| Objective:Studies have shown that the telomere length of gastric mucosa in patients with chronic atrophic gastritis(CAG)is shorter than that in normal subjects,and CAG may be related to the aging of gastric mucosa,but the molecular mechanism has not yet been clarified.According to the theory of "stomach Tiannian",the mentor has achieved good clinical effect in the treatment of CAG with Guben Tongluo decoction,but the mechanism of Guben Tongluo decoction is not clear.In this study,the telomere length,TRF1,TRF2,POT1 mRNA and protein expression levels of gastric mucosa and the expression of p53mRNA,the key factor of cell senescence pathway in patients with CAG were studied.The pathogenesis of CAG and the mechanism of GubenTongluo decoction in the treatment of CAG were discussed from the point of view of telomere and aging.Methods:(1)A case-control study on telomere length,TRF1,TRF2,POT1mRNA,protein expression and p53mRNA expression in patients with CAG.Using the experimental design of case-control study,30 patients with CAG and 30 patients with CNAG were collected as control group.All patients were diagnosed by gastroscopy and pathological biopsies in Guang’anmen Hospital of Chinese Academy of traditional Chinese Medicine.The risk factors of disease in the two groups were determined by filling out the questionnaire.The telomere length,TRF1,TRF2,POT 1 mRNA and protein expression of gastric antral mucosa and the expression of p53mRNA,the key factor of cell aging pathway,were detected to explore the relationship between telomere regulation network and aging and CAG in human gastric mucosa,so as to further clarify the molecular mechanism of CAG.(2)study on the mechanism of Guben Tongluo decoction in the treatment of CAG.30 patients with at least one grade of gastric antral mucosa pathology were treated with Guben Tongluo decoction for 3 months.The gastric antral pathological specimens were resliced before and after treatment,and the telomere length,TRF1,TRF2,POT1mRNA and protein expression,as well as the expression of p53mRNA,the key factors of cell aging pathway,were detected by measuring the telomere length,TRF2,POT1mRNA and protein expression of gastric antral mucosa before and after treatment.To explore the effect of Guben Tongluo decoction on telomere regulatory network and p53,the key gene of cell senescence pathway,so as to clarify the mechanism of Guben Tongluo decoction.Results:(1)A case-control study on telomere length,TRF1,TRF2,POT1mRNA,protein expression and p53mRNA expression in patients with CAG.There was no significant difference in gender and age composition between the two groups,and the baseline was consistent and comparable.Comparison of risk factors between the two groups:compared with CNAG group,CAG patients had more history of Hp infection and longer course of disease,the difference was statistically significant(P<0.05);BMI,smoking history,drinking history,staying up late,overeating,spicy stimulation,cold and high salt diet,family history of gastric cancer and Hp infection.There were 22 cases(73.3%)with atrophy,29 cases(96.7%)with intestinal metathesis and 1 case(3.3%)with dysplasia in CAG group.There was significant difference in pathological score of gastric mucosa between the two groups(t=9.682,P<0.001).Comparison of telomere length between the two groups:the relative telomere length of gastric mucosa in the two groups did not conform to the normal distribution,the median relative telomere length in CAG group was 0.67,the median relative telomere length in CNAG group was 1.06,and the relative telomere length in CNAG group was significantly shorter than that in CAG group(U=297.000,P=0.024)Comparison of TRF1,TRF2,POT1mRNA and protein expression in gastric mucosa between the two groups:the expression of TRF1mRNA in gastric mucosa was high in CNAG group,but there was no significant difference between the two groups(t=0.788,P<0.05).The expression of TRF2mRNA in CAG group was high,but the difference was not statistically significant(t=1.969,P=0.054);POT1mRNA was highly expressed in CNAG group,but the difference was not statistically significant(t’=-1.225,P=0.226).TRF1,TRF2 and POT1 proteins were highly expressed in CAG group,which were significantly different from those in CNAG group(P<0.01).Comparison of p53mRNA expression in gastric mucosa between the two groups:the expression of p53mRNA was high in CAG group,but there was no