Role of post-hemorrhagic shock mesenteric lymph drainage in restoring balance of ACE/ACE2 in kidney of mice

AIM: To study the role of post-hemorrhagic shock mesenteric lymph (PHSML) drainage on the balance of angiotensin-converting enzyme (ACE) and ACE2 in the kidney. METHODS: A hemorrhagic shock model was established and then fluid resuscitation was performed to the animals in shock and shock+drainage groups, and the PHMSL was drained in shock+drainage group after fluid resuscitation. After 6 h of resuscitation, the mRNA expression of ACE, ACE2, angiotensin Ⅱ (Ang Ⅱ) type 1 receptor (AT1R) and Mas-related G-protein-coupled receptor (MasR), and the levels of Ang Ⅱ and Ang (1-7) in the renal tissues were observed. RESULTS: Hemorrhagic shock increased the levels of ACE mRNA, AT1R mRNA and Ang Ⅱ, and decreased the levels of ACE2 mRNA, MasR mRNA and Ang(1-7) in the kidney. PHSML drainage abolished the effect of hemorrhagic shock on ACE2 and AT1R mRNA expression. Meanwhile, PHSML drainage reduced the hemorrhagic shock-induced increases in the ratios of ACE/ACE2, Ang Ⅱ/Ang(1-7) and AT1R/MasR. CONCLUSION: The PHSML drainage restores the balance of ACE/ACE2, which is beneficial to alleviate acute kidney injury following hemorrhagic shock in the mice.     

Differential expression of Ang-1 and Ang-2 proteins contributes to biphasic changes of vascular reactivity after hemorrhagic shock in rats

AIM: To observe the protein expression of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) after hemorrhagic shock, and to explore the role of this difference in the development of the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS: The vascular reactivity of the first class arborization of superior mesenteric artery (SMA) and protein expression of Ang-1 and Ang-2 after hemorrhagic shock were measured via the isolated organ perfusion system and Western blotting technique. The relationship between the protein expression of Ang-1/Ang-2 and the vascular reactivity after hemorrhagic shock was observed. By treatment with Ang-1 and Ang-2 or inhibition of Ang-1 and Ang-2, the effects of angiopoietins on the vascular reactivity of SMA in the early stage (hyperreactivity) and late period (hyporeactivity) of hypoxia were observed to analyze the role of the differential expression of Ang-1 and Ang-2 protein in the biphasic change of vascular reactivity after hemorrhagic shock. RESULTS: (1) The vascular reactivity of SMA showed a biphasic change following hemorrhagic shock, which was increased in the early stage of shock, and decreased in prolonged stage. The E_max of norepinephrin in the shock 10 min group was the highest (129.3% of normal control, P<0.01). The protein expression of Ang-1 in SMA was also increased in the early stage of shock, decreased at prolonged stage of shock, and topped at 10 min shock group (1.85 folds of normal control, P<0.01). The protein expression of Ang-2 in SMA didn't change obviously in the early stage of shock, yet it was increased at prolonged stage of shock, and topped at 4 h shock group (2.90 folds of normal control, P<0.01). (2) In the hyperreactivity stage (hypoxia for 30 min), the extrinsic source of Ang-1 further increased the vascular reactivity, while the extrinsic source of Ang-2 decreased the vascular reactivity (P<0.01). In the hyporeactivity stage (hypoxia for 4 h), Ang-1 improved the vascular reactivity (P<0.01), while Ang-2 further decreased the vascular reactivity (P<0.01). CONCLUSION: The protein expression of Ang-1 and Ang-2 is different after hemorrhagic shock, and the differential expression of Ang-1 and Ang-2 contributes to the biphasic changes of vascular reactivity after hemorrhagic shock in rats.     

