Role And Mechanism Of Resveratrol On The Reactivity In Mice Following LPS Challenge

Endotoxin shock, sepsis, septic shock, which are caused by a variety of serious infections factors, are common clinical pathological processes; A large number of studies have shown that the hypovascular reactivity of endotoxin shock at late stage is an important factor causing the microcirculation disturbance and tissue hypoperfusion. Resveratrol(Res) has a variety of biological activity. In recent years, many studys also found that resveratrol has intervention effect on endotoxic shock, reduceing the structural damage and dysfunction of vital organs of the lung, kidney and liver, reducing oxidative stress and inflammation, improving microcirculation perfusion status. However, whether the Res is involved in the process of vascular hyporeactivity induced by endotoxin shock has not been reported. Therefore, our study established a mice model of LPS challenge and observed the role and mechanism by which Res on vascular reactivity using microvascular tension measurement technique.Firstly, we observed the effect of resveratrol on mice by intraperitoneal injection of LPS. Totally 24 BALB/c mice of SPF level were randomly divided into: sham operation group(Sham), Sham+Res group, LPS group, LPS+Res group. LPS was injected(35 mg/kg, 5 mg/ml) intraperitoneally primarily and Res(30 mg/kg, 300 mg/ml) was injected intramuscularly at 1 h after LPS injection in the LPS+Res group. Res were replaced with an equal volume of solvent- Dimethyl sulfoxide(DMSO) at the same time in the LPS group. In the sham group, the mice received an intraperitoneal injection of equal volume of saline and an intramuscularly injection of DMSO, respectively. In the sham+Res group, the mice received an intraperitoneal injection of equal volume of saline and an intramuscularly injection of Res, respectively. The survival time of each mouse was record. Then, 54 BALB/c male mice were randomly divided into nine groups: normal control group(Control), Sham 6 h, Sham+Res 6 h, LPS 6 h, LPS+Res 6 h, Sham 12 h, Sham+Res 12 h, LPS 12 h, LPS+Res 12 h group, Control group was not given any treatment, other groups dealing with as above description. At 1 h before the corresponding point time, the mouse received general anesthesia by intraperitoneal injection with pentobarbital(70 mg/kg, 1%), under the operating microscope, the mice received a femoral operation for the anticoagulation with heparin sodium(1%, 1 ml/kg) injection via the right femoral artery, the animals’ mean artery pressure(MAP) continuously monitoring using the biological signal collecting and processing systemvia the right femoral artery. Then, the mice received a neck surgery and the intravenous injection of noradrenaline(NE, 4.2 μg/kg) was performed using a syringe via through the right jugular vein. The MAP before and after NE injection were recorded, and MAP in difference(△MAP) between before and after injection of NE injection was used for the evaluation of the pressor response in mice. The other 24 mice were randomly divided into: Sham, Sham+Res, LPS, LPS+Res group, which were treated as above, at 6 h after injection of LPS, clipping the secondary mesenteric arterioles blood vessels of mouse under deeply anesthetic conditions preparing vitro microvascular ring, applying in vitro vascular tone system to detect the change of vascular reactivity with gradient NE contractile.The present results showed that, the survival time of mice in the sham and sham+Res were more than 24 h, meaning long-term survival; the survival time of mice in the LPS+Res group(17.59±2.24 h) were significantly longer than that of the LPS group(14.37±1.96 h, P<0.05). The levels of MAP and △ MAP in the sham 6 h, sham+Res 6 h, sham 12 h, sham+Res 12 h had no significantly difference with the control group(P>0.05); MAP of LPS 6 h and LPS+Res 6 h group were significantly lower than Control, Sham 6 h, Sham+Res 6 h group(P<0.05), △ MAP of LPS 6 h and LPS+Res 6 h group were significantly higher than Control, Sham 6 h, Sham+Res 6 h group(P<0.05); MAP of LPS 12 h and LPS+Res 12 h group were significantly lower than Control, Sham 12 h, Sham+Res 12 h group(P<0.05), △ MAP of LPS 12 h and LPS+Res 12 h group were significantly higher than Control, Sham 12 h, Sham+Res 12 h group(P<0.05); MAP and △ MAP of LPS 6 h group had no significantly difference with LPS+Res 6 h(P>0.05), MAP and △ MAP of LPS 12 h group had no significantly difference with LPS+Res 12 h(P>0.05). The micro-vascular reactivity to the gradient of NE at 1×10-7, 3×10-7 mol/L in the sham+Res group was significant increased than that in the sham group(P>0.05); the microvascular reactivity to the gradient of NE in the LPS group was significantly lower than the Sham group(1×10-6, 3×10-6, 1×10-5, 3×10-5 mol/L, P<0.05) and