significant difference between the two groups(t=0.260,P=0.796).Analysis of the correlation between telomere length and age in gastric mucosa:there was no correlation between telomere length and age in CAG group and CNAG group(CAG group:r=-0.303,P=0.131,CNAG group:r=-0.195,P=0.302)The correlation analysis between telomere length and TRF1,TRF2,POT1,p53mRNA,TRF1,TRF2,POT1 protein expression in CAG group:there was a slight negative correlation between p53mRNA expression level and gastric mucous telomere length in CAG group(r=-0.396,P=0.031),regression equation Y=-0.321X+1.099(F=3.438,P=0.074),suggesting that the regression equation was not statistically significant,so the expression level of p53mRNA could not be used to predict the telomere length of CAG gastric mucosa.There was no correlation between the telomere length of gastric mucosa and the levels of TRF1,TRF2,POT1 and p53mRNA in CNAG group,and the expression of TRF2 and POT1 protein was negatively correlated with the telomere length of gastric mucosa in CAG group,and the linear regression equation between TRF2 protein expression and telomere length was Y=-0.041X+1.249(F=1.741,P=0.198),suggesting that the regression equation was not statistically significant,and the expression of TRF2 protein could not be used to predict the telomere length of CAG gastric mucosa.The linear regression equation between the expression of POT1 protein and telomere length was Y=-0.103X+1.272(R2=0.206,F=7.249,P=0.012).There was no correlation between telomere length and TRF1,TRF2 and POT1 protein expression in CNAG group.The correlation between the pathological score of gastric mucosa and the relative telomere length of gastric mucosa,the expression of TRF1,TRF2,POT1,p53mRNA and the expression of TRF1,TRF2 and POT1 protein:there was a negative correlation between the pathological score of gastric mucosa and the relative telomere length of gastric mucosa(r=-0.410,P=0.001),the linear regression equation is Y=-5.018X+11.54(R2=0.181,F=12.81,P<0.001).There was no correlation between the pathological score of gastric mucosa and the expression of TRF1mRNA(r=-0.054,P=0.682),which was positively correlated with the expression level of TRF2mRNA(r=0.339,P=0.008),there was no correlation with the expression level of POT1mRNA(r=0.052,P=0.695).There was no correlation between p53mRNA expression level and p53mRNA expression level(r=0.037,P=0.778).The pathological score of gastric mucosa was positively correlated with the expression of TRF1,TRF2 and POT 1,and the regression equation with the expression of TRF1 protein was Y=1.974X+0.534(R2=0.437,F=45.04,P<0.001),the regression equation with the expression of TRF2 protein was Y=0.848X-1.457(R2=0.138,F＝9.312,P=0.003),the regression equation with the expression of POT1 protein was Y=1.743X+0.814(R2＝0.294,F＝24.15,P<0.001).Multivariate Logistic regression analysis of CAG showed that the positive expression of TRF 1 and POT1 protein was an independent risk factor for CAG.The risk of CAG in patients with positive expression of TRF1 protein was 16.54 times higher than that in patients with negative CAG(95%CI:1.84-149.03).The risk of CAG in patients with positive expression of POT1 protein was 9.11 times higher than that in patients with negative POT1 protein expression(95%CI:1.64-50.54).(2)study on the mechanism of Guben Tongluo decoction in the treatment of CAG.Comparison of pathological data before and after treatment:there was significant difference in the constituent ratio of atrophy,intestinal metathesis and chronic inflammation before and after treatment(P<0.01).There were significant differences in the scores of atrophy and intestinal metaplastic before and after treatment and the total scores before and after treatment,which were statistically significant.PThere was no significant difference in the score of chronic inflammation before and after treatment(P<0.001).There was no significant difference between before and after treatment(P>0.05);The number of cases of dysplasia was only 1 case,so there was no significant difference before and after treatment.Comparison of telomere length of gastric mucosa before and after treatment:the relative telomere length of gastric mucosa before and after treatment was 0.30±0.16,0.32±0.19 after treatment,and the difference before and after treatment was-0.02±0.20.there was no significant difference(t=-0.549,P=0.587),it can not be proved that Guben