| ObjectiveTo observe the effects of mild moxibustion on testosterone synthesis,testicular cell apoptosis and related signal pathway SIRT1/ NRF2 in naturally aging rats,in order to provide experimental evidence for the clinical treatment of age-related hypotestosterone.MethodMale SD rats were randomly divided into natural aging group,moderate moxibustion group,and 9-month-old rats as young control group.From the age of 14 months,the rats in the MM group were treated with 0.5cm moxa stick to gently moxibustion unilateral "Zusanli(ST36)" point,once a day,20 min each time,and the left and right acupoints were used alternately.The intervention lasted from 6 months to 20 months.At the end of the intervention,testicular tissue and serum were collected.(1)He staining was used to observe the pathological changes of testicular tissue.(2)The levels of total testosterone TT and free testosterone FT in serum and testicular tissue were detected by ELISA,and the expression of testosterone synthase P450 scc was detected by Western blot.(3)The apoptotic level of testis was detected by TUNEL assay.(4)The expression of p53 protein and acetylated p53(Ac-p53)was examined by Western Blot,and the effects of mild moxibustion on the expression and acetylation of p53 in testicular tissues were observed.(4)The protein expressions of SIRT1,Nrf2,GPx4,MnSOD and the mRNA expressions of SIRT1 and Nrf2 were detected by Western blot and PT-PCR.Results1.Histopathological observation of testis: no obvious pathological damage was observed in testis of young control group rats;In the natural aging group,the spermatogenic tubule cells were few and disordered or even missing,the number of cells was significantly reduced,the space appeared in the lumen,and the interstitial cells showed vacuol-like aging changes.The structure of spermatogenic tubule in moxibustion group was clear,the levels and number of cells in the tubule were increased compared with those in natural aging group,and the arrangement of cells at all levels was basically orderly.2.Testosterone level:(1)Compared with the young control group,the serum TT and FT levels in the natural aging group were significantly decreased(P<0.01).Compared with the natural aging group,the serum TT and FT levels in the mild moxibustion group were significantly increased after the intervention of the mild moxibustion "Zusanli" point(P<0.01).(2)Compared with the young control group,the content of testosterone and the expression of testosterone synthetase P450 scc protein in the natural aging group were significantly decreased(P<0.01).After the intervention of mild moxibustion at "Zusanli" point,the testosterone content and the protein expression of testosterone synthase P450 scc in testis were significantly increased(P<0.01).3.Levels of apoptosis: There were fewer apoptotic cells in the spermatogenic tubule and stromal layer of young control rats,and a few apoptotic cells were regarded as normal physiological apoptosis.The apoptotic signal of natural aging group was distributed in spermatogenic cells at all levels in spermatogenic tubule and occasionally in interstitial cells.The number of testicular tissue apoptosis in the mild moxibustion group was significantly less than that in the natural aging group,and some granules were weakly positive.Compared with the young control group,the apoptosis rate of testicular cells in the natural aging group was significantly higher than that in the young control group(P<0.01).Compared with the natural aging group,the apoptosis rate in the mild moxibustion group was significantly decreased(P<0.01).4.Expression of p53 and its acetylated p53(Ac-p53)protein :Western blot results showed that the protein expressions of p53 and Ac-p53 in the natural aging group were significantly up-regulated compared with those in the young control group(P<0.01).The protein expression of Ac-p53 was significantly decreased in mild moxibustion group(P<0.01).5.Expression of SIRT1,Nrf2 protein and gene and GPx4 protein in testicular tissue: Compared with the young control group,the protein expressions of SIRT1,Nrf2,MnSOD and GPx4 in the natural aging group were significantly down-regulated(P<0.01),and the protein expressions of SIRT1,Nrf2,MnSOD and GPx4 in the mild moxibustion group were significantly up-regulated compared with the natural aging group(P<0.05,P<0.01).RT-PCR results showed that the mRNA expression trend of SIRT1 and Nrf2 was consistent with the protein expression trend(P<0.01).Conclusion1.Mild moxibustion at Zusanli(ST36)can effectively improve the testicular tissue structure and cell apoptosis level of natural aging rats,and improve the function of testosterone synthesis;2.The moderate moxibustion of Zusanli(ST36)on testosterone synthesis in naturally aging rats may play a role by regulating the SIRT1/ Nrf2 pathway to improve oxidative stress state. |
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