The Role And Mechanism Of Gut Microbiota In Methamphetamine Addiction

Drug addiction is a chronic brain disease that manifests itself from initially controllable behavioral motivation to later uncontrollable compulsive drug foraging.Cues between the pleasure effects produced by the addict during drug use and the surrounding environment generate associative memories that induce uncontrollable drug-seeking behaviors when prompted by relevant cues.This pathological memory of addiction may persist even after withdrawal from the drug.MA has become the most abused psychostimulant drug,and addicts often suffer irreversible brain damage,which severely affects physical and mental health,and poses a danger to families and society.There is no proven treatment for drug addiction.Recent studies have showed that addictive drugs can cause significant dysbiosis of the gut microbiota,and MA altered the diversity of the gut microbiota in mice,with an increase in Bacillus and Clostridium and a decrease in Mycobacterium.However,the effects of addictive drugs on the abundance as well as the structure and composition of the gut microbiota are complex,and there is no systematic studies on MA addiction and its intrinsic mechanisms of action from a microbiome perspective,as well as combined studies on the changes of gut microbiota in MA addicts and animal models.Based on the above research background,a two-part experiment was designed to investigate the role of gut microbiota in MA addiction and the possible mechanisms.First,the expression profile changes of gut microbiota in MA addicts and addiction model mice were analyzed,and the biomarker bacteria with characteristic changes were predicted by the analysis of MA addicts.Then,the mechanisms of gut microbiota in MA addiction were further analyzed by MA addiction model group mice,including intestinal inflammation,intestinal function,intestinal permeability,changes in neurotransmitter receptors within specific brain regions,and alterations in neuroinflammation,etc.This study provides a new perspective for understanding the mechanisms of MA addiction.Part 1:Profiling of gut microbiota profile in methamphetamine abusers and addition miceFirst,an animal model of MA addiction in C57BL/6 mice was established,and feces were collected at different time points during the modeling period and analyzed by 16S rDNA sequencing.The a diversity analyses showed that the phylogenetic richness of the gut microbiota was significantly higher,and the evenness was lower in MA-addicted mice compared with control mice.The β diversity analysis indicated that the microbiota structure was also significantly changed.The potentially pathogenic bacteria were significantly increased in MA-injected mice as predicted by Bugbase phenotype.During the modeling process,some genera were consistently elevated,such as Candida arthritis and RF39.LEfSe analysis was performed to find the characteristic bacteria with significant differences,and it was found that the characteristic bacteria that remained elevated after 7 days of MA exposure and subsequent discontinuation for 7 and 14 days were F16 Bacteroidetes(genus unknown),Adlercreutzia(red bug family)and Prevotella(Prevotella).The characteristic bacteria that decreased continuously were Bacteroides,Streptococcus,indicating that MA caused a continuous change in some of the microbiota.Second,we examined the changes in gut microbiota in MA addicts and healthy controls.The results showed that the phylogenetic richness of the gut microbiota was also significantly higher in MA addicts compared to healthy subjects,and their flora structure was also significantly altered.the relative abundance of probiotic bacteria(such as Lactobacillus spp.)decreased and the relative abundance of pathogenic bacteria(such as Megacoccus spp.)increased in MA addicts.the LEfSe analysis showed that there were significant differences in several levels of bacteria,such as the relative abundance of Verrucomicrobiaceae,Veillonellaceae,and Ruminococcaceae was increased significantly,while the relative abundance of Lachnospiraceae,some Veillonellaceae,and some Clostridiaceae,etc.showed a significant decrease in relative abundance.Based on the above analysis of the gut microbiota of mice from MA addicts and addiction model groups,we performed a joint analysis of the two across species.Through the overlap analysis,16 common differential bacteria between MA-addicted mice and MA addicts were identified at different taxonomic levels,especially at the genus level.Among them,11 species changed consistently in mice and humans,and 5 species showed opposite performance.The 11 marker bacteria that showed consistent performance,the relative abundance of 4 species increased significantly after MA ingestion,while the relative abundance of 7 species decreased significantly.In addition,we found a correlation between the common characteristic differential bacteriophage genera between the MA addiction model group of mice and MA addicts and the frequency as well as the timing of MA ingestion.In the experiment,the common differential bacteria were further tested for clinical predictive efficacy using them as biomarkers.The clinically significant finding from the analysis of four common differential bacteria,namely Ruminococcus spp.,Oscillospira spp.,Coprococcus spp.of Clostridiaceae,and Dorea spp.of Clostridiaceae,was that using a working curve of