| Objectives:To observe the expression of homocitrulline(HCit)in the epidermis of skin lesions of seborrheic keratosis(SK),skin around SK lesions and normal skin in different age groups,and to explore the correlation between HCit and SK.To observe the expression trend of HCit in peripheral blood of healthy people of different ages,compare the expression level of HCit in the peripheral blood of SK patients or patients with newly developed type 2 diabetes mellitus(T2DM)with that of healthy subjects in the same age group,preliminarily study the correlation between the level of carbamylation and blood glucose in human body.The expression levels of CircPVT1,TGF-?1,P53,P21,IL-6 and Hepcidin mRNAs in peripheral blood of healthy subjects of different ages were detected,and the signaling pathways that may be related to the regulation of HCit expression level were preliminarily discussed.Methods:Skin tissues of patients admitted to the dermatology department of the affiliated hospital of Yangzhou university and undergoing skin biopsy from September 2017 to June 2019 were selected.Archived wax block specimens were collected,including SK specimens in 20 cases and normal skin specimens in 21 cases.Whole blood samples of 60 healthy people who passed the physical examination from the physical examination center of Yangzhou university hospital from June 2019 to November 2019 were collected.At the same time,8 cases of serum samples from SK patients in dermatology outpatient department and 13 cases of serum samples from newly developed T2DM patients in endocrinology outpatient department were collected.Immunohistochemistry(IHC)was used to observe the expression of HCit in SK lesions,skin around SK lesions and normal skin in different age groups.Sandwich enzyme-linked immunosorbent assay(ELISA)was used to observe the expression trend of HCit in peripheral blood of healthy subjects of different age groups,and the expression level in peripheral blood of SK patients and patients with newly developed T2DM was detected.The expressions of CircPVT1,TGF-?1,P53,P21,IL-6 and Hepcidin mRNA in peripheral blood of healthy subjects in different age groups were detected by real-time polymerase chain reaction(RT-PCR).Result:1.IHC results:1)HCit is mainly expressed in the nucleus of the epidermis.Immunohistochemical scores of HCit were 9(4.5,10.5),9(6.75,9),7.5(6,11.25),and 9(8.25,9.75)in SK tissues in four age groups:40-49 years,50-59 years,60-69 years and>70 years.There was no significant trend in the expression of HCit in SK lesions of different age groups,and the difference was not statistically significant(?2=0.605,P>0.05).2)The immunohistochemical scores of HCit were 9(9,10.5),9(9,11.25),9(9,11.25),9(9,11.25),9(9,11.25),and 9(9,9.75)in the skin around SK lesions in the four age groups of 40-49 years,50-59 years,60-69 years and>70 years.There was no significant change trend in the expression of HCit in skin around SK skin lesions at different ages,and the difference was not statistically significant(x2=0.14,P>0.05).3)The immunohistochemical scores of HCit were 1(1,5),3.5(5,6),and 9(6,9)in the normal skin tissues in the three age groups of 20-39 years,40-59 years and>60 years.The expression of HCit in normal skin epidermis of various ages showed a gradual increase trend,the difference was statistically significant(?2=9.123,P<0.05).4)In 20 SK samples,the immunohistochemical scores of HCit in the skin lesions and surrounding skin tissues were 9(6,9)and 9(9,9),respectively.There was no significant difference in the expression of HCit between the two groups(Z=-1.892,P>0.05).5)The expression of HCit in SK lesions was higher than that in normal skin tissues in 40-59 years old,and the difference was statistically significant(Z=-2.326,P<0.05).In?60 years old,there was no significant difference in expression of HCit in SK skin lesions and normal skin tissues,with no statistical significance(Z=-0.747,P>0.05).6)In 40-59 years old,the expression of HCit in skin around SK lesions was higher than that in normal skin tissues,and the difference was statistically significant(Z=-3.374,P<0.001).In?60 years of age,there was no significant difference between the expression of HCit in the skin around SK lesions and the normal skin tissues,with no statistical significance(Z=-2.049,P>0.05).2.Sandwich ELISA results:1)The peripheral blood concentration of HCit was 1.44±0.07 and 1.78±0.41 in healthy subjects aged 30-49 years and?60 years,respectively.The difference between the two groups was statistically significant(t=-2.351,P<0.05).2)The concentration of HCit in the peripheral blood of healthy subjects and SK patients aged 30-49 years was 1.44±0.07 and 1.55±0.11,respectively.The difference between the two groups was statistically significant(t=-2.539,P<0.05).3)The concentration of HCit in peripheral blood of healthy subjects and newly diagnosed T2DM patients aged 30-49 years was 1.44±0.07 and 2.16±0.37,respectively,with statistically significant differences between the two groups(t=-6.952,P<0.05).3.RT-PCR results:1)The relative expression levels of CircPVT1 mRNA were 2.02±0.89,2.93±1.28,and 2.06±0.85 at ages 20-39,40-59,and>60,respectively.The difference between the two groups aged 20-39 years and 40-59 years was statistically significant(P<0.05),the difference between the two groups aged 40-59 years and>60 years was statistically significant(P<0.05),and the difference between the two groups aged 20-39 years and>60 years was not statistically significant(P>0.05).2)The relative expression levels of TGF-?1 mRNA at ages 20-39,40-59 and>60 were 1.97±0.54,2.29±0.58 and 2.8±1.4,respectively.The difference between the groups aged 20-39 years and>60 years was statistically significant(P<0.05),but the difference between the two groups and the group aged 40-59 years was not statistically significant(P>0.05).3)The relative expression levels of P53 mRNA at ages 20-39,40-59 and>60 were 1.4±0.49,1.2±0.34 and 1.7±0.64,respectively.The difference between the groups aged 40-59 years and>60 years was statistically significant(P<0.05),but the difference between the two groups and the group aged 20-39 years was not statistically significant(P>0.05).4)The relative expression levels of P21 mRNA at ages 20-39,40-59 and>60 were 2.51 ±0.53,3.16±0.47 and 3.18± 1.04,respectively.Pairwise comparison between the three groups showed no statistically significant difference(P>0.05).5)The relative expression levels of IL-6 mRNA at ages 20-39,40-59 and>60 were 1.55±0.34,1.55±0.42 and 2.45±1.23,respectively.Pairwise comparison between the three groups showed no statistically significant difference(P>0.05).6)The relative expression levels of Hepcidin mRNA at ages 20-39,40-59 and60 were 1.81±0.5,3.46± 1.43 and 2.94± 1.8,,respectively.The differences between the two groups aged 20-39 years and 40-59 years were statistically significant(P<0.05),but the difference between the two groups and the group aged?60 years was not statistically significant(P>0.05).Conclusion:The occurrence of SK may be related to the accumulation of HCit.Higher blood sugar levels may promote carbamylation,which in turn promotes aging.The accumulation of HCit is not only related to aging,but may also reflect the increased metabolic stress in the body.The aging related CircPVT1,TGF-TGF 1,P53,P21,i1-6 and Hepcidin may be related to the regulation of HCit expression level in the human body,and the specific relevant signaling pathways still need to be further studied.In summary,HCit may be a biomarker for aging. |
PDF Full Text Request