Study On The Role And Mechanism Of Gut Flora Dysbiosis In Patients With Preeclampsia

Background:Preeclampsia is a serious complication of pregnancy and one of the leading causes of maternal and fetal deaths worldwide.Hypertension,proteinuria,and systemic inflammation are the most important features of preeclampsia.However,the pathogenesis of preeclampsia is extremely sophisticated and has not yet been fully elucidated until now.Intestinal flora has been reported to be closely related to hypertension and inflammation.Therefore,it is reasonable to speculate that intestinal flora is involved in the pathogenesis of preeclampsia.However,the experimental evidence for this speculation is inadequate.Seveal researchs have reported the changed composition of intestinal flora in preeclampsia patients,such as the increased abundances of pathogenic bacteria(Bilophila,Fusobacterium)and decreased abundances of beneficial bacteria(Faecalibacterium and Akkermansia).Very recently,two studies showed that gut dysbiosis and SCFA produced by intestinal flora contributed to the development of preeclampsia.However,the animal model used in the study published in gut was not preeclampsia according to the NICE guidelines.The animal experiments demonstrated the influence of intestinal flora on the blood pressure of pregnant mice with hypertension,but not on the development/condition of preeclampsia.Aims:In order to reveal the composition and symbiotic relationship of intestinal bacteria in preeclampsia patients,the role of SCFA in the pathogenesis of preeclampsia and the causal relationship between intestinal bacteria and preeclampsia,we performed studies both in clinical and animal levels.Methods:We totally recruited 54 preeclampsia patients and 62 normal late pregnant women as subjects.All patients with preeclampsia were diagnosed according to the diagnostic criteria.Pregnant women suffering from diabetes,anemia,autoimmune diseases,malignant tumors and gastrointestinal diseases and those who have used antibiotics,probiotics,prebiotics and hormones within 3 months were excluded.The two groups of subjects were matched by age,body mass index(BMI),gestational week,gravidity,parity,number of fetuses carried,diet,and blood lipid levels,etc.The changes in composition and short-chain fatty acid(SCFA)metabolism of intestinal flora in preeclampsia patients were analyzed using 16S rRNA gene sequencing and gas chromatography with mass spectrometric(GC-MS).USEARCH software was used to process sequencing data and then generate amplicon sequence variants(ASVs).Furthermore,the RDP database was used to annotate the intestinal bacteria intestinal bacteria species information.The causal relationship between the intestinal flora and preeclampsia was clarified using fecal microbiota transplantation(FMT)in L-NAME-induced preeclampsia rat models.Serum levels of pro-inflammatory cytokines(IL-1?,IL17)and anti-inflammatory cytokines IL10 and intestinal 2-AG levels were measured by ELISA,and serum LPS levels were determined by LAL method.The intestinal levels of the key propionate-roducing enzymes MmdA and PduP and the key butyrate-roducing enzyme ButA were determined by PCR.The thickness of the intestinal mucus layer and the number of goblet cells were measured using AB-PAS staining.Finally,a random forest model was used to predict intestinal biomarkers for preeclampsia.Results:The composition and symbiotic relationship of intestinal bacteria in preeclampsia patients were obviously reshaped.Especially,the abundances of SCFA-producing bacteria decreased significantly,such as Mitsuokella,Clostridium leptum,A.muciniphila,and Oscillibacter,accompanied by a significant reduction in key enzymes of SCFA production.Symbiotic network analysis found the correlations between beneficial bacteria and harmful bacteria were primarily negative and these negative correlations were markedly increased in the intestine of preeclampsia patients,accompanied with an increase of the positive correlations among harmful bacteria.Correlation analysis between the abundance of bacteria genera and key clinical indicators of preeclampsia(SBP,DBP and 24h-UP)showed that the abundances of Lysinibacillus,Moheibacter and Flavobacterium significantly positively correlated with the levels of blood pressure and/or urinary protein and were markedly increased in preeclampsia patients,while the abundances of Oscillibacter,Akkermansia,and Mitsuokella significantly negatively correlated with the two clinical indicators and were obviously reduced in preeclampsia patients.Among them,the abundance of Akkermansia decreased most significantly,and its abundance was significantly negatively correlated with the above key clinical indicators of preeclampsia.The levels of intestinal SCFA,2-AG,and serum IL-10 were considerably decreased,while the serum levels of LPS and pro-inflammatory cytokines were remarkably increased.FMT results showed that although transplanting the intestinal flora of patients with preeclampsia significantly increased the systolic blood pressure of pregnant rats,this elevation did not meet the criteria for hypertension in pregnant animals.In the rat model of preeclampsia treated with L-NAME,the transplantation of intestinal flora from preeclampsia patients markedly decreased the numbers of goblet cells per crypt and mucus layer thickness of colon of pregnant rats,thus significantly impaired the intestinal mucosal barrier,also it increased inflammatory responses and elevated blood pressure.Additionally,the abundances of intestinal Akkermansia and Oscillibacter differentiated healthy pregnant women and preeclampsia patients at an accuracy of 89.7%,which have potential as inexpensive and noninvasive markers for auxiliary diagnosis of preeclampsiaConclusions:Compared with normal late pregnant women,the intestinal flora of preeclampsia patients is considerably remodeled,and the abnormal intestinal flora significantly correlated with the clinical features such as blood pressure and inflammation of preeclampsia patients.Gut dysbiosis contributed to the enhanced inflammation and blood pressure and impaired gut barrier,thereby aggravated the condition of preeclampsia.Although abnormal intestinal flora alone may not be sufficient to cause the development of preeclampsia,it can significantly aggravate the condition of preeclampsia,indicating that it is still one of the important pathogenic factors of preeclampsia rather than only its consequence and accompanying phenomena.Moreover,intestinal Akkermansia and Oscillibacter could accurately distinguish preeclampsia patients from normal pregnant women and have potential as markers for non-invasive screening of preeclampsia.In general,our findings highlight the significance of intestinal flora in the pathogenesis,diagnosis and treatment of preeclampsia.