Angiotensin Ⅱ Participates In The Pathogenesis Of Hypertension By Regulating Thymic FoxN1

Objective: Hypertension is a multi-factor disease,which a variety of cells or cytokines are involved in the pathogenesis.With the aging of population and the change of lifestyle,the incidence of hypertension has become more and more serious.Hypertension has become an important global public health problem.All kinds of cardiovascular and cerebrovascular complications caused by hypertension are the key problems that seriously threaten the physical and mental health of the people in the world.Previous studies have shown that inflammatory responses and immune systems are involved in the process of hypertension and have been widely recognized.Angiotensin Ⅱ(AngⅡ),a potent vasoactive peptide,is a major mediator of hypertension and target organ damage.In addition,we have also made it clear through previous studies that T lymphocytes are involved in the inflammatory and immune responses,and play an important regulatory role in the pathogenesis of hypertension.Thymus,as a very important immune organ,is an important site for the maturation of T cells.Although the thymus gradually diminishes with age,it still has some physiological functions.In particular,many studies recently revealed that thymus aging can miraculously reverse the overall aging of mice by regulating related transcription factor FoxN1,which has farreaching effect and inspired the researchers unlimited reverie.Previous studies have shown that hypertensive mice may have impaired thymic function.The thymus may have a close correlation with hypertension,the pathological process of the hypertension itself will result in the thymus decline which accelerated aging,as well as their possible related mechanisms are not clear,while by reversing the thymus aging can affect the process of high blood pressure also is not clear.In this study,the changes of thymus function in hypertensive mice was observed by the model of hypertension induced by angiotensin Ⅱ.Then thymus transplantation and other techniques were used to observe the effects of the above operation on the progression of hypertension and its target organs,and through the in vitro cultivation of thymic epithelial cells(TEC)to investigate the possible mechanisms that provide new treatment for clinical treatment and therapeutic targets of hypertension.Methods: In this study mice experiment will be divided into two parts.In the first part,45 C57 BL / 6J male mice of 8 weeks old were randomly divided into normal control group(Con group,n = 15),sham operation(Sham group,n = 15)and angiotensin Ⅱ induced hypertension model group(HTN group,n = 15).Hypertension model group was implanted with angiotensin Ⅱ micro-pump.The third day after the implantation of micro-pump,nonstop mouse tail non-invasive pressure measurement began every morning 8: 00-9: 00.Blood pressure data was obtained for 8 weeks,mice were sacrificed,and organs and tissues such as blood,thymus,spleen,heart and kidney in mice were collected.In the second part,another 45 8-week-old C57 BL / 6J male mice were randomly divided into normal control group(Con group,n = 15)and angiotensin Ⅱ induced hypertension model group(n = 30).After 4 consecutive weeks of non-invasive pressure measurement,the hypertensive model group was randomly divided into hypertensive group(HTN group,n = 15)and transplantation group(HTN + Trans group,n = 15)after successful modeling.Thymus of C57 BL / 6J neonatal rats born at 12-24 h was transplanted into the renal capsule of the transplantation group.After 3 days of transplantation,the mouse tail noninvasive pressure measurement was continued every morning from 8: 00-9: 00 for 5 weeks.The heart function of mice was detected by echocardiography.Then mice were sacrificed and organs or tissues such as blood samples,thymus,spleen,heart and kidney were collected.The blood pressure of mice was measured by noninvasive rat tail pressure measurement.The peripheral blood and spleen T lymphocyte subsets and cTEC / mTEC ratio(cTEC / mTEC)of thymocytes were analyzed by flow cytometry.The expression of FoxN1,Aire,sjTREC and ATRAP in thymus was detected by real-time quantitative PCR.The protein levels of FoxN1,Aire and ATRAP in thymus were detected by Western Blot.HE staining was performed and the thymus FoxN1 level was detected by immunohistochemistry.In vitro experiment: Thymus of C57 BL / 6J neonatal rats born at 12-24 h was removed under aseptic conditions and cultured in thymus epithelial cells(TEC).Thymic epithelial cells were divided into blank control group(no stimulation),Ang Ⅱ group(Angiotensin Ⅱ stimulation 48h)and Co-culture group(Angiotensin Ⅱ stimulation 48 h and non-stimulated thymus epithelial cells co-culture).and then sampled to extract RNA and protein,detect FoxN1 and ATRAP mRNA expression and protein levels.Results: 1.The systolic blood pressure and diastolic blood pressure in HTN group were significantly higher than those in Con group and Sham group(p <0.05).There was no difference in blood pressure between Con group and Sham group.And the blood pressure of HTN+Trans group was lower than that in the HTN group(p<0.05),which was slightly higher than that in the Con group(p >0.05).In the first part,the thymus index of HTN mice was lower than that of Con and Sham groups(p <0.05),but there was no difference between Con and Sham mice.In the second part,the thymus index of HTN mice was significantly higher than that of Con Compared with HTN group,the thymus index of HTN + Trans group was significantly increased(p <0.01),the spleen index and kidney index were increased(p <0.05),and the left ventricular heart ratio was significantly increased(P <0.01).The spleen index and kidney index decreased(p <0.05)and the left ventricular