| Objective:Methamphetamine(METH,surpassed the popularity of traditional drug heroin,has become the most widely used illicit drug in China.METH damages almost every organ and system throughout the body.Most of the METH-induced disruption research is mainly in the nervous system,whereas researchs on digestive system injury are few at present.Many clinical cases have reported that METH induce disruption to the digestive system,such as digestive inflammation,ulcers,perforation,bleeding,obstruction,or enterogenic infection.Our previous htoxylin eosin staining study on intestinal tissue of METH-dependent rhesus monkey showed intestinal inflammatory cell infiltration,mucosa erosion with ulceration,diffuse villous degeneration and necrosis of intestinal mucosa,unclear intestinal structure,and thinning of intestinal wall.Molecular experiments also found that METH induced toxic effects on the blood-brain barrier by producing oxidative stress products such as TNF-? and reducing expression of vascular endothelial tight junction protein.But the underling mechanism of intestinal mucosal damage caused by METH has not been explored.In this study,the expression of peripheral blood intestinal injury markers and pro-inflammatory factor TNF-?,by detecting the clinical characteristics of METH-dependent patients,and combining with the molecular biological experiments of TNF-? in intestinal epithelial cells,the damage of METH on the intestinal tract and the damage mechanism were identified.To study the role of the pro-inflammatory factor TNF-? in the intestinal mucosal disruption caused by METH,and further study the regulation mechanism of the upstream,so as to provide a new idea for the study of the intestinal mucosal barrier injury caused by METH.Methods:1.In the clinical study,207 METH-dependents in the 5th compulsory drug treatment center were screened,including 193 males and 14 females.The general information and whole blood of the patients were collected.The blood samples were used for blood cell analysis,biochemical and intestinal mucosal damage detection,and detect the expression of pro-inflammatory factor TNF-?.The nutritional,inflammatory and immune status of METH-dependents subjects was analyzed,and the relationship between intestinal mucosal barrier injury,METH-dependents history and TNF-?protein was statistically analyzed.2.In vitro experiments2.1 Cell model construction:normal rat intestinal epithelial cells IEC-6 were treated with METH,the effects of METH with different concentrations and different times on the intestinal epithelial barrier were detected by a transmembrane resistance?TEER?meter.Besides,the effects of METH at different concentrations and diferent on cell proliferation activity were detected by cell viability assay.We obtained the working concentration and drug treatment time of this experiment.2.2 To explore the mechanism of intestinal barrier damage caused by METH:apoptosis rate was detected by flow cytometry.The expression of mRNA and protein level of TNF-?,MLCK,ZO-1 was detected by reverse transcription quantitative real-time PCR?RT-qPCR?,ELISA and western blotting.2.3 To verify the role of TNF-? in the METH-induced intestinal barrier damage:vectors for Tnf overexpression or interruption were designed using the sequence from GenBank?accession no.NM012675?.The experiment were divided into six groups as normal group,METH group,TNF-?-group,METH+TNF-?-group,shRNA group and METH+shRNA group.The mRNAand protein expression of TNF-? in the IEC-6 cells which were treated with METH were verified.TEER assay detected the changes of epithelial barrier permeability,and RT-qPCR,ELISA,Western blotting detected mRNA and protein expressions of epithelial tight junction relative molecules TNF-?,MLCK and ZO-1.2.4 Upstream regulatory mechanism of TNF-?:we screened the TargetScan database?www.targetscan.org?,miRDB?www.mirdb.org?,and microRNA website?www.microrna.org?for TNF-? targeting miRNAs.Real-time PCR was used to detect the expression of mircoRNA in METH-treated IEC-6 cells.Duel luciferase reporter gene assay,RT-qPCR and WESTERN BLOTTING were used to verify the targeting relationship between the TNF-? and miRNA.2.5 Elucidate the role of miR-181c-5p in METH-induced intestinal epithelial barrier