Mechanism Of Methamphetamine Altering The Metabolism Of Linoleic Acid By Intestinal Flora Leading To Anxiety During Acute Withdrawal

Objective:Methamphetamine(METH)has become the drug with the most serious abusers in China,seriously endangering social stability and public health.It is extremely difficult to quit METH after addiction,so it is important to study how to improve the withdrawal success rate of METH treatment.Anxiety is the most common symptom during withdrawal of METH,which is not only extremely harmful to the physical and mental health of abstinence patients,but also closely related to METH relapse.Therefore,it is very important to study the causes of anxiety after acute METH withdrawal and to find effective control means.Many studies have shown that the gut flora,through the microbiome-gut-brain axis,affects all events from brain development to neurological disease progression,and one important mechanism is the regulation of the gut flora of many important metabolites in the body.According to the previous research results,drugs will significantly change the composition of gut flora and metabolic spectrum,anxiety symptoms and gut flora changes and metabolism in the body,so METH has broad prospects in the study of the mechanism of regulating body metabolism by changing the composition of intestinal microbes and leading to anxiety symptoms,which can not only open up a new research path for the mechanism of anxiety after acute withdrawal from METH,but also provide new intervention ideas for the acute withdrawal and treatment of METH.Methods:(1)Population study:The Hamilton anxiety scale(HAMA)survey was conducted on patients with acute withdrawal from METH(for 14 days),to assess whether they have obvious anxiety symptoms.Their blood samples and feces were collected,to conduct targeted metabolomics detection of serum fatty acids and 16S rDNA sequencing of fecal microorganisms,respectively,to look for fatty acids and floras related to anxiety symptoms,so as to explore the possible mechanism of METH influencing anxiety symptoms after acute withdrawal by regulating intestinal microecology and metabolism;(2)Animal model study:The anxiety model of mice with acute withdrawal from METH was established,and the anxiety symptoms of mice with acute withdrawal from METH were verified by open field and cross elevated maze experiments.Important signs of anxiety were observed by immunofluorescence:Something wrong happened to the dentate gyrus in hippocampus,and fatty acids were detected in peripheral blood and central hippocampal region of mice,and 16S rDNA of fecal microorganism was sequenced;Endocannabinoid 2-arachidonoylglycerol(2-AG)and Arachidonylethanolamide(AEA)in the hippocampus of mice were detected by ELISA.Finally,the expression of mammalian target of rapamycin(mTOR)signaling pathway related proteins was detected by WB,to understand the effects of metabolic changes in endocannabinoids on the mTOR signaling pathway;(3)Study on intestinal-brain axis mechanism:The gut flora of mice was removed by mixed antibiotics,to establish a mouse model of intestinal microbiota depletion,followed by the fecal microbiota transplantation(FMT)experiment.The flora from the stool of METH abusers and healthy controls and the stool of METH abusers and control mice were transplanted into the recipient mice,for flora determination.Population(patients with acute withdrawal from METH and healthy controls)and mice(mice with acute withdrawal from METH and control mice)were transplanted into recipient mice for colony values.The behavior of recipient mice was,then,examined by open field and cross-elevated maze experiments.Fecal microbial composition of recipient mice was determined by 16S rDNA sequence,and fatty acids were detected by metabonomics in serum and hippocampus of recipient mice.After determining arachidonic acid(AA)as the key fatty acid,we designed supplementary experiments of AA and intervention experiments of Rimonabant(Endocannabinoid CB1 receptor inhibitor).Subsequently,the behavior,endocannabinoid content in hippocampus,hippocampal neurogenesis and activation protein expression of mTOR signaling pathway in each group of mice were determined,to verify the microbiome-gut-brain axis mechanism of anxiety after acute abstinence from METH;Results:(1)Results of population study:① METH addicts had obvious anxiety symptoms during the acute withdrawal period;② Compared to a healthy control population,METH addicts had abnormally altered serum fatty acid metabolism during the acute withdrawal period,especially polyunsaturated fatty acids(PUFAs)was evident.Among them,α-Linolenic Acid(ALA)in essential fatty acids had no significant change,while Linoleic Acid(LA)in essential fatty acids and the serum concentration of its subsequent derived fatty acids significantly decreased;③ The degree of anxiety in METH addicts during the acute withdrawal period showed a clear negative correlation with n-6 PUFAs,particularly AA and LA;④ Compared to the healthy population,METH causes a marked disturbance of the gut flora in the population with acute withdrawal from METH.In particular,the ratio of Firmicutes/Bacteroides increased significantly;(2)Results of Animal model study:① METH can successfully induce addiction in mice,and addicted mice showed significant anxiety-like behavior after getting acute withdrawal from METH;②Compared with healthy control mice,mice with acute withdrawal from METH showed significantly abnormal in serum fatty acid metabolism,and significant disorder of PUFAs.Among them,LA,y-Linolenic Acid(GLA),Dihomo-y-Linolenic Acid(DGLA)and AA significantly decreased.Besides,affected by the METH,the metabolism of hippocampus was also disturbed,and the decrease of AA in peripheral blood resulted in the decrease of AA in hippocampus;③ The levels of endocannabinoid 2-AG in the hippocampus of mice with acute withdrawal from METH decreased with its sole precursor substance,however,the other endocannabinoid AEA was not significantly different;④ METH significantly reduced the expression of activated proteins(p-m TOR,p-p70S6k and p-rpS6)in m-TOR signaling pathway in hippocampus of mice;⑤Compared to the control mice,there was a significant attenuation in dentate gyrus neurogenesis in the hippocampus in mice with acute withdrawal from METH;⑥METH causes a disturbance of the gut flora in mice,of which the most obvious are the increased Firmicutes/Bacteroides ratio;(3)Results of microbiome-gut-brain axis mechanism study:① Recipient mice received the transplantation of bacteria in the stool from the population with acute withdrawal from METH and model mice have anxiety-like behavior,and compared with the control mice,the Firmicutes/Bacterioides ratio of recipient mice in the two groups not only significantly increased,but also had a significant lack of LA and AA in serum and hippocampus;② The concentration of endocannabinoid 2-AG and the protein expressions of p-MTOR,P-P70S6K and PRPS6 in hippocampus significantly increased by administering AA to mice with acute withdrawal from METH,which promoted hippocampal neurogenesis and alleviated the anxiety-like behavior after acute withdrawal from METH.However,the concentration of endocannabinoid 2-AG in hippocampus was not affected by the injection of Rimonabant(RIM)at the same time as the supplementation of AA by gavage,but the protein expressions of P-MTOR,P-P70S6K and P-RPS6 decreased again,and hippocampal neurogenesis was attenuated again.In addition,mice still showed significant anxiety-like behavior after acute withdrawal from METH.Conclusions:(1)Acute withdrawal from METH can lead to disordered composition of gut flora,significantly increased the Firmicutes/Bacteroides ratio,and disordered gut flora can then affect the absorption of essential fatty acids,down-regulating the metabolism of LA and AA in serum and hippocampus;(2)Decreased AA level in hippocampus can lead to a deficiency of endocannabinoid 2-AG,resulting in insufficient activation of mTOR/P70S6K/rpS6 signaling pathway,and reduced neurogenesis in the dentate gyrus of hippocampus,ultimately leading to anxiety symptoms.However,the supplementation of AA can activate CB1 receptor by increasing the content of endocannabinoid 2-AG in hippocampus,then activate the mTOR signaling pathway to promote hippocampal neurogenesis,and ultimately relieve the anxiety symptoms during the acute withdrawal period from METH.