed thatthe interaction of strain and age was not significant. The protein level ofCOX2in testes increased in an age-dependent manner in P8and R1. In P8mice, the level of COX2increased at8,10months than2,4months (P<0.005).The level of COX2in P8became significantly higher at8months old than thatin age-matched R1mice (P<0.005). The immunohistochemistry techniquerevealed the presence of COX2in the cytoplasm of interstitial cells, and theintensity of positive cells increased in an age-dependent manner in P8. In P8 mice, the intensity of COX2immunohistochemistry staining was muchstronger than that in R1mice at8months old.7Age-related increase in transcription factor NF-κB: The result showedthat the interaction of strain and age was not significant. The level of NF-κB inthe nuclear fraction increased in an age-dependent manner in P8, andsignificantly increased at8,10month old compared with2,4months old (p <0.05), which implies that more NF-κB is translocated into the nucleus withaging. However, there was no different in the level of NF-κB in the nuclearfraction at different age in R1. The level of NF-κB in the nuclear fraction in P8appeared to be higher at8,10months old than that in age-matched R1mice.We assessed NF-κB protein level by immunofluorescence staining, andshowed that there was higher NF-κB expression in the testes in P8mice withincreasing age. In P8mice, the intensity of NF-κB immunofluorescencestaining in the nucleus of the testes at8months old was much stronger thanthat in R1mice. Thus, this result supports the alteration of NF-κB protein levelin the nucleus of the testes of P8mice.8Changes of p38MAPK signaling pathways in response to aging: Theresult showed that the interaction of strain and age was not significant. P38MAPK phosphorylation (P-P38) was increased with aging in SAMP8mice.There was no different at different age in SAMR1. Moreover, the level of p38MAPK phosphorylation in SAMP8became significantly higher at8,10months old than that in age-matched SAMR1mice (p <0.05). This supportsthe possibility of a causal impact of p38MAPK on the decline insteroidogenesis in aged mice.Conclusion: Aging, in the absence of other risk factors, is associatedwith oxidative stress and inflammatory changes in the phenotype of testes. It ispossible that the decreased translocation of Nrf2into the nucleus of the testesin SAMP8resulted in reduced antioxidant enzyme expression, which causesvulnerability to oxidative stress. This might lead to accelerated senescence inSAMP8mice.Age-associated ROS induction of NF-κB activation is especially interesting, since it seems to contribute significantly to testosterone reductionin aged mice. Future studies need to elucidate the line relationship ROSâ†'p38MAPKâ†'NF-κBâ†'COX2. Overall, we can expect recent advances in ourunderstanding of oxidative/nitrosative stress and redox sensitive inflammatorymechanisms of testes aging.Part â…¡ Evidence that age-related changes in ROSâ†'p38MAPKâ†'COX2,NF-κ bâ†'COX2and Nrf2signaling pathways contribute to the decreasedtestosterone production by the Leydig cells from old SAMP8miceObjective: The current studies were initiated to investigate age-relatedchanges in ROSâ†'p38MAPKâ†'COX2, NF-κ bâ†'COX2and Nrf2signalingpathway contribute to the decreased testosterone production by the Leydigcells from old SAMP8mouse.Methods:1Isolation of primary SAMP8mouse Leydig cell and measurement oftestosterone:Leydig cell was obtained using differential adhesion speed method. Analiquot was incubated for5min with0.4%Trypan blue and used for cellcounting and viability assay in a light microscope. The purity of cellpreparations from mouse was assessed by determining the percentage of cellsthat immunohistochemical stained and histochemical stained for3β-hydroxysteroid dehydrogenase (3β-HSD). Triplicate samples of cells wereincubated for2h without (basal) or with LH (100ng/ml), and subsequentlysamples of incubation medium were collected, and analyzed for testosteroneproduction by the radioimmunoassay technique.2Measurement of intracellular ROS: The level of intracellular ROS wasquantified by measuring the fluorescence of DCFH-DA. The relative levels offluorescence were measured with a flow cytometer.3MTT Assay: Cell viability was evaluated by using3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra-zoliumbromide (MTT) assay.4pEGFP-N1-Nrf2plasmid construction, transfection and Identification to positive cells: Mouse Nrf2cDNA (1.8kb)was cut at the SacI/KpnI site fromthe pcDNA3/m Nrf2plasmid, and inserted into p-EGFP-N1(kindly providedby Yirao Liu, Life Sciences, University of Electronic Science and Technology,China) vector pre-cut with the SacI/KpnI restriction enzymes.After the sequence of the recombinant plasmid was proved correct, therecombinant plasmid was transferred into the primary cultured Leydig cellsusing the Lipofectamine2000reagentCells with green fluorescent under the confocal