Blockage of mesenteric lymph return promotes the vascular reactivity and calcium sensitivity in hemorrhagic shock rats

AIM: To observe the effects of mesenteric lymph duct ligation and mesenteric lymph drainage on the vascular reactivity and calcium sensitivity in hemorrhagic shock (HS) rats, and to investigate the role of mesenteric lymph on the vascular hyporeactivity during shock. METHODS: Seventy-two male Wistar rats were randomly divided into sham group (only operation), shock (duplicating HS model) group, shock+ligation group (duplicating HS model and mesenteric lymph duct ligation) and shock+drainage group (duplicating HS model and mesenteric lymph drainage). The changes of mean artery pressure (MAP) after injection of norepinephrine (NE, 3 μg/kg) at different time points were recorded. After hypotension (40 mmHg) for 3 h, the vascular ring of superior mesenteric artery (SMA) was made for determining the vascular reactivity and sensitivity to calcium by observing the contraction initiated by NE and Ca²⁺ under depolarizing conditions (120 mmol/L K⁺) in the isolated organ perfusion system. Meanwhile, the effects of angiotensin Ⅱ (AngⅡ) and insulin (Ins) on the vascular reactivity were also observed. RESULTS: Compared to sham group, the △MAP in shock group was increased significantly at 0 h and 0.5 h after shock, and that was decreased markedly at 1.5 h, 2 h, 2.5 h and 3 h after shock, respectively, and that in shock+ligation group and shock+drainage group was increased at 0 h, 0.5 h and 1 h after shock, decreased at 2.5 h and 3 h after shock, respectively. The △MAP in shock+ligation group and shock+drainage group was higher than that in shock group at 0.5 h after shock and all the time points followed. The SMA reactivity to NE and sensibility to Ca²⁺ in shock group, shock+ligation group and shock+drainage group were lower markedly than those in sham group. The vascular reactivity and calcium sensitivity in shock+ligation and shock+drainage groups were higher than those in shock group. The vascular reactivity and calcium sensitivity in shock group, shock+ligation group and shock+drainage group were lower than those in sham group, and those in shock+ligation and shock+drainage groups were increased as compared to shock group, respectively. CONCLUSION: Blockage of mesenteric lymphatic return with the methods of mesenteric lymph duct ligation and mesenteric lymph drainage promotes the vascular reactivity of HS rats. The mechanism may be related to improving the calcium sensitivity in the vasculature.     

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 Rho kinase in blocking the return of mesenteric lymph to improve vascular calcium sensitivity in hemorrhagic shock rats

AIM: To observe the role of Rho kinase in mesenteric lymph duct ligation or mesenteric lymph drainage to improve vascular calcium sensitivity in the rats subjected to hemorrhagic shock. METHODS: Male Wistar rats were randomly divided into sham group, shock group, shock+ligation (shock plus mesenteric lymph duct ligation) group and shock+drainage (shock plus mesenteric lymph drainage) group. After induction of shock (hypotension at 40 mmHg) for 3 h, the vascular rings of superior mesenteric artery (SMA) were prepared and used to measure the response to gradient calcium ions for determining the calcium sensitivity with a wire myograph system. In shock+ligation group and shock+drainage group, the vascular rings were incubated with Rho kinase agonist angiotensinⅡ or antagonist fasudil before the measurement of the response to gradient calcium ions. RESULTS: The calcium sensitivity of vascular rings in shock group was significantly lower than that in sham group, and that in shock+ligation group and shock+drainage group was significantly higher than that in shock group, but still lower than that in sham group. AngⅡ elevated the contractile activity of the vascular rings in response to gradient calcium ions and the pD₂, and fasudil significantly decreased the response to gradient calcium ions and E_max in shock+ligation group and shock+drainage group. At the same time, fasudil decreased the pD₂ in shock+ligation group. CONCLUSION: Rho kinase plays an important role in blocking shock mesenteric lymph return that improves calcium sensitivity.     

