Role Of RAS Disequilibrium In Hyporeactivity And Injure Of Aortic Vascular After Tourniquet Shock

Objectives The internal homeostasis of renin angiotensin system(RAS) plays an important role in a variety of diseases and injuries. This work aims to reveal the pathogenesis of vascular hyporeactivity and to provide new ideas of clinical therapeutic strategies by studying the role of RAS homeostasis in vascular hyporeactivity and injury following tourniquet shock(TS) in mice.Methods 1 Preparation of TS model. 8-month-old male C57B6 mice were used in this study, which hind legs were ligated for 2h by using tourniquets for blocking their blood flow to induce ischemia. Cutting the tourniquets initiated reperfusion. Mice in the first part of the experiment were divided into 7 groups including control group and 6 model groups.Mice in the model groups were respectively condemned to death at reperfusion of 10 min,1h, 2h, 4h, 6h and 12 h. Mice in the control group were not subjected to tourniquet ligation.In the second part of the experiment, animals including wild-type(WT) and angiotensin converting enzyme 2(ACE2) knockout(KO) mice were divided into the following 5groups: the WT control group, WT-TS group, WT-TS-Diminazene Diaceturate(DIZE)group, KO control group and KO-TS group. Mice in the WT-TS-DIZE group were given10mg/kg/d DIZE for 28 days by subcutaneous injection before experiment. Mice in the WT-TS, WT-TS-DIZE and KO-TS groups were killed after 2h ischemia and 4h reperfusion, while mice in the WT and KO groups was subjected to the same treatment as that in the model groups except for ligation and reperfusion. 2 Determination of damage index. The Doppler flowmetry was used to determine the limbs’ blood flow. The carotid artery catheter method was applied to detecting the blood pressure. The isolated vascular tension tester was available to measure the reactivity of aorta. HE staining combined with transmission electron microscope was used to evaluate the aorta’s injury of morphology. 3Determination of RAS components. Western blotting(WB) was used for measuring the expression of AT1, Mas, ACE and ACE2 proteins. At last, serum Ang II /Ang(1-7)content were determined by enzyme-linked immunosorbent assay(ELISA) method.Results In the first part of the experiment, results showed that, compared with that in the control group of mice, blood flow in the model groups of mice decreased gradually with the prolongation of reperfusion time. The blood pressure increased at first 10 min after reperfusion, and then decreased gradually. Accordingly, vascular reaction to norepinephrine(NE) rised at 10 min time point and then descended. The vascular reactivity reached the lowest level at 4h reperfusion time point. Morphological injury score gainedgradually. Vascular AT1 receptor and ACE2 protein expressions reduced, Mas receptor and ACE protein expressions added. Content of Ang II in serum gained, content of Ang(1-7) subtracted. In the second part of the experiment, results showed that reactivity of aorta to NE in WT-TS-DIZE group mice elevated significantly compared with that in the WTTS group. The degree of vascular injury alleviated obviously. AT1 receptor, Mas receptor and ACE2 expressions reduced, ACE expression was not found to change. Serum Ang II decreased, and Ang(1-7) increased. Compared with that in WT mice, vascular reactivity in KO group mice added, the vascular lesion was not obvious. Aortic expressions of AT1,MAS, ACE protein were elevated, and ACE2 expression was not found. Serum Ang II gained and serum Ang(1-7) did not change. Compared with that in WT-TS mice, aortic reactivity in KO-TS mice subtracted apparently. The morphological damage added slightly(P>0.05). Aortic AT1 and ACE expressions rised slightly(P>0.05), and Mas expression promoted distinctly. There was no expression of ACE2 in vascular tissue. Serum Ang II and Ang(1-7) did not change clearly.Conclusions 1 RAS system was activated after tourniquet shock in mice. With the prolongation of reperfusion time, vascular hyporeactivity and damage degree promoted,accompanied by different expressions of ACE/Ang II/AT1 and ACE2/Ang(1-7) /Mas in aortic tissue. The results suggest that internal homeostasis of RAS may be related to vascular hyporeactivity and injury. 2 Targeting the ACE2, improving the imbalance of RAS may reduce hyporeactivity and injury of aorta, which is expected to become a new way in the clinical therapy of vascular hyporeactivity in shock.