Role of hydrogen sulfide in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats

AIM：To investigate the role of hydrogen sulfide (H2S) in alleviation of liver injury by mesenteric lymph drainage in hemorrhagic shock rats. METHODS：A hemorrhagic shock model was established in male Wistar rats. DL-propargylglycine (PPG), an inhibitor of cystathionine γ-lyase (CSE) which is a synthase of H2S, or sodium hydrosulfide (NaHS), a donor of H2S, was administered to the hemorrhagic shock rats with mesenteric lymph drainage. The rats were randomly divided into sham, shock, shock+drainage, shock+drainage+PPG (45 mg/kg, ip, 0.5 h before hemorrhage) and shock+drainage+NaHS (28 μmol/kg, ip, 0.5 h before hemorrhage) groups. Fluid resuscitation was performed 1 h after hypotension, and then mesenteric lymph was drained in the rats of shock+drainage, shock+drainage+PPG and shock+drainage+NaHS groups for 3 h. The hepatic histomorphology was observed. The biochemical indexes of hepatic function in plasma, and H2S, CSE, Toll-like receptor 4 (TLR4), interleukin (IL)-10, IL-12 and tumor necrosis factor α (TNF-α) in hepatic homogenate were also examined. RESULTS：The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bile acid (TBA) in plasma, and H2S, CSE, TLR4, IL-10, IL-12 and TNF-α in hepatic homogenate in shock group were significantly higher than those in sham group. Mesenteric lymph drainage obviously reduced these indexes in shock rats, except for TLR4. PPG further decreased these indexes except for CSE, while NaHS increased these indexes except for TBA and CSE. Morphological observation showed that liver injury appeared in the rats from shock and shock+drainage+NaHS groups, and there was nearly normal hepatic structure in the rats from sham, shock+drainage and shock+drainage+PPG groups. CONCLUSION：The mechanism of mesenteric lymph drainage alleviating liver injury in hemorrhagic shock rats is related to reducing the production of H2S and alleviating the H2S-mediated inflammation.     

Role of A3 adenosine receptor on vascular reactivity of superior mesenteric artery in hemorrhagic shock in rat

AIM: To explore whether A3 adenosine receptor plays a role in the modulation of vascular reactivity after hemorrhagic shock in rat, and to find out the prospective drug target to restore the decreased vascular reactivity following hemorrhagic shock. METHODS: The hemorrhagic shock (40 mmHg) model was established in rat, and the reactivity of superior mesenteric artery (SMA) to norepinephrine (NE) was observed. A3AR expression at protein level and mRNA level were measured by Western blotting and RT-PCR respectively. RESULTS: The vascular reactivity of SMA to NE after hemorrhagic shock (40 mmHg) was decreased significantly in a biphasic response manner. The expression of A3AR mRNA in SMA after hemorrhagic shock decreased without significant difference. The expression of A3AR protein has a slight increase without statistical difference after 30 min of hemorrhagic shock and then has a significant decrease (especially at 2 h and 4 h after hemorrhagic shock). The usage of IB-MECA, a selective A3AR agonist, significantly increased the responsiveness of SMA to NE in hemorrhagic shock in rat. MRS1523, the selective A3AR antagonist, significantly abolished the restoration of the vascular reactivity to NE by IB-MECA in hemorrhagic shock in rat. CONCLUSION: A3AR plays a role in the modulation of vascular responsiveness to NE in hemorrhagic shock in rat, and the selective agonist of A3AR could restore the reactivity of SMA to NE in hemorrhagic shock in rat.     

Changes of RyR-mediated Ca2+ release in VSMCs is involved in the development of vascular hyporeactivity in hemorrhagic shock rats