Effect of ischemic preconditioning on vascular reactivity and calcium sensitivity in hemorrhagic shock rats

AIM: To investigate the effect of ischemic preconditioning (IPC) on vascular reactivity and calcium sensitivity during hemorrhagic shock. METHODS: Appropriate method of IPC was selected by observing the effect of different strategies of IPC on the survival time and the survival rate in hemorrhagic shock rats. The effect of IPC on the pressor effect of norepinephrine (NE, 3 μg/kg) and the contractile response of superior mesenteric artery (SMA) to NE and calcium in vivo and in vitro were observed. RESULTS: Among 3 strategies of IPC, 3 cycles of abdominal aorta occlusion for 1 min and loosing for 5 min increased the survival time and 24 h survival rate significantly, which was superior to the other two IPC methods. In vivo, IPC significantly increased the pressor response to NE and the contractile response of SMA to NE (P<0.01). In vitro, IPC significantly improved the reactivity of SMA to NE and Ca²⁺. The E_max values of SMA to NE and Ca²⁺ in IPC group were significantly higher than that in shock control group (P<0.01). CONCLUSION: Ischemic preconditioning reverses Shock-induced vascular hyporeactivity via improving calcium sensitivity of the vasculatures.     

Pinacidil pretreatment increases vascular reactivity and calcium sensitivity after hemorrhagic shock

AIM: To observe the protective effects of protein kinase Cα(PKCα) and protein kinase Cε(PKCε) activated by pinacidil pretreatment on vascular reactivity and calcium sensitivity after hemorrhagic shock in rats. METHODS: The changes of the pressor effect(the change of mean arterial pressure) and vasoconstriction response(the changes of diameter) of superior mesenteric artery(SMA) to norepinephrine(NE) were observed. The vascular reactivity and calcium sensitivity of the first class arborization of SMA induced by pinacidil pretreatment with different volume and at different time points before shock were determined. The effects of PKCα and PKCε antagonists on the protection of pinacidil pretreatment, and the effects of pinacidil pretreatment on the translocation of PKCα and PKCε were also measured. RESULTS: (1) The pressor effect and vasoconstriction response of SMA to NE, and the vascular reactivity and calcium sensitivity of the first class arborization of SMA in 2 h shock group were significantly decreased as compared to those in normal controls(P<0.01). Pinacidil(25 μg/kg) pretreated at 30 min before shock attenuated the above changes.(2) The inhibitors of PKCα and PKCε suppressed the protective effects of pinacidil pretreatment(25 μg/kg pinacidil pretreated at 30 min before shock) on the vascular reactivity and calcium sensitivity. The E_max of NE was decreased by 42.9% and 62.9%, respectively(P<0.01). The E_max of Ca²⁺ was decreased by 31.1% and 56.1%, respectively(P<0.01). Pinacidil(25 μg/kg) pretreated at 30 min before shock increased the protein expression of PKCα and PKCε on the membrane, and decreased the protein expression in the cytoplasm as compared to those in 2 h shock group(P<0.01). CONCLUSION: Pinacidil pretreatment activates PKCα and PKCε, and induces the increasing effects of vascular reactivity and calcium sensitivity after hemorrhagic shock in rats.     

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.     

Estrogen treatment enhances mesenteric lymphatic contractility in rats after hemorrhagic shock

