Regulation Of CYP450 And Drug Transporter On Gut Microbiota Under High-Altitude Hypoxia

Objective: We analyzed the changes in gut microbiota and the characteristic intestinal microorganisms under high-altitude hypoxic environment.A preliminary investigation of the potential mechanism of CYP450 and drug transporter regulation by gut microbiota under high-altitude hypoxia was conducted.Based on the analysis,the key role of gut microbiota in drug metabolism affected by the high-altitude hypoxia was delineated.Methods: In this study,we elucidated the effects of high-altitude hypoxia on gut microbiota by comprehensively analyzing changes in the structure and diversity of gut microbiota under high-altitude hypoxic conditions.Male Sprague Dawley rats were randomly divided into plain control group,moderatealtitude hypoxic group,and high-altitude hypoxic group,where rats in the hypoxic group were exposed to hypoxia for 3,7,15 and 30 days,respectively.Fecal samples were collected from each group of rats and 16 S r RNA gene sequencing was used to detect changes in the gut microbiota,and the effect of hypoxia on the gut microbial of rats was further verified by combining the hypoxic fecal transplantation experiment and hypoxic pseudo sterility experiment.To investigate the changes of CYP450 and drug transporter expression under high-altitude hypoxic conditions,and to systematically elucidate the correlation between the changes in the gut microbiota and expression of CYP450/drug transporter under high-altitude hypoxia using fecal transplantation and pseudo sterile treatment.Sprague Dawley rats were randomly divided into plain control group,moderate-altitude hypoxic group,and highaltitude hypoxic group.Western blotting and RT-q PCR were used to detect changes in the expression of CYP450 and drug transporters in the rat liver under high-altitude hypoxia.The correlation between changes in protein and m RNA expression of CYP450 and drug transporter and gut microbiota under highaltitude hypoxia was analyzed in combination with hypoxic fecal transplantation experiments and hypoxic pseudo-sterile experiments.A preliminary investigation of the mechanism of CYP3A4(homologous rat CYP3A1)and MDR1 regulation by gut microbiota was formulated based on the characteristics of high-altitude hypoxic bacteria and their extracellular vesicles under similar conditions.Screening for sensitive microorganisms under high-altitude hypoxia was performed through correlation analysis of the relative abundance of intestinal bacteria with the environmental factors related to high-altitude hypoxia.Ultracentrifugation was used to isolate and extract the extracellular vesicles which from hypoxic intestinal bacteria and sensitive microorganisms.Electron microscopy(scanning and transmission)and nanoparticle tracking analysis were used to identify the enriched extracellular vesicles and characterize their number and integrity under high-altitude hypoxic conditions.The lipophilic fluorescent dye PKH67 was used to label the extracellular vesicles,and their fusion with Caco-2 cells was observed under a confocal microscope.Western blot and RT-q PCR were used to detect the effects of extracellular vesicles from hypoxic intestinal bacteria,sensitive microorganisms,and its extracellular vesicles on the expression of CYP3A4 and MDR1,as well as to elucidate the regulatory effects of these vesicles on drug metabolism.To preliminarily analyze the mechanism related to drug metabolism mediated by gut microbiota under high-altitude hypoxic conditions.Results: The diversity and structure of the rat gut microbial community dynamically changed under high-altitude hypoxic conditions.The significant decrease in the relative abundance of intestinal symbiotic bacteria such as Firmicutes,Verrucomicrobia,Akkermansia,and Lactobacillus,and the significant enrichment of conditionally pathogenic bacteria Clostridium,Enterococcus and Alistipes,suggested that the changes in the intestinal environment of rats in the high-altitude hypoxic environment may be closely related to the changes in the relative abundance of intestinal symbiotic bacteria and conditionally pathogenic bacteria.In addition,the proportions of aerobic,parthenogenic and anaerobic bacteria showed different trends with changing altitude and hypoxic treatment time,and we hypothesized that a correlation possibly exists between a high-altitude hypoxic environment and a decrease and increase in the proportion of aerobic and anaerobic bacteria,respectively.Notably,the most significant effects on rat gut microbial diversity were observed on the 7th day of both moderate-and high-altitude hypoxic treatments,indicating that the influence of hypoxia treatment time on microbiota is rapid,persistent,and directional.Under high-altitude hypoxic conditions,the protein expression of OCT1 was significantly increased(P < 0.05),whereas the CYP3A1 and MDR1 protein expression were significantly decreased(P < 0.05).Meanwhile,the m RNA expression of Cyp1a2 and Cyp2e1 were significantly increased(P < 0.05),while the m RNA expression of Cyp2b1,Cyp3a1,Abcb1,and Slco2b1 were significantly decreased(P < 0.05).With the transplantation of fecal bacteria from rats under high-altitude hypoxia to plain rats,the changes in the protein and m RNA expression of CYP3A1 and MDR1 in the rat liver were consistent with the hypoxia treatment,suggested that the effect of hypoxia on CYP3A1 and MDR1 protein,and m RNA expression may be transferred through the gut microbiota.In addition,the effect of gut microbiota on CYP450 and drug transporter expression under high-altitude hypoxia was further confirmed with plateau pseudo sterile assays,and gut microbiota was found to be involved in regulating the protein and m RNA expression of CYP3A1 and MDR1.Hypoxic intestinal bacteria from high-altitude hypoxic environment can secrete extracellular vesicles with a spherical bilayer membrane structure and particle size in accordance with this range,and Caco-2 cells can effectively take up these vesicles.The protein and m RNA expression of CYP3A4 and MDR1 in Caco-2 cells were significantly increased by the extracellular vesicles of intestinal bacteria from high-altitude hypoxia.Further analysis showed that the abundance of Akkermansia muciniphila and Lactobacillus reuteri differed significantly,suggested that Akkermansia muciniphila and Lactobacillus reuteri are characteristic intestinal microorganisms in hypoxic environment.Moreover,Akkermansia muciniphila and Lactobacillus reuteri can secrete extracellular vesicles with a spherical bilayer membrane structure and particle size in accordance with this range,and Caco-2 cells can effectively take up these vesicles.Extracellular vesicles secreted by Akkermansia muciniphila significantly increased the protein and m RNA expression of CYP3A4 and MDR1 in Caco-2 cells.Conclusion: Therefore,high-altitude hypoxia is an important environmental factor that regulates the structure of the gut microbiota by altering the diversity of intestinal microorganisms and affecting the balance of the host intestinal microecology.The gut microbiota plays a key role in the regulation of drug metabolism under high-altitude hypoxic conditions,where reduced protein and m RNA expression of CYP3A1 and MDR1 are significantly associated with changes in the structural composition and diversity of the gut microbiota.The gut microbiota mediates the regulation of CYP3A4 and MDR1 expression under high-altitude hypoxia,which in turn affects the metabolic processes of drugs in vivo.The gut microbiota may mediate the up-regulation of CYP3A4 and MDR1 protein and m RNA expression through the release of extracellular vesicles under high-altitude hypoxia.Thus,regulation of CYP3A4 and MDR1 expression by Akkermansia muciniphila derived extracellular vesicles-based gut microbiota is key to the effects associated with high-altitude hypoxia on drug metabolism.