Correlation Between Testosterone And Coronary Artery Calcification In Men And Its Effect On Vascular Calcification In Rats

BackgroundVascular calcification is primarily a phenomenon of calcium phosphate deposition that occurs in the intima and media of the arteries.Calcification of the intimal layer can occur in atherosclerotic fibrosis plaques and surrounding lesions,which can be observed by intravascular ultrasound techniques.Calcification occurs in the middle layer（also known as Monckeberg calcification）and is more common in patients with chronic kidney disease,type 2 diabetes,and advanced aging,and is associated with all-cause mortality and cardiovascular mortality in these patients^[1-2].Vascular calcification involves the progression of many clinical diseases.Intima calcification in the aorta,such as the aorta,can lead to increased pulse rate and systolic hypertension,which increases cardiac load.In addition,the calcified aorta also causes an increase in blood flow to the left ventricle during systolic reflux,ultimately leading to coronary perfusion.Left ventricular hypertrophy.Endothelial calcification can lead to coronary artery stenosis or rupture of coronary atherosclerotic plaque,eventually leading to myocardial infarction or myocardial ischemia,and may lead to sudden cardiac death,calcification of small arteries in the skin and other organs can lead to local infarction and ischemia^[3].All Framingham risk factors,including hypertension,diabetes,hyperlipidemia,age,smoking,etc.may exacerbate intimal and medial calcification.Vascular calcification is an independent predictor of cardiovascular mortality and an important predictor of coronary heart disease and stroke incidence.Decreased testosterone levels with age are a well-known condition in older men called"low T","menopause"or hypogonadism.Observational studies have shown a link betweenlowendogenoustestosteronelevelsandcardiovascularmetabolic characteristics（high blood pressure,dyslipidemia,insulin resistance,atherosclerosis,thrombosis）and increased overall and cardiovascular（CV）death rate.Testosterone decline and CVD disease are mixed with various influencing factors,such as diabetes,high blood pressure,tobacco,alcohol,etc.,which makes the correlation between testosterone and cardiovascular disease more complicated.At present,the correlation between testosterone levels and the cardiovascular system in multiple experimental animal models is multiple,and the experimental results are contradictory.Some cross-sectional studies have shown that lower endogenous testosterone levels are associated with increased intimal and medial thickness,independent of traditional CV risk factors.In the case-control study,testosterone levels were significantly lower in patients with confirmed coronary heart disease compared with controls without coronary heart disease.And the severity of coronary lesions increases with decreasing testosterone levels.In contrast,studies have found that testosterone replacement therapy（TRT）increases cardiovascular risk.However,there is currently no uniform standard for the definition of low testosterone.The potential confounding factors and sample size in relevant experiments are the main factors affecting the accuracy of the experimental results.This study mainly explores the relationship between testosterone and coronary artery calcification by detecting testosterone levels in patients with coronary artery calcification and non-CAC.At the same time,the relationship between potential confounding factors（such as hypertension,diabetes,blood lipids,uric acid,etc.）and CAC was detected,and the effects of testosterone on cardiovascular disease were further analyzed.The rat vascular calcification model was established by nicotine and vitamin D3.Exogenous testosterone was used to intervene the rats,and the expression of OPN and BMP4 bone-related protein was detected by Western blot.To study the effects of exogenous testosterone replacement therapy on aortic calcification induced by nicotine and vitamin D3.Aims1.To explore the relationship between serum testosterone levels and coronary artery calcification in men2.To elucidate the effects of testosterone on vitamin D3 and nicotine-induced aortic calcification in rat.Methods1.Collect patient serum samples to test testosterone levels.Collection of Serum samples of hospitalized male patients who underwent coronary CTA examination from December 2016 to September 2017.centrifuged at 3000 rpm for10 minutes,Stored in a refrigerator at-20°C to tested testosterone levels.According to the results of coronary CTA examination,they were divided into two groups,including32 cases without coronary artery calcification（which is control group）and 40 cases with coronary artery calcification.General clinical data（such as age,hypertension,diabetes,smoking,uric acid,total cholesterol,LDL,HDL,triglycerides,creatinine,CRP,BMI）of the 72 male patients were collected.SPSS23.0 software was used to