| Background and Object:Morphine is one of the most abused drugs in the world.Previous studies on morphine addiction have focused on the central nervous system.The results show that there is two-way regulation between brain and intestinal microbiota,suggesting that gut microbiota dysbiosis plays an important role in several central nervous system diseases.However,whether there is an association between gut microbiota and morphine dependence remains unclear.Sinomenium acutum and Uncaria rhynchophylline are commonly used medicines for detoxification of traditional Chinese medicine.In combination with a large number of previous clinical and basic studies of our research team,the important alkaloid components of Sinomenium acutum and Uncaria rhynchophylline are selected:sinomenine and isorhynchophylline for anti-morphine dependence mechanism.Based on the intestinal microbiota-gut-brain axis(MGBA),this project intended to analyze the characteristics of intestinal flora of morphine-dependent zebrafish under normal and disordered intestinal flora,study the interaction between flora and morphine dependence and the intervention effects of two alkaloids.What is more,the mechanism of anti-morphine dependence of two alkaloids was further studied,by in vitro morphine-activated BV2 microglia model.Methods:1.Conditional position preference(CPP)method was used to eatablished morphine-dependent zebrafish CPP model.16S rRNA sequencing was used to observe the effects of morphine dependence on the characteristic,composition and function of zebrafish intestinal mirobiota and the effect of sinomenine and isorhynchophylline on morphine dependence.The effects of morphine on the expression of MGBA-related genes in zebrafish and the intervention effects of sinomenine and isorhynchophylline were measured by qPCR.2.Antibiotics were used to reduce the intestinal flora of zebrafish,and then established the morphine-dependent zebrafish CPP model.The experimental method and observation index are the same as the method 1 above.3.BV2 cells were treated with morphine to establish the activation model.Immunofluorescence and western blotting were used to observe the effects of sinomenine and isorhynchophylline on morphine-induced activation;qPCR was used to observe the effect of morphine on the expression of MGBA-related genes in BV2 cells and the intervention effects of sinomenine and isorhynchophylline.Results:1.After morphine treatment,the residence time and road map of zebrafish in drugpair box in morphine group increased significantly,which indicated that the CPP model of zebrafish was successfully established.At this time,the diversity of gut microbiota of zebrafish decreased significantly,the relative abundance of Firmicutes decreased significantly and the B/F value increased significantly,and the gene metabolism of intestinal flora changed significantly.After morphine addiction,the expression of opioid receptor genes and tight junction protein genes in brain and intestine of zebrafish were significantly decreased,the expression of neurologic factor genes and inflammatory factor genes were significantly increased.However,the administration of sinomenine and isorhynchophylline can reverse the above changes caused by morphine.2.After antibiotic treatment,zebrafish intestinal flora decreased significantly.The residence time and road map of zebrafish in drug-pair box in antibiotic+morphine group increased significantly.However,the diversity,composition and metabolism functions of zebrafish gut microbiota did not change significantly;the expression of opioid receptor genes and tight junction protein genes in zebrafish were decreased significantly,and the expression of neurologic factor genes and inflammatory factor genes were significantly increased.Interestingly,the effects of sinomenine and isorhynchophylline on morphine dependence of zebrafish were weakened.3.It was found that BV2 cells were activated after chronic morphine treatment which was manifested by the increased expression of IBA-1.In the process of activation,the expression of opioid receptor genes,neurologic factor genes and inflammatory factor genes in BV2 cells were changed significantly.After treatment with sinomenine and isorhynchophylline,the activation and the changes in the expression of these genes in B V2 cells caused by morphine could be inhibited.Conclusion:Morphine dependence caused changes in zebrafish gut microbiota.Antibioticdriven changes in the zebrafish gut microbiota could also affect the formation and treatment of morphine dependence.Gut microbiota and morphine dependence may be bidirectionally regulated through MGBA.Sinomenine and isorhynchophylline may exert anti-morphine dependence through MGBA. |
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