Research On The Role Of Intestinal Microorganism In The Course Of High Altitude Polycythemia

Objective: High altitude polycythemia(HAPC),as a chronic disease of high altitude,has long been affecting the health of people in high altitude areas.There have been a lot of researches on it,but there is still no clear discussion to elucidate its pathogenesis.With the deepening of research on intestinal microflora in recent years,researchers have found that intestinal microflora have close relationship with human health and diseases.This study was to explore the role of intestinal microorganisms in the course of high altitude polycythemia and to provide a theoretical basis for elucidate the pathogenesis of the disease.Methods:1.Establishment of HAPC rat model:Female SD rats aged 7 weeks were randomly divided into normoxic group and hypoxic group.Hypoxic group rats were placed in a low-pressure oxygen chamber to simulate low-pressure hypoxic exposure at an altitude of 6000 meters for 8 hours a day.The rats were observed for 28 days,and then sacrificed.Blood,tissue and organ samples were collected for hematological and pathological analysis.The levels of hypoxia-inducing factor-1(HIF-1α and HIF-1β),vascular endothelial growth factor(VEGF),erythropoietin(EPO),interleukin-6(IL-6)and other cytokines in serum were detected by ELISA.The structures,functions and metabolic levels of intestinal flora were analyzed by 16 S r DNA ampland sequencing,metagenomic sequencing and non-targeted metabolomics.2.Experiment of fecal microbiota transplantation:Female SD rats aged 7 weeks were randomly divided into normoxia group,hypoxia group,antibiotic induced flora disorder group(hereafter referred to as antibiotic normoxia group),antibiotic induced flora disorder with hypoxia exposure group(hereafter referred to as antibiotic hypoxia group)and faecal bacteria transplantation group(hereafter referred to as transplantation group).Normoxic group was fed normally in plain normoxic environment.Hypoxic group was exposed to hypobaric hypoxia daily.Rats in antibiotic normoxia group,antibiotic hypoxia group and transplantation group were first added with compound antibiotics in their drinking water to induce the disturbance of intestinal flora.The antibiotic hypoxic group was then subjected to hypobaric hypoxia exposure.The transplantation group returned to normal drinking water and underwent HAPC intestinal flora colonization for 4 weeks,while the antibiotic normoxic group kept feeding.Samples of blood,tissues and organs,and feces were collected for hematology,pathology,cytology,and 16 S r DNA ampland sequencing.The rat model of iron deficiency anemia was established by feeding 4-week-old female SD rats with low iron diet and deionized water.Then the changes of hematopoietic function of rats were observed by colonization of HAPC intestinal flora.Results:1.Establishment of HAPC rat model:Compared with the normoxic group,the hypoxic exposed rats showed clinical manifestations of HAPC,the hemoglobin content greater than 190g/L meets the diagnostic criteria of HAPC;Intestinal tissue structures were damaged;The levels of HIF-1α,HIF-1β,VEGF,EPO and other factors related to the pathogenesis of HAPC in serum were significantly increased(p < 0.05);The inflammatory factor interleukin-6was significantly elevated;The α diversity of intestinal flora decreased,while the βdiversity began to differ between the two groups from the beginning of hypoxia exposure.The intestinal microflora’s structures and functions of HAPC rats were further analyzed by metagenomics.We found 31,328 and 1142 species that differ between groups at phylum,genus and species levels.It was found that HAPC rats showed high expression of genes related to metabolism,genetic information processing,cellular process and organic system.The results of non-targeted metabonomics analysis showed that the metabolic pathways of intestinal flora in HAPC rats,including glycolysis and glycogenesis,fructose and mannose metabolism,tyrosine metabolism and thyroid hormone synthesis,had significant changes compared with those in the normoxia group.2.Experiment of fecal microbiota transplantation:The intestinal flora of HAPC rats was normally colonized in the intestinal tract of transplantation group rats through faecal bacteria transplantation experiment.After colonization of HAPC intestinal flora,the levels of HIF-1α,HIF-1β,VEGF,EPO and other factors related to HAPC in serum were significantly increased.The inflammatory factors interleukin-1β,2,6 and 8 were also significantly increased.Serum ferritin(SF),transferrin(TRF),total iron binding force(TIBC)were increased,and ferritin(HEPC)was decreased.Differentiation of lymphocytes in spleen and mesenteric lymph nodes in rats was observed after antibiotic induction of intestinal flora disturbance or colonization of HAPC intestinal flora(p<0.05).The anemia and iron deficiency were improved in iron deficiency anemia rats after HAPC intestinal flora colonized.Conclusion:1.During the course of HAPC,the structures of intestinal microflora in rats will change significantly,and the gene functions of the microflora is highly expressed in metabolism,genetic information processing,cellular process and organic system.Metabolomics analysis indicated that the metabolic pathways of glucose,tyrosine and thyroid hormone were changed.2.After the colonization of HAPC rat intestinal flora,the levels of HAPC pathogenesis-related factors and some interleukin in serum of rats were increased in normal plain environment.Lymphocyte differentiation in mesenteric lymph nodes and spleen were affected.These results indicate that the disorder of intestinal flora plays an important role in the pathogenesis of HAPC in hypobaric hypoxia environment,but its specific mechanism still needs to be further studied.3.After the colonization of HAPC rat intestinal flora,anemia and iron deficiency were improved,suggesting that HAPC intestinal flora contains species that can improve the body’s hematopoietic function and iron metabolism,and subsequent studies should focus on the mining of specific species.