AIM: In order to investigate the mechanisms involved in the vascular hyporeactivity after hemorrhagic shock, the changes of Ca²⁺ release from calcium store in vascular smooth muscle cells (VSMCs) with hypoxia were observed and the role of Ca²⁺ release from calcium store in the occurrence of vascular hyporeactivity to norepinephrine (NE) after hemorrhagic shock in rats was further explored.METHODS: A hemorrhagic shock model (40 mmHg for 2 h) in rats and a VSMCs hypoxic model were established. The changes of intracellular Ca²⁺ concentration in VSMCs were evaluated by fura3-AM and the role of IP₃R and RyR mediated Ca²⁺ release from calcium store was further observed. The role of IP₃R and RyR mediated Ca²⁺ release from Ca²⁺ store in the development of vascular hyporeactivity was measured with an isolated organ perfusion system. RESULTS: In the absence of extracellular Ca²⁺, NE upregulated by mobilizing Ca²⁺ release through calcium store. Compared to the normal control, the VSMCs had a slight increase when treated with hypoxia and NE-induced intracellular down-regulated, both without significant difference. Compared to the normal control cells, there was a significant change of Ca²⁺ release from calcium store in hypoxia-treated VSMCs, characterized by the significant increase in triggered by RyR-sensitive Ca²⁺ releasing activator caffeine. However, the increase in triggered by IP₃R-mediated Ca²⁺ release agonist adenophostin A (10^-5 mol/L) and ATP-Na₂ (10^-4 mol/L) had no significant difference in hypoxic VSMCs. Furthermore, the vascular reactivity to NE decreased in abdominal aorta in hemorrhagic shock (40 mmHg, 2 h) rats. The activation of IP₃R mediated Ca²⁺ release with ATP-Na₂ (10^-4 mol/L) did not improve the vascular reactivity to NE, while inhibition of IP₃R mediated Ca²⁺ release with heparin (10⁴ U/L) significantly antagonized the vascular reactivity to NE in hemorrhagic shock rats. In addition, in normal K-H solution (with about 2.2 mmol/L) and Ca²⁺-free K-H solution, RyR antagonist ryanodine (10^-5 mol/L) partly restored the vascular reactivity to NE in hemorrhagic shock rats, while RyR agonist caffeine(10^-3 mol/L) further decreased the vascular reactivity. CONCLUSION: The over-activation of RyR-mediated Ca²⁺ release from calcium store is partly involved in the development of vascular hyporeactivity after hemorrhagic shock in rats.     

Mesenteric lymph drainage alleviates spleen injury in hemorrhagic shock rats

AIM：To observe the effects of post-shock mesenteric lymph (PSML) drainage on histopathology, apoptosis, cell cycle and proliferation of the spleen in rats with hemorrhagic shock. METHODS：Eighteen Wistar rats were randomly divided into sham, shock and shock+drainage groups (n=6 in each group). The hemorrhagic shock model was established in the shock and shock+drainage groups. Fluid resuscitation for 30 min was performed 1.5 h after hypotension, and PSML was drained in the rats in shock+drainage group from 1 h after hypotension to 3 h after resuscitation finished. The fixed spleen tissue was harvested from each rat for histological observation with HE staining. The apoptosis of splenocytes was observed by Hoechst 33258 staining. The expression of Bcl-2 and Bax proteins was detected by immunohistochemical staining. The cell cycle and the expression of p53 protein were measured by flow cytometry, and the proliferation index (PI) was calculated. RESULTS：Compared with sham group, splenic tissue injury appeared in the shocked rats. The apoptotic cells and the expression of Bax and p53 in shock group were increased, while Bcl-2 expression was decreased. The percentage of G2/M cells in shock group was decreased. Compared with shock group, the splenic tissue damage in shock+drainage group was significantly attenuated. Moreover, the number of apoptotic cells, the percentage of G0/G1 cells, and the expression of Bax and p53 were obviously decreased, and the G2/M cells, Bcl-2 protein expression and PI were significantly increased in shock+drainage group. CONCLUSION: PSML drainage alleviates splenic injury in hemorrhagic shock rats, which may be related to reducing the apoptosis of splenocytes.     