heart ratio increased(p <0.05).The left ventricular heart ratio of HTN + Trans group was higher than that of Con group(p <0.05).2.Compared with Con group,LVEF,FS and SV of HTN group decreased(p<0.05),and ESV increased(p<0.05).LVEF,FS and SV of HTN+Trans group were higher than HTN group,ESV was lower than HTN group(p<0.05),no difference with the Con group.3.In the first part,the expression of FoxN1 and Aire in thymus of HTN mice was downregulated(P <0.05),the expression of sjTREC was decreased in HTN mice(p <0.05),and the other groups had no Significant statistical difference.In the second part,the expression of FoxN1 and Aire in thymus of HTN group was decreased(p <0.05),and the content of sjTREC in thymus of HTN group was also decreased(p <0.05),however,the content of sjTREC in spleen of HTN group was increased(p <0.05)compared with Con group and HTN + Trans.What’s more,the expressions of ATRAP and Thymosin beta4 in thymus of HTN group were also down-regulated(p <0.05).4.Flow analysis results show that TEM of peripheral blood and spleen in HTN group was obviously higher than that of Con group and Sham group(p < 0.05),but spleen naive T cells is lower(p < 0.05)in the first part.In the second part,the peripheral blood and spleen of HTN group were significantly higher than that of Con group and HTN + Trans group(p <0.05).The number of na?ve T cells in HTN group was lower than that in Con group and HTN + Trans group <0.05).The ratio of cTEC/mTEC in HTN group was higher than that in Con group and lower than that in HTN + Trans group(p <0.05).5.Compared with Con group and HTN + Trans group,FoxN1 and Aire,and ATRAP protein expression in HTN group were significantly down-regulated(p <0.01),and FoxN1 protein level in HTN + Trans group was decreased(p <0.05).6.HE staining of the thymus showed that all groups of mice thymus appeared different degree of degradation atrophy,however,it is most serious that stereotypical deterioration of the thymic epithelial compartment,reduced distinction between the cortical and medullary regions and a reduction in the number of medullary islets per thymic lobe in the HTN group.But there is no significant difference between thymus and thymus in hypertensive group,and the degeneration of thymus is less.HE staining of the kidneys showed that there was obvious demarcation between the lesion area and the normal tissue in the HTN group,tubular atrophy and interstitial fibrosis in some areas,infiltration of inflammatory cells with lymphocytes and plasma cells;Some vasopressin embolization,individual glomerular atrophy,cystic dilation,thickening of the cyst wall.Spleen HE staining showed that the spleen tissue of the normal mice mild structural abnormalities,no significant reduction in the number of splenic nodules,a few splenomegaly atrophy,decreased lymphoid number,a small part of the red pulp white pulp boundaries are unclear,but still visible splenic artery,splenic tissue showed a large number of neutrophil infiltration;The HTN group of mice spleen organization overall structural abnormalities,most of the regional structure disorder,no normal splenic nodules of structures,red pulp white pulp line is not clear,visible multicore macrophages and neutrophils increase;The overall structure of the spleen tissue in the HTN + Trans group was abnormal,but it was relatively mild compared with the HTN group,the number of splenic nodes decreased.Partial regional splenic contraction;There was a decrease in lymphatic numbers,and in some areas,there was no clear line of red medulla and no obvious inflammatory cell infiltration.Heart HE staining showed cardiac hypertrophy,left ventricular hypertrophy index,left ventricular wall thickness increased and fibrosis increased;The aortic wall of HTN group was thickened(p<0.05),(p <0.05),no significant difference between Con group and HTN + Trans group.7.The difference between thymus cortex and medulla area in immunohistochemical HTN group was decreased,the FoxN1 staining was decreased,the cumulative optical density of FoxN1 fluorescence in HTN group was significantly decreased(p <0.01),and HTN + Trans group was significantly improved.8.The results of in vitro experiments showed that,compared among blank group stimulated with Ang Ⅱ and Co-culture group,the mRNA expression of FoxN1 and ATRAP were down-regulated after stimulated with 1 μmol / L angiotensin Ⅱ(p <0.05),what’s more,the protein level of FoxN1 and ATRAP were also down-regulated.But there is no difference between the blank group and Co-culture group.Conclusion: In vitro experiments show that angiotensin Ⅱ can down-regulate the expression of ATRAP,which in turn down-regulates FoxN1 expression in thymus,leading to hypothyroidism,however co-culture could improve its effect.In vivo experiments showed that angiotensin Ⅱ-induced hypertensive mice had dysfunction of thymic,characterized by downregulation of mRNA and protein levels of FoxN1,Aire and ATRAP,as well as a decrease in recent output function,an imbalance of percentage of T cell subsets and downregulation of thymosin β4,leading to or aggravating the process of hypertension.Through thymus transplantation,the expression of FoxN1 of the thymus was improved,and the heart function of mice was significantly improved,and the increase of blood pressure was restored and suppressed.The potential mechanisms underlying this may be that thymus transplants can upregulate the expression of transcription factor FoxN1,which is essential for thymus development,in turn leads to the upregulation of ATRAP,thus affecting the effects of angiotensin Ⅱ on thymus function,and cause the increase of thymosin β4 and sjTREC,as well as restoration of normal percentages of T-cell subsets,thereby improving and inhibiting the pathogenesis of hypertension.