subjects with only one differential bacteria,Ruminococcus spp.AUC was 0.858 for Ruminococcus and 0.941 for Dorea in combination with four common differential organisms.Part 2:The mechanism of gut microbiota in methamphetamine-induced addictionMechanistic studies were conducted based on the analysis of the altered gut microbiota caused by MA.This part of the experiment started with both antibiotic(ABX)destruction and fecal bacteria transplantation(FMT)to observe the effects of the destruction of gut microbiota and after supplementation on addictive behavior in MA model mice.The results showed that receiving ABX treatment alone did not affect the conditioned place preference(CPP)in mice.In contrast,if MA treatment was given before ABX treatment,the formation of MA addiction memory was inhibited and CPP scores were significantly lower than those of the control group.Whereas,after supplementation of gut microbiota by FMT treatment,CPP scores increased,indicating that the formation of MA addiction memory was partially restored.This suggests that gut microbiota plays an important role in the formation of MA addiction.Then,we analyzed the inflammatory infiltration in the colon and ileum of mice in the MA model group after ABX and FMT treatments by HE staining,and observed the cellular junctions in the colon and the arrangement of the intestinal epithelium by transmission electron microscopy.The results showed that the structural damage of intestinal mucosa and inflammatory cell infiltration was reduced in the ABX-treated group compared with the model mice,and the inflammatory infiltration score of the intestine was positively correlated with the CPP score.FMT increased the inflammatory cell infiltration and disordered the arrangement of microvilli in the colonic region compared with the ABX group.Next,to further enhance the reliability of the conclusions,and intestinal permeability assay was performed,using isothiocyanate fluorescently labeled dextran(FD4)by gavage and detecting the change of fluorescein in blood samples after absorption through the intestinal epithelium,which can visually reflect the change of intestinal permeability.The changes in systemic inflammation levels after the destruction and supplementation of intestinal flora were also analyzed.The results showed that the concentration of fluorescein(FITC)in serum was higher in the MA group than in the control group.After antibiotic treatment,serum FITC concentrations decreased in the ABX group,while FMT treatment restored them to levels similar to those of mice in the normal MA model group.disruption of intestinal flora by ABX decreased serum inflammatory levels,such as IL-1β and TNF-α,in model mice,while FMT treatment increased serum inflammatory factor levels.Finally,considering that gut microbiota may affect brain function through the gut-brain axis,which in turn affects addictive behaviors caused by MA,we selected three brain regions that are closely related to MA addiction,namely,prefrontal cortex(PFC),nucleus ambiguus(NAc)and hippocampus(Hip),and examined the microglia-mediated inflammatory responses in these brain regions in MA-addicted rats after disruption of gut microbiota and supplementation,as well as the neurotransmitter 5-HT receptors 5-HT2cR and 5-HT4R.The results showed that microglia in Hip and NAc brain regions of ABX group mice were mostly resting,and the level of neuroinflammation in the brain was low.The FMT group showed the increased cytosolic volume of microglia in NAc compared with the ABX group,indicating an increase in activated microglia.ELISA showed increased IL-1β level in PFC,but the difference of TNF-α level between the two groups in Hip and PFC was not significant.The results of real-time fluorescence quantitative PCR showed that the expression levels of 5-HT4R in NAc and Hip were significantly higher in the FMT group compared with the ABX group,especially in Hip.5-HT2cR expression levels in PFC,NAc,and Hip were not significantly different from those in the ABX group.Correlation analysis showed that there was a significant correlation between 5-HT2cR expression in NAc and 5-HT4R expression levels in Hip and CPP scores.CONCLUSIONThe following conclusions were drawn from the above two parts of the experiment:(1)The composition of gut microbiota was significantly altered in both MA addicts and addiction model groups of mice.Fifty-five significantly differential genera were detected in MA addicts and healthy controls,and 35 significant differential genera were detected in addicted mice.The cross-species bioinformatics analysis screened for differential flora common to both,such as Ruminococcus,Oscillospira,Coprococcus,and Dorea,which can be used as characteristic indicators for clinical MA addiction detection.(2)Destruction of gut microbiota by ABX can inhibit MA addiction memory formation,while FMT supplementation of flora can partially restore MA addiction memory.The possible mechanisms of action of MA-related gut microbiota are the regulation of intestinal permeability,the induction of the intestinal inflammatory response,and the regulation of microglia-mediated inflammatory response in the brain through the intestine-brain axis;and the elevated expression of 5-HT4R in NAc and Hip is also associated with the regulation of addiction memory by FMT.This study provides a new way of understanding drug addiction from the perspective of gut microbiota.