Liver protection by Sini decoction in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide

AIM:The work was designed to explore protective effects of a traditional Chinese medicine-sini decoction (SD) on liver in hemorrhagic shock and its mechanism relating to oxygen free radical and nitric oxide.METHODS:Anesthetized Wistar rats were subjected to a hemorrhagic shock protocol for 60 min followed by intravenous injection with normal sodium chloride solution or SD solution. Superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) in liver were examined. The inducible nitric oxide synthase (iNOS) was determined immunohistochemically. RT-polymerase chain reaction (RT-PCR)was used to assay the mRNA, which were corresponding to eNOS (endothelial nitric oxide synthase) and iNOS.RESULTS:The activity of SOD decreased, while the concentration of MDA increased in liver during hemorrhagic shock. SD enhanced SOD activity and inhibited a increase in MDA level in liver (P＜0.01). The NO concentrations in liver in SD group increased at three hours after resuscitation (P<0.01). In addition, it was found that the expression of iNOS was upregulated in sodium chloride-treated group, while SD upregulated the expression of eNOS.CONCLUSION:SD reduces the liver injury caused by oxygen free radicals during hemorrhagic shock. The increasing NO concentration by SD is through upregulation of endothelial NOS expression.     

Effects of hemorrhagic shock on BKCa channel tyrosine phosphorylation level in mesenteric artery in rats

AIM: To study the effects of hemorrhagic shock on BK_Ca channel tyrosine phosphorylation in rat superior mesenteric artery and the role of nitric oxide (NO) in BK_Ca channel tyrosine phosphorylation. METHODS: The hemorrhagic shock model [(35±5)mmHg] was established in rats and the whole lysate of superior mesenteric artery were extracted and analyzed by immune precipition (IP) and immunoblotting. The tyrosine phosphorylation levels of BK_Ca channel alpha-subunit in mesenteric artery in hemorrhagic shock rats were investigated, and the modulation of BK_Ca channel alpha-subunit tyrosine phosphorylation by NO and its dose-and time-dependended relationships were observed. RESULTS: The tyrosine phosphorylation level of BK_Ca channel alpha-subunit in mesenteric artery in rats increased significantly after hemorrhagic shock 2 h and 4 h (P<0.01 ), and L-arginine (5×10^-5-5×10^-4 mol/L) up-regulated BK_Ca channel alpha-subunit tyrosine phosphorylation in primary cultured VSMC in a 30 min incubation and without significant decrease after 2 h; L-arginine induced BK_Ca channel alpha-subunit tyrosine phosphorylation in a dose-dependent manner. CONCLUSION: Hemorrhagic shock enhances BK_Ca channel tyrosine phosphorylation in resistant artery in rats, and NO is involved in this process.     

The signal transducers involved in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock

AIM: To observe the regulatory effects of Rho-kinase, PKC and PKG on calcium sensitivity of vascular smooth muscle in hemorrhagic shock in rats. METHODS: The superior mesenteric artery (SMA) from hemorrhagic shock model of rat was adopted to assay the calcium sensitivity via observing the contraction initiated by Ca2+ under depolarizing conditions (120 mmol/L K+) with isolated organ perfusion system. Rho-kinase agonist Ang-Ⅱ and inhibitor fasudil, PKC agonist PMA and inhibitor staurosporine, PKG agonist 8Br-cGMP and inhibitor KT-5823 were used as tool agents to study the regulatory effect of Rho-kinase, PKC and PKG on the calcium sensitivity of SMA following shock. RESULTS: Ang-Ⅱ, PMA and KT-5823 improved the calcium sensitivity of SMA and made the cumulative dose-response curve of SMA to Ca2+ shift to the left, their Emax of Ca2+ (at 3×10-2 mol/L) was 0.630 g/mg, 0.595 g/mg and 0.624 g/mg, respectively, which were all higher than that in shock control (0.377 g/mg) (P<0.05, P<0.01). Fasudil, staurosporine and 8Br-cGMP delimitated the calcium sensitivity of SMA and made the cumulative dose-response curve of Ca2+ shift to the right, their Emax at 3×10-2 mol/L of Ca2+ was 0.242 g/mg, 0.230 g/mg and 0.256 g/mg, respectively, which were all lower than that in shock control (0.377 g/mg) (P<0.05, P<0.01). CONCLUSION: Rho-kinase, PKC, PKG play important roles in the regulation of calcium sensitivity of vascular smooth muscle in hemorrhagic shock.     