statistically analyze the correlation between various indexes and coronary artery calcification,and logistic multivariate statistical analysis was used to correct the influence of confounding factors on the study,and to explore the correlation between male serum testosterone level and coronary artery calcification.2.Establishing a classic rat vascular calcification model.Male SD rats were randomly divided into four groups（calcification group,calcification+low testosterone group,calcification+high testosterone group,control group）,with 8rats per group.1.Except the control group,the other three groups were given intramuscular injection of vitamin D3（30 0000 U/kg）at 8:00 on the first day of the experiment,and nicotine（25 mg/kg）was dissolved in peanut oil and administered again at 18:00 in the afternoon.The same dose of nicotine was administered once;2.Control group:the same amount of absolute ethanol was given intramuscularly and peanut oil was intragastrically administered;after 8 weeks,the heart was collected and anesthetized with 5%chloral hydrate,and sacrificed.The pathological sections of rat aortic vessels were prepared to observe the deposition of calcium salts and to detect calcification related factors.3.Detection of calcification related indicators after exogenous testosterone intervention.From the second day,1 mg/kg（calcification+low testosterone group）,2 mg/kg（calcification+high testosterone group）testosterone intramuscular injection were given every other day,and the calcified group and the control group were given the same amount of absolute ethanol intramuscular injection.A total of 8 weeks of administration.After 8 weeks,the rats were sacrificed（ibid.）and serum testosterone and bone morphogenetic protein-4（BMP-4）levels were determined by Elisa method.The contents of calcium and alkaline phosphatase（ALP）in blood vessels were detected by kit.Western blot analysis was performed.Western blot was used to detect the protein expression levels of BMP-4 and osteopontin（OPN）in aortic vascular tissues,and vascular calcification was observed by Vonkossa staining.Results1.Male testosterone levels are negatively correlated with coronary artery calcification.The basic clinical information of 72 hospitalized male patients was analyzed.Compared with the control group,the differences in age,smoking,uric acid were statistically significant（P<0.05）.The other indicators（hypertension,diabetes,total cholesterol,LDL,creatinine,BMI,HDL and CRP）were not statistically significant.Serum testosterone levels were significantly lower in patients with coronary artery calcification than in patients without coronary artery calcification（P<0.01）.Logistic single factor regression analysis showed a correlation between testosterone and coronary artery calcification（P<0.01）.The difference was statistically significant.Logistic multivariate regression analysis and after adjusting for confounding factors,the correlation between testosterone and coronary artery calcification was P<0.05,and the difference was statistically significant.3.Successful establishment of rat vascular calcification model.Both vonkossa staining and alizarin red staining showed a large amount of black calcium deposits in the calcified aortic vascular media,while the control group’s vascular wall was smooth and intact,and no obvious black calcium deposits were observed.Calcium ion detection kit was used to detect calcium ion content in aortic blood vessels.Calcium ion content in calcification group was significantly higher than that in the control group（P<0.01）.That is,the calcification model was established successfully.4.Exogenous testosterone can alleviate vitamin D3 and nicotine-induced vascular calcification in rats.Vonkossa staining and alizarin red staining showed a large amount of black granular calcium deposits in the vascular media of the calcified group,while a small amount of calcium salt was deposited in the low testosterone group and the high testosterone group,and no calcium salt deposition in the control group.The calcium content and ALP content in the kit were measured,and the calcification group,the calcification+low testosterone group,the calcification+high testosterone group,and the control group were sequentially decreased（P<0.05）.Western blot was used to detect the expression levels of calcification-related proteins BMP-4 and OPN.The expression level of calcification group was the highest.The low testosterone group and high testosterone group were the lowest,and the control group was the lowest.The difference between the two groups was statistically significant（P<0.05）.In summary,exogenous testosterone supplementation can alleviate vitamin D3 and nicotine-induced aortic vascular calcification in rat to some extent.Conclusion1.Male testosterone levels are negatively correlated with coronary artery calcification.2.Exogenous testosterone can alleviate vitamin D3 and nicotine-induced aortic calcification in rats to some extent.