AIM To investigate the effects of 17β-estradiol （E2） treatment on the mesenteric lymphatic microcirculation and isolated lymphatic contractility in rats after hemorrhagic shock, and to explore the relationship between contractility and the difference between intra- and extracellular calcium ion concentrations （［Ca²⁺］） of lymphatic smooth muscle cells （LSMCs）. METHODS Male Wistar rats were divided into sham group, shock group and shock+E2 group. The rats were subjected to hemorrhage ［（40±2） mmHg for 90 min］ and resuscitation with or without subcutaneous injection of E2 （2 mg/kg）. After resuscitation for 3 h, the mesenteric lymphatic microcirculation in vivo was observed. Moreover, the isolated mesenteric microlymphatic rings were prepared for the observations of lymphatic contractility evaluated by the indexes including end-systolic diameter, end-diastolic diameter, contraction frequency （CF） and passive diameter. Meanwhile, the difference between intra- and extracellular ［Ca²⁺］ of LSMCs was recorded during lymphatic contraction. RESULTS Treatment with E2 significantly enhanced the CF, total contractile fraction and lymphatic dynamics index in vivo in the rats after hemorrhagic shock, and increased the CF, the fractional pump flow and the difference between intra- and extracellular ［Ca²⁺］ of LSMCs in isolated lymphatics from the shocked rats （P<0.05）. CONCLUSION Estrogen treatment enhances lymphatic contractility in rats after hemorrhagic shock, which is related to enhancement of difference between intra- and extracellular ［Ca²⁺］ of LSMCs.     

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.     

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.     

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 myosin-light-chain kinase in biphasic contractile activity of lymphatics isolated from hemorrhagic shock rats

AIM: To elucidate the mechanism by which myosin-light-chain kinase (MLCK) modulates the biphasic contractile activity of lymphatics isolated from the rats subject to hemorrhagic shock (HS). METHODS: Male Wistar rats were randomiz to control group and HS group. In HS group, the rats were subject to HS and then further divided into HS 0 h, 0.5 h, 1 h, 2 h and 3 h subgroups. Thoracic ducts of control and shock rats were isolated and used to determine the protein levels of phosphorylated MLCK (p-MLCK). In addition, thoracic ducts obtained from control, 0.5 h- and 2 h-shocked rats were used to observe the contractile properties of lymphatics by a pressure myograph in vitro . Lymphatic rings were prepared and incubated with ML-7 (a specific inhibitor of MLCK) or substance P (SP, an agonist of MLCK). During the experiment, the contractile frequency (CF), end-diastolic diameter, end-systolic diameter and passive diameter in Ca²⁺-free PSS buffer were measured and used to calculate the lymphatic tonic index (TI), contractile amplitude (CA) and fractional pump flow (FPF) as the indexes of lymphatic contraction activity. RESULTS: The levels of p-MLCK in lymphatics in 0 h- and 0.5 h-shocked rats were significantly increased compared with the control rats, and it was gradually decreased with the development of shock. The values of CF, TI and FPF in 0.5 h-shocked lymphatics were significantly increased at transmural pressure of 1, 3 and 5 cmH₂O compared with those in control group, and significantly blunted by ML-7. SP obviously increased the suppressive effects induced by ML-7 and restored the values of CF, TI and FPF to the levels of HS 0.5 h group. CF, TI and FPF in 2 h-shocked lymphatics significantly declined under different transmural pressure as compared with those in control group, and significantly elevated by SP. Similarly, ML-7 depressed the effects of SP. No significant difference was found in CA between 0.5 h- and 2 h-shocked lymphatics. SP decreased the CA of lymphatics obtained from 2 h-shocked rats and this effect was suppressed by ML-7. However, both agents had no effects on CA of 0.5 h-shocked lymphatics. CONCLUSION: MLCK, as an essential enzyme that influences the contraction of lymphatic smooth muscle cells, involves in the modulation of biphasic changes of lymphatic contractile activity during the process of HS.     

Changes of aortic contractile reactivity and RAS components after tourniquet shock