Mechanism of mesenteric lymph reperfusion aggravates multiple organ injury in superior mesenteric artery occlusion shock rats

AIM: To explore the mechanism of mesenteric lymph reperfusion (MLR) aggravates multiple organ injury in superior mesenteric artery occlusion (SMAO) shock rats. METHODS: Male Wistar rats were randomly divided into 4 groups: in sham group, only anesthetization and operation were performed; in MLR group, occlusion of mesenteric lymphatics (ML) for 1 h followed by 2 h of reperfusion; in SMAO group, occlusion of superior mesenteric artery (SMA) for 1 h followed by 2 h reperfusion; in MLR+SMAO group, occlusion of SMA and ML for 1 h followed by 2 h of reperfusion. The homogenates of liver, kidney, myocardium and lung were prepared for determining the activities of free radical, nitric oxide, myeloperoxidase (MPO) and cell membrane ATPase. RESULTS: The MDA, NO contents and NOS, MPO activities of multiple organic homogenate in SMAO and MLR+SMAO group were higher than those in sham and MLR group, and these indexes in MLR+SMAO were increased significantly than those in SMAO group. The SOD and ATPase activities of muliple organic homogenate in SMAO and MLR+SMAO group were lower than those in sham and MLR group, and those in MLR+SMAO group was decreased obviously than those in SMAO group. CONCLUSION: The MLR enhances the multiple organ free radical injury, NO synthesis and release, PMN detention and decreases the activity of cell membrane ATPase, aggravating the major organs injury in SMAO shock rats. Intestinal lymphatic pathway plays an important role in the pathogenesis of SMAO shock.     

Mechanism of RhoA on vascular reactivity following hemorrhagic shock in rats

AIM: To observe the mechanisms of RhoA on vascular reactivity following hemorrhagic shock (HS) in rats. METHODS: The superior mesenteric artery (SMA) in rats subjected to hemorrhagic shock was adopted to assay the vascular reactivity via observing the contraction initiated by norepinephrine (NE) with isolated organ perfusion system. Meanwhile, the effects of Rho kinase, myosin light chain phosphatase (MLCP), myosin light chain kinase (MLCK) on RhoA regulating vascular reactivity were observed. The effects of RhoA agonist U-46619 and inhibitor C3 enzyme on the activities of Rho kianse, MLCP, MLCK and phosphorylation of MLC20 in the vascular smooth muscle cells (VSMC) with hypoxia were also measured. RESULTS: As compared to control group, the cumulative dose-response curves of SMA to NE at 2 h after shock shifted to the right, the maximal contractions (Emax) of NE was significantly decreased. RhoA agonist U-46619 increased the vascular reactivity in the late period of shock. C3 enzyme abolished U-46619 induced the increase in the contractile response of SMA to NE. Rho kinase inhibitor Y-27632 decreased U-46619-induced the increase in the vascular reactivity, MLCP inhibitor calyculin further promoted the increase in the vascular reactivity. However, MLCK inhibitor had no effect on the U-46619-induced change of vascular reactivity. After hypoxia, the activities of Rho kinase and MLCK, and the level of MLC20 phosphorylation were decreased, MLCP activity was increased. RhoA agonist U-46619 increased the activity of Rho kinase and phosphorylation of MLC20, decreased the activity of MLCP, but had no effects on MLCK activity. CONCLUSION: RhoA plays an important role in the regulation of vascular reactivity following shock. The mechanism is closely related to regulating the activities of Rho kinase and MLCP, and increasing the phosphorylation of MLC20 in VSMC.     