Tongluo decoction has an effect on the telomere length of gastric mucosa before and after treatment.Comparison of the expression of TRF1,TRF2,POT1 mRNA and protein in gastric mucosa before and after treatment:the expression level of TRF1 before and after treatment was significantly lower than that before treatment(P<0.05).There was no significant difference in the expression of TRF2 and POT1mRNA before and after treatment,so it could not be proved that Guben Tongluo decoction had an effect on the expression of TRF2 and POT1mRNA,but there was no significant difference in the expression of TRF1,TRF2 and POT1 protein in gastric mucosa before and after treatment(P>0.05).It could not be proved that Guben Tongluo decoction had an effect on the expression of TRF1,TRF2 and POT1 protein.Comparison of p53mRNA expression level in gastric mucosa before and after treatment:the expression level of p53mRNA after treatment was lower than that before treatment,the difference was statistically significant(P<0.05).Conclusion:A case-control study on telomere length,TRF1,TRF2,POT1mRNA,protein expression and p53mRNA expression in patients with CAG showed that:(1)The telomere length of gastric mucosa in CAG patients is shorter than that in CNAG patients,and the shortening of telomere is closely related to the occurrence of CAG.(2)It has not been found that the telomere length of gastric mucosa in CNAG is shortened with the increase of age.(3)The expression of TRF1,TRF2 and POT1 protein in gastric mucosa of CAG patients was significantly higher than that of CNAG patients.(4)The expression of TRF2 and POT1 protein in gastric mucosa of patients with CAG was negatively correlated with the telomere length of gastric mucosa.It was considered that the shortening of telomere in gastric mucosa of patients with CAG was related to the overexpression of TRF2 and POT1 protein.(5)There was a slight negative correlation between the expression of p53mRNA in CAG patients and the telomere length of gastric mucosa.The shortening of telomere length was accompanied by the increase of p53mRNA expression in CAG patients.The activation of p53 pathway was considered as mediating cell aging,preventing telomere from being recognized as DNA damage,causing chromosome instability and carcinogenesis,which needs to be confirmed by further study.(6)The pathological score of gastric mucosa was negatively correlated with the relative telomere length of gastric mucosa,and positively correlated with the expression of TRF2mRNA and the expression of TRF1,TRF2 and POT1 protein,which was statistically significant.Therefore,the telomere length,TRF2mRNA expression level,TRF1,TRF2 and POT1 protein expression of gastric mucosa could predict the pathological changes of gastric mucosa.(7)The positive expression of TRF1 and POT1 protein was an independent risk factor for CAG.The risk of CAG in patients with positive expression of TRF1 protein was 16.54 times higher than that in patients with negative expression of CAG,and the risk of CAG in patients with positive expression of POT1 protein was 9.11 times higher than that in patients with negative expression of CAG.The clinical detection of TRF1 and POT 1 protein in gastric mucosa can be used as a reference index for the diagnosis and prognosis of CAG.Through the self-control study of the patients with pathological improvement of gastric mucosa treated with Guben Tongluo decoction,it was found that:(1)There was no significant difference in the relative telomere length of gastric mucosa before and after treatment with Guben Tongluo decoction,which could not indicate that Guben Tongluo decoction had an effect on the telomere length of gastric mucosa.(2)Guben Tongluo decoction could down-regulate the expression of TRF1mRNA.(3)There was no significant difference in the expression of TRF1,TRF2 and POT1 protein before and after treatment with Gubentongluo decoction,which could not indicate that Gubentongluo decoction had an effect on the expression of TRF1,TRF2 and POT 1 protein.(4)Guben Tongluo decoction can down-regulate the expression of p53mRNA.It may be through delaying the shortening of telomere,providing an opportunity for the repair mechanism of DNA damage and blocking the activation of cell senescence pathway mediated by p53,so as to delay gastric aging and reverse CAG,but its mechanism needs to be further verified by binding cell experiments in vitro and in vivo. |
