The Mechanism Study Of Hypertensive End-Organ Injury Induced By High Salt Diet In ANP-Deficient Mice

Objective:Hypertension is the primary risk factor for cardiovascular disease.Sustained high blood pressure causes severe damage to organs and tissues such as the heart and kidneys.Evidences have shown that hemodynamic changes are not the only cause of hypertension and end-organ damage,which may be synergistic with intestinal flora and immune regulation.ANP gene deletion may induce high blood pressure through a sustained effect on hemodynamic changes which promotes end-organ damage development.We used ANP knockout(ANP^-/-)salt-sensitive hypertension model to simulate persistent hemodynamic abnormalities and terminal organ damage in order to investigate the role of intestinal flora and immune regulation in end-stage tissue damage of hypertension.Methods:Thirty wild-type and ANP^-/-male mice（8 weeks old）were randomly divided into three groups:（1）Con group and ANP^-/-group were feed normal diet（containing 0.5%NaCl）with distilled water for four weeks.（2）HC and HA group were given high salt diet（including 8%NaCl）with distilled water for four weeks.（3）HC+A group and HA+A group were feed high salt diet plus mixed antibiotics in drinking water（ampicillin（1g/L）,vancomycin（500mg/L）,kanamycin（1g/L）and metronidazole（1g/L））for four weeks.The arterial blood pressure was measured on day 0,day7,day 14,day 21 and day 28,respectively.Four weeks later,HE staining was used to evaluate the pathological changes in aorta,heart,kidney,liver and intestine of each group of mice.Masson staining,Alcian blue staining and Lysozyme immunofluorescence staining were performed to further observe intestinal histological changes.The number of T cell subsets in blood vessels and intestines of mice in each group were detected by flow cytometry.The expression of antimicrobial peptide and bile acids metabolism related receptors were determined by QPCR.Results:（1）Four weeks later,the blood pressure of ANP^-/-group and HC group was significantly higher than that of Con group,respectively（P<0.001）,and the wall of aorta in ANP^-/-group was thickened（P<0.01）compared to Con group.Meanwhile,compared to the ANP^-/-group,the blood pressure of HA group was significantly increased（P<0.001）and the wall of blood vessel was markedly thickened（P<0.001）,indicating that ANP deletion enhance the blood pressure of mice and induced hypertension after high-salt diet.（2）Contrary to Con group,the proportion of goblet cells in the small intestine villi of ANP^-/-group and HC group was reduced meanwhile the expression of antimicrobial peptide-related genes was also significantly decreased（P<0.01）.The ratio of goblet cells in HA group was apperantly lower than that in ANP^-/-group（P<0.01）,and the expression of antibacterial peptide-related genes also decreased（P<0.001）.The changes of intestinal goblet cells and antimicrobial peptides in ANP^-/-group,HC group and HA group suggested that the intestinal flora involved in the process.（3）There were no significant changes in blood pressure between the HC+A group and the HC group after high salt diet,but the blood pressure in the HA+A group was significantly lower than that in the HA group（P<0.001）.Moreover,antibiotics reversed the terminal organ damage in the HA group,such as the thickening of the blood vessel wall,myocardial hypertrophy and glomerular atrophy,and repaired intestinal atrophy and fibrotic lesions,indicating that the elimination of intestinal flora reverses the end-organ damage of hypertension.（4）The number of TH17 cells in the vascular and intestinal tract of the HA group was significantly higher than that of the ANP^-/-group（P<0.05）.The vascular and intestinal TH17 cells in the HA+A group marked decreases compared with the ANP^-/-group（P<0.05）,which showed that the intestinal flora may regulate the intestinal immune system.（5）Compared to ANP^-/-group,CYP7A1 expression in liver significantly enhanced in HA group（P<0.001）,while the intestinal FXR expression was no significant increased.The expression of CYP7A1 in the HA+A group significantly decreased（P<0.001）.Compared to the HA group,and the expression of FXR in the intestine significantly increased（P<0.001）,which indicated that intestinal flora may regulate bile acid synthesis and bile acid-activated receptors.Conclusion:ANP deficiency may affect intestinal immune system and bile acid metabolism by dysregulating intestinal flora,which may contribute to damages of end-organs in salt-sensitive hypertensive model.