Role of Rho kinase in the dysfunction of myocardium contraction in hemorrhagic shock rats

AIM: To explore the role of Rho kinase in myocardium contraction in hemorrhagic shock rats. METHODS: The rat cardiac papillary muscle and rat heart at different time points during hemorrhagic shock were isolated. The contraction of papillary muscle in response to isoprenaline and the hemodynamic parameters of the isolated hearts in vitro including left intraventricular systolic pressure (LVSP), the maximal change rate of left intraventricular pressure (±dp/dtmax) were measured. RESULTS: The activity of Rho kinase, the contractile response of cardiac papillary muscle to isoprenaline and the hemodynamic parameters of the hearts were decreased gradually at the 1 h, 2 h and 4 h after shock. Compared to the normal controls, the activity of Rho kinase, the contractile response of cardiac papillary muscle and the hemodynamic parameters of the hearts were decreased significantly. The change of Rho kinase was positively correlated with the contractile response and the hemodynamic parameters of the heart. Stimulation with U-46619 at concentration of 10-8 mmol/L increased the activity of Rho kinase and improved the contractile response of cardiac papillary muscle to isoprenaline and the hemodynamic parameters of the isolated heart. The effects of U-46619 on the above parameters were abolished by Rho kinase specific inhibitor Y-27632 at concentration of 10-5 mol/L. CONCLUSION: Rho kinase plays an important role in the regulation of cardiac contractility during hemorrhagic shock.     

Effects of dexamethasone on acute lung injury by inhibiting renin-angiotensin system in rats

AIM:To explore the relationship between renin-angiotensin system (RAS) and acute lung injury (ALI) in rats, and the effect of dexamethasone (DEX) on RAS was observed.METHODS: The rat model of ALI was induced by hemorrhagic shock and LPS administered intraperitoneally. The changes of MAP and the effects of DEX on MAP were observed. The mRNA expressions of ACE, AGT, AT1 and AT2 in lung tissue were assayed by RT-PCR. The changes of Ang I and Ang II in the serum and the effects of DEX on them were observed.RESULTS: The increasing of MAP was statistically obvious. MAP in hemorrhagic shock+LPS (HL) group recovered more slowly than that in HL+DEX(HLD) group. ACE, AGT, AT1 and AT2 mRNA expressions in HL group were increased, and higher than those in HLD group. The change of Ang II in serum in HL group was obviously higher than that in HLD group, while that of Ang I was not obvious.CONCLUSION:ALI activates RAS in rat lung, and promotes the production of Ang II then aggravates the injury of lung through increasing the expression of ACE and AGT. DEX decreases expression of Ang II.     

CO2 与养分交互作用对番茄幼苗养分利用的影响

以番茄(Lycopersicon esculentumMill.)‘合作906’为材料进行溶液培养试验,设2个因子:CO2和营养液浓度;CO2浓度设正常(360μL/L)和倍增(720μL/L)2个水平;营养液浓度设基本营养液(日本山崎番茄营养液),微量元素采用阿农营养液配方的1/2、1/4、1/8、1/164个水平,完全试验方案8个处理,3次重复。pH为6·0±0·2,3d更换1次营养液。移植到1·2L盆(2株/盒)中,植株在CO2生长箱(VS-3DMC)中培养,全天施放CO2,白天25℃,晚上15℃,光照为14h/d,光照强度11000lx,相对湿度60%。46d时收获,根、茎、叶经蒸馏水冲洗吸干水分后,放入纸袋105℃杀青,75…     

Akt-eNOS-NO signal pathway mediates regulatory effect of Ang-1 and Ang-2 on vascular hyperreactivity in early hemorrhagic shock rats

