Study On The Relationship Between GHSR Gene Methylation Changes And Carotid Arteriosclerosis In Male Tobacco-dependent Patients

Objective:To observe the relationship between GHSR gene methylation levels and carotid arteriosclerosis in male tobacco-dependent subjects.Methods:Part I: A total of 130 men who underwent physical examination in our hospital from September 2020 to January 2022 were selected as the study subjects,including 64 cases in the tobacco dependence group with an average age of(58.88±10.17)years,and 66 cases in the non-smoking control group with an average age of(58.94 ± 10.63 years).The names,ages,length of education,tobacco use history,and past medical history of the study subjects were collected,including previously diagnosed drugs well controlled and newly discovered hypertension,diabetes,and hyperlipidemia.Then scale tests were performed,including: a mini-mental state examination(MMSE)for assessing the cognitive level of the subjects;Fagerstorm test for nicotine dependence(FTND)for the diagnosis of tobacco dependence and assessment of tobacco dependence;Professionals collected 5-6ml of peripheral venous blood in the basic state of all subjects(early morning,fasting for 8-12 hours,in a quiet and relaxed state)and placed it in an EDTA anticoagulation tube,and the samples were stored in the-80°C refrigerator according to the date of blood collection,and finally combined with Methyl Target technology by multiplex polymerase chain reaction(MPCR)technology.Assays for GHSR gene methylation levels were performed in all samples.The methylation differences of 64 Cp G sites and 4 fragments of GHSR gene in the tobacco-dependent group and nonsmoking control group were compared.SPSS26.0 was used for statistical analysis and Shapiro-Wilk was used for normality testing.Quantitative data for skewed distributions,expressed as median(interquartile range),were analyzed using the Mann-Whitney rank sum test.For normal distribution measurement data,expressed as mean ± standard deviation(’x±s),t-test is used for analysis;The counting data is expressed in frequency(frequency),and the counting data is analyzed by chi-square test,and the P<0.05 is statistically significant.Part II: The thickness of the carotid intima at the proximal end of the common carotid artery bifurcation was measured in the longitudinal profile of 130 subjects using GELOGIQ E9 ultrasound diagnostic instrument,and recorded.According to the median methylation value of the site,the methylation degree of subjects was divided into low methylation and high methylation,and the subjects were divided into tobacco dependence and non-tobacco dependence according to whether they were tobacco dependent,and 2*2 factorial ANOVA was used to explore the relationship between the methylation degree of GHSR gene Cp G site and carotid intima thickness in tobacco dependent patients(P<0.05 was statistically significant),and the relationship between tobacco dependence and GHSR gene methylation level and carotid arteriosclerosis was analyzed.Results:Part I: GHSR gene methylation analysis1.There was no significant difference between the tobacco dependence group and the non-smoking control group in terms of average age and years of education(P>0.05);There was no significant difference in the history of hypertension,diabetes mellitus,and hyperlipidemia between the two groups(P>0.05).Analysis of tobacco dependence in tobacco dependence group: age of initial tobacco use 24.47 ± 7.70 years,duration 31.67 ± 9.79 years,daily tobacco use 21.08 ± 9.18 sticks,FTND score 6.38 ± 1.40 points.2.The methylation level of Cp G204 site in the GHSR-4 fragment of the tobacco dependent group was higher than that in the control group,and the difference was statistically significant(P<0.05).Cp G29,Cp G32,Cp G39,Cp G56,Cp G100,Cp G108,Cp G112,Cp G194 in GHSR-1 fragment,Cp G60,Cp G82,Cp G201 in GHSR-2 fragment,Cp G37,Cp G47,Cp G67,Cp G78 in GHSR-3 fragment;The methylation levels of 29 sites in GHSR-4 fragments,including Cp G30,Cp G42,Cp G62,Cp G89,Cp G108,Cp G116,Cp G120,Cp G123,Cp G153,Cp G174,Cp G201,Cp G218,Cp G233,and Cp G237,were higher than those in the non-smoking control group,but the difference was not statistically significant(P> 0.05,see Table 3).3.The mean methylation values of GHSR-1 and GHSR-2 fragments in the tobacco dependent group were lower than those in the non-smoking control group,and the average methylation values of GHSR-3 and GHSR-4 fragments were higher than those in the non-smoking control group,but the difference was not statistically significant(P>0.05).Part II: Study on the relationship between GHSR gene methylation changes and carotid arteriosclerosis in tobacco-dependent patientsThe median methylation of Cp G204 sites in GHSR-4 was 0.202976,and the carotid intima thickness of 4 groups: 22 cases in the tobacco-dependent hypomethylation group had a carotid intimal thickness of 1.241±0.469,42 cases in the tobacco-dependent hypermethylation group had a carotid intimal thickness of 1.493±0.593,43 cases in the control hypomethylation group had a carotid intimal thickness of 1.095±0.497,and 23 cases in the control hypermethylation group had a carotid intimal thickness of 1.243 ± 0.537;2×2 The results of factorial design ANOVA showed that the main effects of tobacco dependence: F=4.041,P=0.047,the difference was statistically significant.The main effects of methylation degree at Cp G204 sites: F=4.147,P=0.044,and the carotid intimal thickness of those with hypermethylation changes was significantly higher than that of low-methylation changes,and the difference was statistically significant.The interaction effect between the two factors: F=0.279,P=0.598,the difference was not statistically significant.In summary,the results showed that the thickness of the carotid intima increased significantly when the hypermethylation of Cp G204 in GHSR-4 increased,the hypermethylation change of Cp G204 in GHSR-4 was related to the degree of carotid artery sclerosis,and tobacco dependence was closely related to the occurrence of carotid artery sclerosis.Conclusion:1.This study showed that the methylation level of Cp G204 site in GHSR-4 was significantly increased in tobacco dependent patients.2.Increased methylation of Cp G204 sites in GHSR-4 and tobacco dependence were associated with carotid arteriosclerosis.