Study On The Effect Of Intermittent Hypoxia On Male Testosterone Metabolism And The Regulatory Mechanism Of Yijing Formula

Testosterone plays a key role in maintaining male sexual characteristics,sexual function,reproductive function,and quality of life.With increasing age,testosterone tends to decline in men.Late-onset hypogonadism(LOH)is a clinical syndrome associated with male aging,with testosterone deficiency or abnormal metabolism as its pathological basis.In recent years,relevant studies have focused on the effects of intermittent hypoxia on testosterone metabolism in men.Snoring predicts narrowed upper airway and increased respiratory resistance,and frequent snoring may be associated with the risk of intermittent hypoxia.Therefore,this study investigated the relationship of snoring frequency with testosterone and free testosterone in men using data from the National Health and Nutrition Examination Survey(NHANES).The"Yijing formula" of Chinese herbal medicine for tonifying the kidneys and invigorating the blood was used in this study,which is the experienced formula of Professor Jia Jinming,a nationally famous senior Chinese medicine practitioner.Using Gene Set Enrichment Analysis(GSEA)and network pharmacology,as well as an intermittent hypoxia animal model,this study aimed to investigate the potential mechanism of the Yijing formula to improve testosterone metabolism in men under intermittent hypoxia conditions.Part 1 Investigating the relationship of snoring frequency with testosterone and free testosterone in men based on the NHANES databaseObjective:Our study was designed to assess the relationship of snoring frequency with serum testosterone and free testosterone levels in men.Method:NHANES data from 2015 to 2016 were analyzed.According to the selfreported frequency of snoring,the participants were classified as "never snoring group","rarely snoring group(1-2 nights/week)","occasional snoring group(3-4 nights/week)"or "frequent snoring group(≥5 nights/week)".Univariate and multivariate regressions were used to explore the relationship of snoring frequency with serum testosterone and free testosterone levels in men.In addition,subgroup analyses stratified by age were performed in this study.Results:A total of 1928 adult men were included.In the unadjusted model,compared with the never snorers,the rare snorers(β:-0.06,95%CI:-0.12 to-0.01,P=0.027),the occasional snorers(β:-0.15,95%CI:-0.21 to-0.09,P<0.001)and the frequent snorers(β:-0.22,95%CI:-0.27 to-0.17,P<0.001)had lower testosterone levels;while only the occasional snorers(β:-0.08,95%CI:-0.14 to-0.03,P=0.004)and the frequent snorers(β:-0.13,95%CI:-0.17 to-0.08,P<0.001)had lower free testosterone levels.After adjusting for age,race,BMI,hypertension,diabetes,and total cholesterol,compared to the never snorers,testosterone levels were lower in the occasional snorers(β:-0.07,95%CI:-0.12 to-0.01,P=0.013)and the frequent snorers(β:-0.09,95%CI:-0.14 to-0.04,P<0.001).And only the frequent snorers(β:-0.05,95%CI:-0.09 to-0.01,P=0.02)had lower free testosterone levels.In the subgroup analysis stratified by age,the results from multivariate regression models(adjusting for race,BMI,hypertension,diabetes,and total cholesterol)showed that only in those aged 4059 years,compared to never snorers,occasional snorers(β:-0.09,95%CI:-0.19 to<0.001,P=0.0497),and frequent snorers(β:-0.13,95%CI:-0.21 to-0.04,P=0.004)had lower testosterone levels.As for free testosterone,subgroup analysis showed that only in those aged 40-59 years,frequent snorers(β:-0.08,95%CI:-0.16 to-0.01,P=0.034)had lower free testosterone levels compared to never snorers.Conclusion:Middle-aged and older men aged 40-59 years who snore frequently were at risk for decreased testosterone and free testosterone.Meanwhile,testosterone deficiency or abnormal metabolism is the pathological basis of LOH,and frequent snoring predicts increased respiratory resistance and risk of intermittent hypoxia.Therefore,the findings suggested that frequent snoring accompanied by intermittent hypoxia may be a risk factor for LOH.Part 2 Exploring the potential mechanism of Yijing formula in intermittent hypoxic conditions to intervene LOH based on Gene set enrichment analysis and network pharmacologyObjective:To investigate the potential mechanism of Yijing formula under intermittent hypoxic conditions to intervene in LOH and improve male testosterone metabolism.Method:GSEA was performed on the GEO database GSE71356 microarray data.The Hallmark and KEGG databases were used to enrich the analysis and screen the critical signaling pathways for the effects of intermittent hypoxia on male biological functions,respectively.The TCMSP and TCMID databases were searched and screened for the active ingredients of Yijing formula and the corresponding targets.We screened LOH-related disease targets in GeneCards,DisGeNET,and Drugbank databases.The intersection of the Yijing formula and LOH targets was taken,and a PPI network was constructed using the String database to screen out the core targets.GO enrichment analysis was performed on the core targets.Combining the GSEA and network pharmacology results,we obtained the overlap between the potential mechanism of Yijing formula intervention in LOH and the possible mechanism of the effect of intermittent hypoxia on male testosterone metabolism.Finally,we explored the potential mechanism of Yijing formula in intermittent hypoxic conditions to intervene in LOH and improve male testosterone metabolism.Results:After screening the pathways analyzed in the Hallmark database,a total of 14 pathways related to the effect of intermittent hypoxia on male biological function were identified.A total of 10 pathways related to the effects of intermittent hypoxia on male biological functions were identified after screening the pathways enriched by the KEGG