| BACKGROUND:Methamphetamine(Meth),one of the main components of "Ice",is an extensively used and often abused drug.Meth is well-known as a potent central nervous system(CNS)stimulant.Meth has central and peripheral toxic effects,increasing the risk of neuropsychiatric disorders in addicts,such as anxiety,depression,as well as cognitive impairment.Due to the unclear pathological mechanism of Meth,there is no effective treatment in clinic.In recent years,clinical and animal experiments have found that intestinal microorganisms were very important in the physiological functions such as emotion control and cognition,as well as the pathological development of depression and schizophrenia.Drug abuse such as Meth,cocaine and morphine caused abnormal intestinal microbial composition and changes in intestinal microenvironment,suggesting that intestinal flora may be the key toxic target of emotional abnormalities caused by drugs.Prefrontal cortex(PFC)is a high-level executive brain area that regulates emotion and cognition behaviors,also known as the target brain area of Meth toxicity.Gegen Qinlian Decoction(GQD),derived from Shang Han Lun,is a classic prescription composed of Gegen,Huangqin,Huanglian and Gancao.GQD has the effect of clearing away heat and annoyance,which is opposite to the hyperactivity of fire and poison,mental disturbance,delirium and excitement caused by Meth addiction.At the same time,modern medicine has proved that berberine,baicalin and other active components in GQD can maintain intestinal homeostasis;Puerarin can enter the brain and has brain protective function.The regulation of peripheral gut microbiota has strong operability and small side effects.Therefore,taking gut microbiota as the starting point,targeting "bacteria","intestine" and "brain",and looking for multi-target drugs to prevent and treat abnormal behavior during Meth withdrawal may be one of the effective strategies for the treatment of Meth addiction.In conclusion,GQD may become a multi-target effective compound traditional Chinese medicine for the treatment of abnormal behavior during Meth withdrawal.AIMS:(1)To clarify the intervention effect of GQD on anxiety-like behavior and cognitive ability of Meth withdrawal mice;(2)To clarify the intervention effect of GQD on the colonic microbiota of Meth withdrawal mice and bacterial target screening;(3)To clarify Intervention effect of GQD on colonic microenvironment and screening of metabolic targets in Meth withdrawal mice;(4)Screening of brain regions and PFC transcriptome targets for GQD to improve anxiety like behavior in meth withdrawal mice.METHODS:(1)The Meth withdrawal model was constructed in mice,and the elevated plus maze(EPM)and open field test(OFT)were used to detect the anxiety like behavior;(2)Morris water maze(MWM)and Y maze was preformed to examine the cognitive ability;(3)16s absolute quantitative sequencing was used to analyze the intestinal microbial structure,abundance and metabolome;(4)The growth of Akkermansia was observed by anaerobic culture in vitro;(5)FITC dextran,immunofluorescence,western blot(WB)and non-targeted metabonomics were used to detect the intestinal permeability,inflammatory factors and metabolic level;(6)The transcriptional activity of PFC was observed by PCR and sequencing.RESULTS:(1)GQD effectively reduced the anxiety like behavior and improve the impairment of spatial learning and memory in Meth withdrawal mice;whereas nether the germ free mice nor the antibiotic mice showed anxiety-like behavior during Meth withdrawal.(2)Meth withdrawal mice showed increased abundance of intestinal flora and the changed structure of microbial diversity,especially the decreased abundance of Akkermansia.(3)GQD improved the abundance and structure of intestinal flora in Meth withdrawal mice,and increased the abundance of Akkermansia strains;(4)Meth treatment decreased the growth level of Akkermansia in vitro,while GQD effectively increased the growth of Akkermansia treated with Meth;(5)Meth withdrawal mice showed increased intestinal permeability,decreased colonic tight junction protein,increased colonic proinflammatory factor,decreased colonic anti-inflammatory factor and,as well as changed colonic metabolome.GQD can effectively improve the intestinal injury in Meth withdrawal mice and effectively reverse the expression levels of cortisol,kynurenine,L-threonine and lysophosphatidylserine;(6)Meth withdrawal enhanced the activities of the OFC,NAC,Hb and Amy neurons,whereas the activitys of PFC neurons decreased as a whole,as measured by the inhibited pyramidal neurons(PN)but excited GABAergic interneurons(GABA IN).GQD effectively reversed these changes of neuronal activities in Meth withdrawal mice;(7)Meth withdrawal mice showed changes in the expression profile of PFC transcriptome,which was realized as up regulation of 189 genes and down regulation of 110 genes.GQD effectively reversed 55 up regulation genes and 17 down regulation genes,involving heat shock protein family related genes,immediate related genes,and GABA related genes.CONCLUSION:(1)GQD effectively improved the anxiety like behavior and spatial learning and memory impairment in Meth withdrawal mice;(2)GQD can improve the abnormal structure and abundance of intestinal flora in Meth withdrawal mice,and Akkermansia is its key intervention target bacterium;(3)GQD can improve the abnormalities of colonic microenvironment and metabolic level in Meth withdrawal mice.Cortisol,kynurenine,L-threonine and lysophosphatidylserine may be its main metabolic targets;(4)PFC brain region is the key brain region for GQD to improve the anxiety like behavior of Meth withdrawal mice.Heat shock protein family related genes,immediate early related genes and GABA related genes may be the key genes for GQD’s intervention. |
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