AIM:To observe the role of endothelial nitric oxide synthase(eNOS) in the regulatory effect of angiopoietin-1(Ang-1) and angiopoietin-2(Ang-2) on the biphasic change of vascular reactivity after hemorrhagic shock in rats. METHODS:The protein expression of eNOS was measured in the superior mesenteric artery(SMA) after hemorrhagic shock by Western blotting. The effect of eNOS inhibitor on the vascular reactivity of SMA treated with Ang-1 and Ang-2 in the early(hyperreactivity) and late(hyporeactivity) periods of hypoxia were observed via an isolated organ perfusion system. The protein levels of eNOS in the hypoxic mixture of vascular endothelial cells(VECs) and vascular smooth muscle cells(VSMCs), and the concentration of nitric oxide(NO) in the medium supernatant of the mixture cells treated with Ang-1, Ang-2 and the inhibitors of Tie-2, Akt, p38 MAPK and ERK were measured. RESULTS:The protein expression of eNOS in SMA was low in normal control group, and increased significantly after hemorrhagic shock, which was 1.84, 3.55, 4.75, 5.96 and 6.33 folds of the normal control level in shock 10 min, 30 min, 1 h, 2 h and 4 h groups, respectively(P<0.01). Inhibitor of eNOS decreased the vascular hyperreactivity in hypoxia 10 min group, in which the E_max of norepinephrine(NE) was decreased from 13.479 mN to 9.043 mN(P<0.05). It also repressed the maintenance effect of Ang-1 on vascular reactivity in hypoxia 10 min group, in wihich the E_max of NE was decreased from 15.283 mN to 11.219 mN(P<0.01). The effect of Ang-2 on the vascular hyperreactivity in hypoxia 10 min group, the vascular hyporeactivity in hypoxia 4 h group, or the effect of Ang-1 or Ang-2 on the vascular reactivity in hypoxia 4 h group did not change. The protein expression of eNOS was increased 10 min after hypoxia as compared with the normal control, which was decreased by Ang-2 and the inhibitors of Tie-2 and Akt(P<0.01), but was not decreased by p38 MAPK and ERK inhibitors. The concentration of NO in the medium supernatant was increased 10 min after hypoxia, and was significantly decreased by Ang-2 and the inhibitors of Tie-2, Akt and eNOS, while the inhibitors of p38 MAPK and ERK had no influence on it. CONCLUSION:Ang-1 and Ang-2 regulate the vascular hyperreactivity in the early hemorrhagic shock rats through Akt-eNOS-NO pathway.     

Protective effect and mechanism of ulinastatin on rats with hemorrhagic shock

AIM: To investigate the protective effect of ulinastatin on rats with hemorrhagic shock. METHODS: A prospective, controlled animal study was designed. The model of hemorrhagic shock in rats was produced by Chaudry method. After 60 min, rats were resuscitated by transfusion of shed blood and normal saline, but a half of them were treated with ulinastatin. At different time points after reperfusion, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected. RESULTS: The levels of TNF-α, IL-6 and MDA significantly increased and the activity of SOD decreased. In the ulinastatin-treated groups, the blood pressure and heart rate were obviously improved; the levels of TNF-α, IL-6 and MDA significantly decreased and the activity of SOD had little change after hemorrhagic shock and reperfusion. CONCLUSION: Ulinastatin has a protection effect on rats with hemorrhagic shock by suppressing the production of inflammatory factors and reducing oxidative damage.     

Role of post-hemorrhagic shock mesenteric lymph in enhancement of vascular permeability

AIM：To investigate the role of post-hemorrhagic shock mesenteric lymph (PHSML) in the enhancement of vascular permeability. METHODS：Eighteen Wistar rats were randomized into sham group, shock group, and shock plus mesenteric lymph drainage (shock + drainage) group. The rats in shock group and shock + drainage group were routinely subjected to hemorrhagic shock and hypotension ［(40±2） mmHg］ was maintained for 90 min, and then the fluid resuscitation was performed. Mesenteric lymph was drained in the rats in shock+drainage group from resuscitation finished to 6 h, for the observation of PHSML drainage on the vascular permeability in multiple tissues of hemorrhagic shock rats. Afterwards, human umbilical vein endothelial cells (HUVECs) were incubated with the PHSML in vitro to observe the effects of PHSML on the morphology and permeability of HUVECs. RESULTS：The degree of blue color and concentrations of Evens blue in the lung, myocardium, kidney, liver, spleen and small intestine were significantly increased in the shocked rats than that in sham group, while the ratios of the dry weight to the wet weight were decreased. The mesenteric lymph drainage reversed these changes. Meanwhile, 4% and 10% of PHSML at 0~3 h and 3~6 h after resuscitation, and lipopolysaccharide (10 mg/L) all caused the damage of HUVECs, decreased the viability and trans-endothelial electrical resistance of HUVECs, and increased the permeability of HUVECs to fluorescein isothiocyanate-labeled albumin. CONCLUSION：PHSML is a vital factor in the enhancement of vascular permeability.     