Sham+Res group(1×10-7, 3×10-7, 1×10-6, 3×10-6, 1×10-5, 3×10-5 mol/L, P<0.05); the curve the microvascular reactivity on the gradient of NE in the LPS+Res group shifted to the left relative to the LPS group, and significantly higher than LPS group at 3×10-6, 1×10-5, 3×10-5 mol/L(P<0.05).Then, we observed the role of resveratrol on mice by intravenous injection of LPS-challenge. First, 24 male BALB/c mouse were randomly divided into Sham, Sham+Res, LPS, LPS+Res group, where: LPS and LPS+Res group underwent neck surgery, then injected LPS(5 mg/kg, 5 mg/ml) in jugular vein, sutured wounds, 1 h later, mouse of LPS+Res group injected Res(30 mg/kg, 300 mg/ml) intramuscularly, mouse of LPS group only injected with an equal volume of DMSO; Sham and Sham+Res group injected an equal volume of saline in jugular vein, 1 h later, mouse of Sham+Res group injected Res(30 mg/kg, 300 mg/ml) intramuscularly, mouse of Sham group only injected with an equal volume of DMSO, recording survival time. Second, another 24 male BALB/c mouse were randomly divided into Sham, Sham+Res, LPS, LPS+Res group, undergoing thigh surgery, undergoing the right femoral artery, connecting biological signal acquisition system to record the MAP; then treated in the same manner as described above. 6 h after injection of LPS, via the jugular vein injected NE(4.2 μg/kg), according to the aforementioned method to evaluate the overall pressor response in mouse. Thirdly, the 24 mice were divided into Sham, Sham+Res, LPS, LPS+Res group, which were treated in the same method as described above, at the appropriate point time, clipping the secondary mesenteric arterioles blood vessels of mouse under anesthesia, preparing vitro microvascular ring, applying in vitro vascular tone system to detect the change of microvascular reactivity with gradient NE contractile, applying tool medicine of Rho A、ROCK and MLCP to observe the microvascular tension of Sham, Sham+Res, LPS and LPS+Res group mouse, and discuss the of Rho A in Res improve the vascular reactivity of LPS-challenged mice.The results show that, the survival time of Sham and Sham+Res mouse were more than 24 h, meaning long-term survival; the survival time of LPS+Res group mouse(20.45±2.81 h) were significantly longer than LPS group(13.89±2.37 h, P<0.05). MAP and △ MAP of Sham 6 h had no significantly difference with Sham+Res 6 h(P>0.05). MAP of LPS 6 h and LPS+Res 6 h group were significantly lower than Sham 6 h, Sham+Res 6 h group(P<0.05), △ MAP of LPS 6 h and LPS+Res 6 h group were significantly higher than Sham 6 h, Sham+Res 6 h group(P<0.05); MAP and △ MAP of LPS 6 h group had not significantly different with LPS+Res 6 h(P>0.05). Sham group had no significant difference in microvascular on the gradient of NE reactivity with Sham+Res group excepted for 10-6 mol/L(P>0.05); the microvascular reactivity on the gradient of NE of LPS group was significantly lower than the Sham group(3×10-5, 10-5, 3×10-6, 10-6, 3×10-7, 10-7mol/L, P<0.05), Sham+Res group(3×10-5, 10-5, 3×10-6, 10-6 mol/L, P<0.05); the curve the microvascular reactivity on the gradient of NE of LPS+Res group shifted to the left relative to the LPS group, and significantly higher than LPS group at 3×10-5, 10-5, 3×10-6 mol/L(P<0.05).At the same time, we found that U-46619, agonist of Rho A, could improve the microvascular reactivity in the Sham and Sham+Res groups, OA, inhibitor of MLCP, could improve the microvascular reactivity of the Sham group, Y-27632, inhibitor of Rho A, inhibitors of Rho A, could reduce the microvascular reactivity of the Sham and Sham+Res groups at multiple concentration points of NE(P<0.05). U-46619 improved the microvascular reactivity of LPS group at multiple concentration points of NE, but this effect was inhibited by Y-27632(P<0.05). OA had no significant effect on the microvascular reactivity of LPS group, but united Y-27632 could reduce the microvascular reactivity of LPS group(P<0.05). Y-27632 reduced the microvascular reactivity in the LPS+Res group at multiple concentration points of NE, which was abolished by the U-46619 treatment(P<0.05).These results showed that resveratrol could inhibit the vascular hyporeactivity in mice following LPS-challenge with intraperitoneal injection and intravenous injection, along with prolonging the survival time. However, it had no significant improvement on the level of arterial blood pressure and the overall reaction in mice. After LPS attack, the reactivity of pressor response on mice and the microvascular in vitro appeared inconsistency, it may related to the role of LPS playing on sympathetic responses, whose mechanisms need to be further studied. The effect of Res which could improve the vascular reactivity of LPS-challenged mice by intravenous injection was related to Rho A-ROCK-MLCP signaling pathway.