| Objective(s):Drug abuse and dependence are still a global public health problem,and the occurrence and development of related comorbidities are important factors that lead to the further deterioration of social problems and the inability of abusers to completely quit.Methamphetamine(METH),commonly known as methamphetamine,is currently the most widely used new synthetic drug.Its abuse and dependence can seriously damage the peripheral immune system of the body and cause intestinal barrier damage.This study intends to detect the intestinal barrier damage,immune cells and cytokines and other indicators of peripheral venous blood in different withdrawal periods of METH and healthy controls,to find immune cells and cytokines that may be related to intestinal barrier damage,and to further pass animal experimental models To verify the relationship between peripheral immune disorders and intestinal barrier damage,explore the possible peripheral immune markers for predicting intestinal barrier damage,and provide a certain theoretical basis for finding new targets for immunotherapy and providing precise immune intervention in the later stage.Methods:First,recruit people with different withdrawal periods in METH:acute withdrawal(withdrawal 0-15 days,Acute abstinence syndrome,ABS,n=22)and late withdrawal(withdrawal 12 to 18 months,Post abstinence syndrome),PABS,n=29),and recruit healthy controls(Healthy Controls,HCs,n=38)matching gender,age,and body mass index(BMI).Peripheral venous blood serum samples of the above three groups of people were tested for related indicators of intestinal barrier damage(Diamine Oxidase(DAO),D-Lactic Acid(D-LC),bacterial endotoxin(Lipopolysaccharide,LPS))And cytokines(TNF-α,IL-10,IL-4,IL-5,IL-12,IL-6,IL-17A,IFN-y,etc.)and immune cells in peripheral venous blood(CD3+T,CD3+CD4+T,CD3+CD8+T,CD3+CD4+CD8+T,CD16+CD56+NK,CD19+B cells,etc.),and statistically analyze the differences in the detection indicators of different groups of people And the correlation between the above test indicators for all populations.Secondly,conduct animal modeling experiments to verify that the experimental animals that have specific preferences for the environment are excluded from the baseline,and then C57BL/6J mice are randomly divided into four groups:control group(Control,n=8)and experimental group.The experimental group includes METH addiction group(Addiction,n=8),METH acute withdrawal group(ABS,n=8)and METH late withdrawal group(PABS,n=8),the experimental group of mice in this study used four cycles Alternate intraperitoneal injection(Intraperitoneal,ip)5.0mg/Kg METH or equivalent normal saline for training every other day.After training,the conditioned place preference(CPP)experiment is used to determine animal addiction,in order to better simulate Based on the characteristics of the mode of drug use in the real society,the experimental group subsequently adopted the method of gradually increasing doses to make animal models.During the experiment,the weight of the mice was regularly monitored and weighed.After the model was completed,the mouse serum samples and anticoagulated peripheral blood were collected by eye-catching methods for follow-up experiments(BD combined with immunofluorescence method to detect the expression level of cytokines in mouse serum and flow cytometry analysis of peripheral blood Immune cell count percentage),and then immediately killed the mouse by cervical dislocation method,carefully cut the mouse abdominal wall,cut the mouse intestinal tissue from the gastric cardia and anus,take out the intact intestine and place it naturally(without pulling)Place the mouse on a sterile towel and measure the length of the mouse’s colon.After the measurement is completed,three sections of colon tissue with a length of about 0.5 cm are collected from the site about 1.0 cm from the end of the anus to the site near the ileocecal area.For subsequent paraffin-embedded hematoxylin-eosin(Hematoxylin-Eosin,HE)staining and frozen section immunofluorescence experiments.Results:1.Compared with the healthy control population,the METH withdrawal period had elevated DAO,D-LC and LPS related indicators of peripheral blood-gut barrier damage,and the difference was statistically significant(P<0.001);2.Compared with healthy controls,CD3+T,CD3+CD4+T,CD3+CD8+T,CD3+CD4+T/CD3+CD8+T,CD3+CD4+CD8+T in the METH acute withdrawal group The absolute counts of immune cells such as CD16+CD56+NK,CD19+B decreased.And the absolute value of CD3+T and CD3+CD4+T counts in the METH acute withdrawal period were significantly lower than the healthy control population,the difference was statistically significant(P<0.001);3.Compared with the healthy control population,the expression levels of TNF-α,IL-6,IL-12,IL-7 and IL-10 in the people with different withdrawal periods of METH all have a rising trend.In addition,the expression levels of TNF-α,IL-6,IL-7 and IL-10 were significantly higher than those of healthy controls in people with different withdrawal periods of METH,and the difference was statistically significant(P<0.05);4.Spearman correlation analysis between the intestinal barrier damage index and the absolute count of immune cells in all populations shows that the DAO level and the absolute count of CD3+T,CD3+CD4+T,CD19+B,CD16+CD56+NK There was a negative correlation(P<0.05).D-LC level was negatively correlated with CD3+CD4+T absolute value count(P<0.05),and D-LC level was positively correlated with CD3+CD8+T absolute value count(P<0.05).The LPS level was negatively correlated with the absolute counts of CD3+T,CD3+CD4+T(P<0.05);5.Spearman correlation analysis between the intestinal barrier damage indicators and the expression levels of peripheral cytokines in all populations shows that DAO levels are correlated with TNF-α,IL-6,IFN-γ,IL-1α,IP-10,IL-10.The expression level of IL-7 was positively correlated(P<0.05),and the level of DAO was negatively correlated with the expression levels of IL-1β and IL-9(P<0.05).The level of D-LC was positively correlated with the expression levels of TNF-α,IL-12,IL-2 and IL-5(P<0.05).LPS level and TNF-α,IL-6,IFN-y,IL-1α,IL-2,IP-10,IL-4,IL-8,IL-7,IL-12,IL-17A and IL-10 The expression level of is positively correlated(P<0.05);6.During the continuous administration of METH,the weight growth rate of the mice decreased,especially during the acute withdrawal period of METH,the weight growth rate of the mice increased gradually.Among them,the intestinal barrier damage of the mice in the METH addictive period was not obvious,and the colon tissue injury score of the mice in the acute withdrawal period of METH increased,the ratio of villus length to the depth of the lacuna decreased,the length of the colon was shortened,and the tight junction of the intestine(Tight Junction,TJ))The immunofluorescence expression of proteins ZO-1,Claudin-1 and E-cadherin decreased;7.The immunofluorescence expression of TJ proteins ZO-1,Claudin-1 and E-cadherin in the colon of mice after METH withdrawal decreased,and the percentages of CD3+T,CD3+CD4+T,CD3+CD8+T counts in peripheral blood were compared with those of the control group Significantly lower,and the difference is statistically significant(P<0.05),the expression level of serum TNF-α is higher than that of the control group;Conclusions:1.The intestinal barrier of the people in the acute withdrawal period of METH is severely damaged,the absolute count of CD3+CD4+T lymphocytes decreases and the expression level of TNF-α increases;2.The levels of DAO,D-LC and LPS in the METH withdrawal period were negatively correlated with the absolute level of CD3+CD4+T(P<0.05),and the levels of DAO,D-LC and LPS were positive with the expression level of TNF-α Correlation(P<0.05);3.Acute withdrawal of METH will result in a significant decrease in the weight of mice,an increase in the score of colon tissue damage,a shortened colon length,and damage to the structure of the intestinal tight junction barrier.The mice in the late-stage METH withdrawal group will have intestinal barrier damage and CD3+CD4+The percentage of T lymphocytes decreased and the expression level of TNF-α increased;... |
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