Changes of lymphatic vessel response to norepinephrine in hemorrhagic shock rats

AIM: To observe the changes of lymphatic vessel response to norepinephrine (NE) in hemorrhagic shock (HS) rats, and to explore the role of lymphatic reactivity in the pathogenesis of shock. METHODS: The lymphatic vessel pressure was observed through intubating into abdomen thoracic duct in 8 rats in sham group and HS group (which was bled from femoral artery until the mean arterial pressure to 40 mmHg). The changes of lymphatic vessel pressure response to NE at different time points were observed by injection of NE (5 μg/kg) through femoral vein. The spontaneous contraction frequency (F), maximal contraction diameter (a), maximal diastolic diameter (b) and static diameter (c) of mesenteric lymphatic (ML) living samples in 8 rats of each group were recorded through microcirculation video systems continuously. The changes of lymphatic fractional contraction index (index I), total contractile activity index (index II) and lymphatic dynamic index (LD-index) (to show the value using △F, △index I, △indexⅡ, △LD-index) were calculated after injection of NE at different time points. RESULTS: The changes of lymphatic boosting pressure response to NE in HS group was started to diminish 30 min after shock, and showed a progressive decreasing trend which significantly reduced than that in sham group at all time points of shock 1 h-3 h. In HS group, the △F, △indexⅡ, △LD-index at shock 1 h, the △F, △index I, △indexⅡ, △LD-index at shock 1.5 h and 2 h were significantly lower than those in sham group, and the △F, △index I, △indexⅡ, △LD-index at all time points were significantly decreased as compared to the values of pre-shock. CONCLUSION: Lymphatic vessel reactivity in shock rats is progressive declined in the process of hemorrhagic shock. The lymphatic vessel hypo-reactivity might play an important role in the pathogenesis of shock.     

Role of ZIPK in the regulatory effects of PKCα and PKCε on calcium sensitivity during hemorrhagic shock in rats

AIM: To observe the role of zipper-interacting protein kinase (ZIPK) in the regulatory effects of protein kinase Cα (PKCα) and protein kinase Cε (PKCε) on calcium sensitivity during hemorrhagic shock(HS) in rats. METHODS: The skinned first class arborization of superior mesenteric artery (SMA) from HS rats were adopted to observe the influence of inhibitor of ZIPK on the effects of PKCα and PKCε agonists on calcium sensitivity after shock via measuring the contraction initiated by Ca2+ with isolated organ perfusion system, hypoxic vascualr smooth muscle cells (VSMCs) were adopted to measure the protein expression and activity of ZIPK after applying PKCα and PKCε agonists following hypoxia via Western blotting. RESULTS: (1) The calcium sensitivity of SMA was decreased after 2 h shock, and increased by agonists of PKCα and PKCε. Emax of Ca2+ was increased from 47.2％to 66.5％ (P<0.01) and 66.3％ (P<0.01) of normal control respectively as compared with 2 h shock group. The increasing effects of PKCα and PKCε agonists on calcium sensitivity of SMA after 2 h shock were weakened by the inhibitor of ZIPK. The cumulative dose-response curve of Ca2+ was shifted to the right, the Emax of Ca2+ was decreased to 42.6％ and 47.5％ of normal control (P<0.01), respectively. (2) The protein expression and activity of ZIPK in VSMCs were decreased after 2 h hypoxia, and were increased by the agonists of PKCα and PKCε following 2 h hypoxia. CONCLUSION: PKCα and PKCε regulate the calcium sensitization probably through changing the protein expression and activity of ZIPK following HS in rats.