AIM: To investigate the role of renin-angiotensin system(RAS) disequilibrium in hyporeactivity and injury of aorta after tourniquet shock(TS) by observing the changes of aortic contractile reactivity and RAS components after TS. METHODS: Male C57BL/6 mice(8 months old) were divided into 7 groups including control group and 6 model groups. The mice in model groups were sacrificed at reperfusion of 10 min, 1 h, 2 h, 4 h, 6 h and 12 h. The mice in control group were not subjected to tourniquet ligation. The Doppler flowmetry was used to determine the limb blood flow. The carotid artery catheter was applied to detect the blood pressure. The isolated vascular tension tester was available to measure the reactivity of the aorta. HE staining combined with transmission electron microscopy was used to evaluate the morphology of injured aortas. The protein expression of AT1 receptor, Mas receptor, ACE and ACE2 was measured by Western blot. The serum contents of Ang Ⅱ and Ang(1-7) were detected by ELISA. RESULTS: Compared with control group, the blood flow in model groups decreased gradually with the prolongation of reperfusion time. The blood pressure increased at 10 min after reperfusion, and then decreased gradually. Accordingly, vascular reaction to norepinephrine(NE) increased at 10 min and then descended. The vascular reactivity reached the lowest level at 4 h. Morphological injury score increased gradually. Vascular AT1 receptor and ACE2 proteins were reduced, while Mas receptor and ACE proteins were up-regulated compared with control group. The content of Ang Ⅱ in the serum elevated, while the content of Ang(1-7) was reduced. CONCLUSION: The mechanism of aortic reaction to NE increased temporarily in the early stage of shock and then decreased. It may be related to the morphological injury of aorta and the imbalance of RAS.     

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.     

Mechanism of mesenteric lymph duct ligation against the renal insufficiency in hemorrhagic shock rats

AIM: To observe the effect of mesenteric lymph duct ligation on free radical and inflammatory mediator in serious hemorrhagic shock rats at different periods, and explore the mechanism of intestinal lymphatic pathway on renal insufficiency. METHODS: 78 male Wistar rats were divided into the sham group, shock group, and ligation group. The model of serious hemorrhagic shock was established in shock group, ligation group, and mesenteric lymph was blocked by ligating mesenteric lymph duct in ligation group after resuscitating. All rats were executed and kidneys were taken out for making homogenate of 10 percent to determine levels of MDA, SOD, NO, NOS, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and myeloperoxidase (MPO) at time points after shock 90 min, after transfusion and resuscitate 0 h, 1 h, 3 h, 6 h, 12 h and 24 h. The expression of inducible nitric oxide synthase (iNOS) mRNA in kindey was detected by RT-PCR. RESULTS: The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expressions in renal homogenate of shock group were increased after transfusion and resuscitation, and were higher at 6 h and 12 h, and was significantly higher than that in sham group. The acvitity of SOD was significantly lower than that in sham group (P<0.01, P<0.05). The contents of MDA, NO, NOS, TNF-α, IL-6, MPO and iNOS expression in renal homogenate of ligation group after transfusion and resuscitation 6 h, 12 h and 24 h were significantly lower than those in shock group at same points, and the SOD activity was higher (P<0.01, P<0.05). CONCLUSION: The results demonstrate that the ligation of mesenteric lymph duct can antagonise the development of renal failure in serious hemorrhagic shock rats, and its mechanism might relate to reduce the PMN sequestration, decrease the levels of TNF-α and IL-6, inhibit NO production and expression of iNOS mRNA, suppress the release of free radical and consumption of SOD.     

Effect of Qiliqiangxin granule on apoptosis of renal tissues in rats with cardiorenal syndrome