laser scanningmicroscopy were positive.5Immunofluorescence staining was used to examine the changes ofCOX2, Nrf2and NF-κB.6Semi-Quantitative Reverse Transcriptase-and Real-Time PolymeraseChain Reactions were used to examine the changes of COX2and StARmRNA expression7Western blotting was used to detect the expression of StAR, Nrf2,NF-κB, COX2, P-P38and P38.Results:1The purity of isolated Leydig cells was more than95%in accordancewith the test requirements.2Aging and testosterone production by isolated mouse Leydig cells: Theaddition of a maximally stimulating dose of LH to cells from control animals(adequate circulating levels of testosterone) resulted in a general increase inhormone production over basal levels, but showed a significant reduction(~70%) in old vs. young Leydig cells.3Effect of age on ROS production: The levels of ROS significantlyincreased in the old Leydig cells compared with the young Leydig cells (p <0.05).4The effects of p38MAPK and/or NF-κB inhibitors on testosteroneproduction, StAR, COX2and Nrf2in aged SAMP8Leydig cells:Pretreatment with SB203580(10μM), a highly potent inhibitor of p38MAPK, restored testosterone production in old Leydig cells to~40%of the level seen in Leydig cells from young animals. Likewise, use of Bay, NF-κBinhibitors, partially restored testosterone production in old Leydig cells (p<0.05). In contrast, treatment of cells with SB203580+BAY did produceadditive or synergistic effect suggesting that two types of inhibitors exert theirstimulatory action respectively.We found that treatment of old Leydig cells with Bay induced tosignificant decrease in COX2protein expression by doubleimmunofluorescence. Bay or SB203580led to a significant decrease inCOX2mRNA and protein expression (p <0.05), and a significant increase inStAR mRNA and protein expression (p <0.05). Treatment of cells withSB203580+BAY did produce additive or synergistic effect on COX2andStAR gene expression. Therefore, the two pathways of p38MAPK-COX2andNF-κB-COX2played important roles in the reduction of testosteroneproduction with aging. Moreover, treatment with Bay increased nuclearaccumulation of Nrf2.5The effects of Transient transfection of Nrf2on testosterone production,StAR and COX2in aged SAMP8Leydig cells:The aged Leydig cells with transfection pEGFP-N1-Nrf2had higherability to produce testosterone compared with control. We found that theLeydig cells with transfection of pEGFP-N1-Nrf2led to a significant decreasein COX2protein expression by double immunofluorescence (p <0.05).Transfection of pEGFP-N1-Nrf2induced to decrease in COX2mRNA andprotein expression (p <0.05), and a significant increase in StAR mRNA andprotein expression (p <0.05).Conclusion: We describe three key findings as follows. First,COX2-mediated inhibition of testosterone production during aging isaccompanied by a selective activation of p38MAPK. Second, the inhibitoryactions of enhanced COX2on testosterone production are mediated by theNF-κB pathway. Third, Nrf2plays a positive role in age-related loss ofsteroidogenic function. Part â…¢ Effects of moderate exercise over different phases of life onage-related physiological dysfunction in testes of SAMP8miceObjective: The effect of moderate exercise over different phases onage-related changes, including serum testosterone levels and biomarkers ofinflammation and oxidative stress were analyzed. The expression and activityof Nrf2, a transcriptional regulator of the cellular anti-oxidant system, andNF-κB, a mediator of inflammatory molecules were also detected.Methods:1In this study, male SAMP8mice, a senescence-accelerated strain, weremaintained as sedentary (sed) or subjected to daily15-min periods ofswimming exercise between ages of2–7months (lifelong),2–4months(earlier) or5–7months (late).2Serum testosterone concentrations from individual mice were assayedby radioimmunoassays (RIA). The sensitivity and intra-assay and inter-assaycoefficients of variation of the RIA were13g/tube,8.9%and13.6%,respectively.3Immunohistochemical and immunofluorescence staining were used toexamine the changes of COX2, Nrf2and NF-κB.4Biochemical analyses were used to examine the oxidation levels ofMDA and protein carbonyls and the activity of SOD, CAT and GPX.5Luminex multi-analyte cytokine profiling technology was used todetect the levels of pro-inflammatory TNF-α, IL-1β and anti-inflammatoryTGF-α, IL-10.6RT-PCR was used to detect the expressions of SOD1, SOD2, GPX,CAT.7Western blotting was used to detect the expression of StAR, Nrf2,NF-κB, COX2, P450scc.8EMSA assays were used to detect the activity of Nrf2and NF-κB.Results:1Effects of exercise over different phases of life on testosterone levelsand expression of steroidogenic enzymes: The testosterone levels in serum and testes were significantly higher in the exercise groups compared with thesedentary group (P <0.05). Lifelong and earlier phase exercise resulted inmore effective