database.In both,peroxisome-related biological functions were involved.In the network pharmacology,89 active ingredients of Yijing formula were involved in 353 related targets;and 168 LOH-related targets were retrieved.After the screening,we obtained 34 core targets of the active ingredients of Yijing formula acting on LOH and performed GO enrichment analysis.The results of GO enrichment analysis showed that the top 15 entries of biological processes(BP)and molecular biological functions(MF)were repeatedly associated with oxidative stress.Conclusion:The potential mechanism of Yijing formula to intervene with LOH and improve male testosterone metabolism under intermittent hypoxic conditions may be related to the regulation of oxidative stress.Part 3 Experimental study on the regulation of testosterone metabolism in mice based on intermittent hypoxia-oxidative stress-autophagy pathway by Yijing formulaObjective:A male mouse model was used to observe the effects of intermittent hypoxia on testosterone metabolism,oxidative stress,and autophagy in mice,as well as the regulatory effects of Yijing formula.Method:Intermittent hypoxia modeling was performed on 6-month-old male C57BL/6 mice using an atmospheric pressure high-low oxygen environment simulation chamber.After 4 weeks of modeling,the intermittently hypoxic mice were randomly divided into the model group and the high,medium,and low doses of Yijing formula group,as well as the positive drug(N-Acetylcysteine,NAC)group.And 6-month-old normal male mice were selected as the negative control group.The experimental time was 4 weeks.The sexual behavior of mice was observed by the mating experiment.ELISA was used to detect serum T,FT,LH,and HIF-α.WST-1 method and TBA method were used to detect SOD and MDA,respectively.The protein and mRNA expression of testosterone enzymes P450scc and STAR,as well as ATG5 and LC-3Ⅱ,were detected by real-time PCR and Western blot,respectively.The expression of LC3Ⅱ protein in testicular tissues was observed by immunofluorescence.The autophagic vesicles in testicular tissues was observed by transmission electron microscopy.The pathology of testicular tissues was observed by HE staining.The apoptosis of testicular tissues was observed by TUNEL fluorescence.Results:1.Compared with the negative group,mice in the intermittent hypoxia model group showed a decrease in sexual behavior(P<0.05).Compared with the model group,there was an increase in the sexual behavior of mice in the high-dose and the medium-dose Yijing formula group,as well as in the NAC group,and the increase was most significant in the high-dose group(P<0.05).2.Compared with the negative group,mice in the intermittent hypoxia model group showed statistically decreased T,FT,and LH(P<0.05).Compared with the model group,Yijing formula high,medium,and low doses as well as NAC administration improved T,FT,and LH levels in mice(P<0.05).3.Compared with the negative group,the mice in the intermittent hypoxia model group showed a statistically significant decrease in both StAR protein and mRNA expression(P<0.05).Compared with the model group,only the mice in the high-dose Yijing formula group showed a simultaneous statistically significant increase in StAR protein and mRNA expression(P<0.05).In terms of P450scc protein expression,there was no statistically significant difference between the negative group,the model group,the high-dose,and the medium-dose Yijing formula group(P>0.05).Compared with the negative group,the mRNA of P450scc in mice in the intermittent hypoxia model group showed a statistical decrease(P<0.05);however,the mRNA levels of P450scc in mice in each intervention group were not statistically different from those in the model group(P>0.05).4.HE staining showed the overall structural abnormalities of testicular tissues in the mice in the intermittent hypoxia model group,and the testicular pathological tissue in the high and medium dose Yijing formula groups and the NAC group showed improvement.5.Compared with the negative group,the rate of apoptosis in the testicular tissue of mice in the intermittent hypoxia model group was significantly increased(P<0.05).Compared with the model group,the apoptosis rate of mice in the medium-dose and high-dose Yijing formula groups showed a statistically significant decrease(P<0.05).6.Compared with the negative group,intermittent hypoxic mice showed a statistically significant decrease in SOD and increase in MDA(P<0.05).Compared with the model group,the increase in SOD and the decrease in MDA were statistically significant(P<0.05)after the administration of high and medium doses of Yijing formula as well as the antioxidant NAC.7.Compared with the negative group,mice in the intermittent hypoxia model group showed a statistical increase in ATG5,and LC-3Ⅱ protein expression(P<0.05).Compared with the model group,ATG5,and LC-3Ⅱ protein expression appeared statistically decreased in mice in the high,medium,and low doses of Yijing formula as well as the NAC group(P<0.05).The differences in mRNA expression levels of ATG5 and LC-3Ⅱ among all groups were not statistically significant(P>0.05).8.Both immunofluorescence and electron microscopy results suggested that there was an increase in the degree of autophagy in the intermittent hypoxia model group.Compared with the model group,the degree of autophagy was reduced in the high,medium,and low doses of Yijing formula as well as in the NAC group.Conclusion:Yijing formula could improve intermittent hypoxia-induced reduction in sexual behavior,decrease in testosterone,free testosterone and testosterone enzyme StAR,and testicular tissue abnormalities and apoptosis in male mice.The specific mechanism may be related to Yijing formula alleviating intermittent hypoxiainduced oxidative stress,down-regulating autophagy,and improving cell damage in testicular tissues.