disruption:phenotype of intestinal barrier injury was detected in METH-treated cell models?TEER,CCK8?.The influence of miR-181c-5p on intestinal mucosal barrier were also determined by flow cytometry,real-time PCR,ELISA and western blotting.At last,we test the expression of the influence of mir-181c on inflammatory factors IFN-?,IL-1 and IL-8.Results:1.Clinical study results:METH dependents showed significantly more digestive symptoms of anorexia,abdominal pain,abdominal distension,constipation and abnormal bowel sounds,compared with normal healthy people.The contents of peripheral blood red blood cell count,hemoglobin and albumin were decreased.In the inflammatory index,C-reactive protein,white blood cell and platelet count,and the percentage of neutrophils%increased.The percentage of lymphocytes decreased,the percentage of basophil increased,and the percentage of NK cells decreased by flow detection.The body mass index?BMI?of the METH group was lower than that of the normal control group.Serum markers of intestinal mucosal barrier injury,such as diamine oxidase?DAO?,d-lactic acid?DLC?and endotoxin?BT?,were significantly higher than those in the healthy control group?P<0.0001?,showing significant statistical differences.The expression level of TNF-? in the serum of methamphetamine dependent patients was significantly higher than that of the normal control group.Increased TNF-? level was positively correlated with DLA level?P=0.001?.2.Cell experiment results2.1 With the increasing of METH concentration and the extension of time,the transmembrane resistance and cell proliferation activity of intestinal epithelial barrier gradually decreased.When cells treated with METH for 0.25mm/L,48 h,TEER was significantly decreased.Cell proliferation activity showed the same dramatically decreased trend at the same concentration and time point.Besides,cell viability of this point is ensured for following experiments.2.2 METH can promote the apoptosis of intestinal epithelial cells,increase the expression of pro-inflammatory factor TNF-? and actin kinase myosin light chain kinase?MLCK?,and reduce the expression of tight junction protein ZO-1,thus leading to the damage of intestinal epithelial mucosal barrier.2.3 Overexpression and interference of TNF-? significantly affected the level of intestinal epithelial barrier transmembrane resistance and expression of tight junction raletive proteins MLCK and ZO-1,indicating that TNF-? was involved in the process of intestinal epithelial mucosal barrier disruption caused by METH.2.4 We screened three microRNA databases for TNF-? targeting miRNAs,and found that rat TNF-? 3'-UTRs have conserved sites for rno-miR-211-5p,rno-miR-204-5p,rno-miR-181 a-5p,rno-miR-18 1 b-5p,rno-miR-18 1 c-5p,rno-miR-181 d-5p,rno-miR-27a-3p,and rno-miR-27b-3p.Among them,miR-181c-5p expression was decreased in the METH-treated cell model.Duel-luciferase reporter assay and overexpression of miR-181c-5p reduced the mRNA and protein expression of TNF-?,confirming the targeting relationship between miR-181c-5p and TNF-?.2.5 MiR-181c-5p overexpression keep intestinal barrier transmembrane resistance and cell proliferation activity and reverse the decrease of TEER and proliferation caused by METH.Overexpression of miR-181c-5p can decrease the apoptosis of epithelial cells,reduce the expression of TNF-? and MLCK,and increase ZO-1 expression.It also reduced inflammatory cytokines release,as IFN-?,IL-1 and IL-8.In conclusion,miR-181c-5p is involved in regulating damage of the intestinal epithelial mucosal barrier caused by TNF-? mediated METH.Conclusion?s?:1.The digestive injury symptoms and signs of METH-dependents are increased,and the indicators of intestinal injury significantly increased also.2.METH increased the release of the pro-inflammatory cytokine TNF-? cytokines and was positively correlated with DLA level.3.METH increased the expression of TNF-? and damaged the intestinal mucosal barrier through the TNF-?/MLCK/ZO-1 axis in vitro.4.METH could inhibit miR-181c-5p expression,which can directly target and regulate TNF-?,and in turn induced cell apoptosis,damaged tight junction,eventually led to intestinal barrier lesion formation in IEC-6 cells. |
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