elevations of testosterone, and StAR and P450scc levels thanwith late phase exercise (P <0.05).2Effects of exercise over different phases of life on levels of oxidativestress and anti-oxidative enzymes: The level of MDA, a marker of lipidperoxidation, was significantly lower in animals of the exercise groupscompared with the sedentary ones, and it was significantly lower in thelifelong and earlier groups than the late group (P <0.05).Levels of protein carbonyl (a biomarker of protein oxidation) weresignificantly lower in the lifelong and earlier groups compared to the late andsedentary groups (P <0.05), and no differences were observed between thelifelong and earlier groups or between the late and sedentary groups.The activity of total SOD in mice with exercise was significantly higherthan that of sedentary ones (P <0.05). The mRNA level of SOD1and SOD2were significantly higher in lifelong, earlier groups compared to late andsedentary ones (P <0.05).The activity of CAT was significantly higher in the exercise groupscompared with the sedentary group (P <0.05), and no difference was foundamong the three exercise groups. However, the mRNA level of CAT did notdiffer among groups.Activities of GPX were significantly higher in animals of the lifelong andearlier groups compared to late and sedentary ones (P <0.05). The mRNAlevel of GPX was significantly higher in the training groups compared withthe sedentary one (P <0.05), moreover, the groups differed statistically fromeach other.3Effects of exercise over different phases of life on Nrf2nucleartranslocation: Immunofluorescence for Nrf2showed the localization in Leydigcells and spermatogenic cells in all the animals studied. The resultsconsistently showed that the levels of nuclear Nrf2were significantly higher inthe exercise groups compared with that of the sedentary group, and they were significantly higher in the lifelong and earlier groups than in the late group (P<0.05). An EMSA revealed that lifelong and earlier exercises increased thebinding activity of nuclear extract to ARE-containing oligonucleotidescompared with sedentary and late groups (P <0.05).4Effects of exercise over different phases of life on testicularmacrophages, inflammatory and anti-inflammatory cytokine levels: Thequantification of macrophages by immunohistochemistry staining for CD68,CD68is a macrophage marker. The density of CD68+macrophages weresignificantly lower in the exercise groups compared with that of the sedentarygroup, with significantly lower levels in the lifelong and earlier groupscompared with the late group (P <0.05), and the IL-1β levels was consistentwith macrophages. The level of TNF-α was significantly lower in the lifelongcompared with the other groups, and it was significantly lower in the earliergroup compared with the late and sedentary groups (P <0.05). TGF-α andIL-10levels were significantly higher in the lifelong and earlier groupscompared with the late and sedentary groups (P <0.05), and the level ofTGF-α was significantly higher in the lifelong group compared with the earliergroup (P <0.05).5Effects of exercise over different phases of life on COX2expression:COX2was significantly lower in the exercise groups compared with thesedentary group, and they differed statistically significantly among theexercise groups (P <0.05). These results indicated that lifelong exercise andearlier phase exercise could decrease COX2levels more significantly than latephase exercise.6Effects of exercise over different phases of life on NF-κB nucleartranslocation: Immunohistochemistry for NF-κB showed the localization inLeydig cells in all the animals studied. The results of both analyses showedconsistently that the levels of NF-κB were significantly lower in the exercisegroups compared with that of the sedentary group, and they differedstatistically significantly among the exercise groups (P <0.05). An EMSArevealed that lifelong and earlier exercises decreased the DNA binding activity of NF-κB compared with sedentary and late groups (P <0.05).Conclusion: We conclude that moderate exercise over different phases oflife variably influences age-related physiological dysfunction in the testes.Lifelong or earlier initiation of exercise may be more beneficial than exercisein late life by reversing the age-related decline in steroidogenic enzymes andin restoring testosterone production by increasing the resistance of cells tooxidative stress and inflammation. This study suggests that moderate exerciseover a lifetime or during a period before the normal decline in testosterone canactivate the cellular anti-oxidant system earlier in life to confer the greatestprotection of testes tissue and function. The responses of Nrf2and NF-κB tolifelong or earlier phase exercise may be counterbalanced in their critical rolesto lower levels of inflammation and oxidative stress. |
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