AIM: To investigate the effect of Qiliqiangxin granule on the apoptosis of renal tissues in rats with cardiorenal syndrome (CRS) and its possible mechanism. METHODS: A rat model of CRS was established by ligation of the left anterior descending coronary artery and acute renal ischemia/reperfusion injury. After operation, the rats were divided into 6 groups:2-week sham operation (2w sham) group, 2-week model (2w CRS) group, 2-week drug (2w CRS-Q) group, 4-week sham operation (4w sham) group, 4-week model (4w CRS) group and 4-week drug (4w CRS-Q) group. The rats in 2w CRS-Q group and 4w CRS-Q group were given Qiliqiangxin granule (4 g·kg^-1·d^-1) by gavage for 2 weeks and 4 weeks, respectively. The levels of serum cystatin C (Cys-C), plasma angiotensin Ⅱ (Ang Ⅱ), urine neutrophil gelatinase-associated lipocalin (NGAL) and urine microalbumin (UMA) were measured by ELISA. The serum level of creatinine (Cre) was detected by sarcosine oxidase method. The renal histopathological changes were observed by HE staining. The mRNA and protein expression levels of Ang Ⅱ, Bcl-2 and Bax were evaluated by RT-qPCR and Western blot, respectively. The apoptosis rate of renal cells was assessed by TUNEL staining. RESULTS: The levels of serum Cys-C, serum Cre, plasma Ang Ⅱ, urine NGAL and UMA were significantly increased in 2w CRS group and 4w CRS group compared with 2w sham group and 4w sham group after modeling (P<0.05). The mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues of CRS rats were significantly up-regulated (P<0.05), while Bcl-2 was significantly down-regulated (P<0.05) compared with 2w sham group and 4w sham group. Compared with 2w sham group and 4w sham group, the damage of renal tissues in 2w CRS and 4w CRS group was severe, and the apoptotic rates of renal cells were significantly increased. Compared with 2w CRS group and 4w CRS group, Qiliqiangxin granule greatly decreased the levels of Cys-C, Cre, Ang Ⅱ, NGAL and UMA, down-regulated the mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues, and up-regulated the expression of Bcl-2 at mRNA and protein levels at 2 and 4 weeks. In addition, Qiliqiangxin granule also greatly attenuated the damage and apoptosis of the renal tissues. CONCLUSION: Qiliqiangxin granule significantly inhibits the apoptosis of renal tissues and improves the renal function of CRS rats, and its mechanism may be related to the inhibition of Ang Ⅱ expression.     

Role of nitric oxide in reactivity of isolated lymphatics to substance P in shock rats

AIM: To observe the role of nitric oxide (NO) in the reactivity of isolated lymphatics to substance P (SP),which presents a biphasic change, in the hemorrhagic shock (HS) rats with the technique of lymphatic perfusion in vitro. METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and shock group (the rats were further divided into shock 0.5 h and shock 2 h groups after the HS model was established). A segment of lymphatics was pressed and perfused in vitro at transmural pressure of 3 cmH₂O after thoracic ducts were separated from the rats at the corresponding time points in each group. The lymphatics of shock 0.5 h and shock 2 h were incubated with different drugs for changing the activity of No and nitric oxide synthase (NOS), respectively. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured, while the contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were calculated after stimulated with gradient SP. Different values between pre-and post-administration of SP in CF, CA, TI and FPF were calculated and expressed as ΔCF, ΔTI, ΔCA and ΔFPF for further assessing the reactivity of lymphatics. RESULTS: NO donor L-Arg reduced ΔCF, ΔTI and ΔFPF of 0.5 h-shocked lymphatics treated with different concentrations of SP. The effect of L-Arg was obviously suppressed by a soluble guanylate cyclase inhibitor ODQ. ΔCF, ΔTI and ΔFPF increased strikingly compared with shock 0.5 h+L-Arg group in the presence of SP at certain concentration, and ΔCF and ΔFPF increased remarkably compared with control group. NOS inhibitor L-NAME elevated ΔCF, ΔTI and ΔFPF of 2 h-shocked lymphatics treated with different concentrations of SP and the manifestation of lymphatics exceeded the values of control levels. In the experiment of 2 h-shocked lymphatics treated with L-NAME+phosphodiesterase inhibitor aminophylline (AP), the effect of L-NAME was suppressed significantly, which manifested by the decrease in ΔCF, ΔTI and ΔFPF as compared with the values of shock 2 h+L-NAME group in the presence of SP at the concentrations of 1×10^-8 mol/L and 3×10^-8 mol/L. CONCLUSION: These data indicate that NO involves in the biphasic modulation of shocked lymphatics and the effect might be involved in the action of cyclic guanosine monophosphate.     

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.     

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.     

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.