| Continuous ischemia and inappropriate resuscitation after hemorrhagic shock cause excessive inflammation, vascular hyporeactivity,vascular hyperpermeability, abnormal blood rheology, dysfunction of myocardial systolic dysfunction, and even lead to organ injury, at last cause multiple organ dysfunction syndrome and multiple organ failure.Among them, microvascular hyporeactivity to vascular active substances caused stubborn low blood pressure is the key link of refractory shock.Some research showed that estrogen could improve the tolerance of experimental animal to hemorrhagic shock, its mechanisem maybe related to improve myocardial contractility, reduce mitochondrial damage,increase ATP production, improve cell energy metabolism disorders,inhibit oxidative stress, reduce inflammation, improve immune function and eventually reduce the organ damage caused by uncontrolled hemorrhagic shock. However, whether the effect of estrogen intervention on hemorrhagic shock is associated with improvement of vascular reactivity? And the research is less. Some studies have shown that low expression of small G protein RhoA closely related to vascular hyporeactivity following hemorrhagic shock. So, whether the mechanism of estrogen improves vascular reactivity following hemorrhagic shock is related to RhoA? It needs further study.Therefore, we apply 17β- estradiol(E2) as an intervention factor, and observe the effects of E2 treatment on blood flow changes of fixed position intestinal mucosa in vivo and microvascular hyporeactivity in vitro following with hemorrhagic shock. Then, we observe the role ofsignal pathway RhoA-Rho kinase(ROCK)- MLCP on improvement of vascular reactivity in female mice following hemorrhagic shock by using some related tool agents, which will provide experimental evidence for clinical application of E2 to prevent hemorrhagic shock.36 SPF level C57BL/6 healthy female mice were randomly divided into ovariectomized group(OVX group, 24, ovarian was ligated and removal), ovary intact group(OVI group, 12, no ovarian ligation and removal, only resect small amount of fat tissue around the ovary). A week later, 12 in OVX group were given E2(every night at 8:00, administrated drug to neck back subcutaneous, 140 μg.kg-1), the other 12 minc(in OVX group) and OVI group were given the same amount of vehicle only for a week. After the administration, OVX+vehicle group, OVX+E2 group,OVI group were divided into Shock group(animals shocked for one hour,maintain the blood pressure at 40±2 mmHg and then resuscitation) and Sham group respectively. Therefore, the mice were divided to 6 groups,Sham+OVX+vehicle group, Shock+OVX+vehicle group, Sham+OVX+E2group, Shock+OVX+E2 group, Sham+OVI+vehicle group,Shock+OVI+vehicle group. After resuscitation for 4 h or at the corresponding time points, an abdominal surgery was performed for the observation of the blood flow volue in a fixed location of intestinal loop.Then, the blood sample was collected for the detection of the E2 contents,separating secondary arteriole at the fixed position in the bowel by using surgical microscope, prepare microvascular ring, then hang them into the four-chamber linear actin instrument perfusion bath tank with PSS cool liquid and fill into the mixed gas of 95% O2 and 5% CO2. When the temperature rose to 37。C, zero and normalize. Finally, observe the reactive of microvascular to NE at different concentration points, later, observe the microvascular reactions to NE after adding the tool agents of the signal pathway of RhoA-ROCK-MLCP, U-46619, OA, Y-27632, and C3 transferase.Results showed that the level of E2 in the plasma in mice decreased significantly after OVX, and elevated significantly after treatment with E2.As the results of microvascular reactivity to NE, we found that the microvascular reactivity of Sham+OVX+E2 group was higher than Sham+OVI+vehicle and Sham+OVX+vehicle groups at multiple concentration points of NE significantly. The reactivity of Shock+OVI+Vehicle, Shock+OVX+Vehicle, Shock+OVX+E2 groups were lower than the Sham+OVI+vehicle, Sham+OVX+vehicle,Sham+OVX+E2 groups at multiple concentrations of NE significantly.The reactivity of the Shock+OVI+Vehicle group was higer than the Shock+OVX+Vehicle at the 1×10-7 mol·L-1 concentration of NE, and the reactivity of Shock+OVX+E2 groups was higer than the Shock+OVI+Vehicle, Shock+OVX+Vehicle groups at multiple concentrations of NE significantly.U-46619, agonist of RhoA, increased the microvascular reactivity of Sham+OVI+Vehicle, Sham+OVX+Vehicle, Sham+OVX+E2 groups at multiple concentration points of NE significantly, OA, MLCP inhibitor,inhibited microvascular reactivity only at 3×10-5 mol·L-1 in Sham+OVI+vehicle group and Sham+OVX+vehicle group significantly;RhoA inhibitor, C3 transferase, inhibited microvascular reactivity at multiple of concentration points of NE; Y-27632, ROCK inhibitor,reduced microvascular reactivity of Sham+OVI+vehicle group and Sham+OVX+vehicle group significantly at multiple concentration points of NE.U-46619 increased microvascular reactivity to NE in the Shock+OVI+vehicle group(NE each concentration point) and Shock+OVX+vehicle(A plurality of concentration points) significantly,and this effect was abolished by Y-27632, a ROCK inhibitor, treatment.Meanwhile, OA, a MLCP inhibitor, has no effect on the microvascularreactivity in the Shock+OVI+vehicle and Shock+OVX+Vehicle groups;The combination of OA and Y-27632 reduced significantly reactivity to NE in the Shock+OVI+vehicle and Shock+OVX+vehicle groups at multiple concentration of NE. Y-27632 inhibited microvascular reactivity in the Shock+OVX+E2 group at each concentration point of NE significantly, which was abolished by the U-46619 treatment; RhoA inhibitor, C3 transferase, has no obvious effect on microvascular reactivity in the Shock+OVX+E2 group, but significantly improved microvascular reactivity at multiple concentration points of NE by joint application with U-46619 in the Shock+OVX+E2 group.Lastly, results showed that the blood flow volume of intestinal loop in the Shock+OVX+Vehicle group was remarkably decreased than that in the Sham+OVX+Vehicle group. However, E2 treatment increased the blood flow volume of intestinal loop and that in the Shock+OVX+E2group was observably higher than the Shock+OVX+Vehicle group.The results demonstrated that: microvascular reactivity reduced following hemorrhagic shock and OVX further reduced the microcirculation reactivity in hemorrhagic shock female mice, estrogen therapy significantly improved the microvascular reactivity in mice, its mechanism maybe related to RhoA-ROCK-MLCP signaling pathway. |
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