Mechanism of calcium sensitivity in lymphatic hypo-reactivity in hemorrhagic shock rats

AIM: To observe the changes of lymphatic reactivity to norepinephrine (NE) and calcium sensitivity in vitro in hemorrhagic shock (HS) rats. METHODS: Male Wistar rats were randomly divided into sham group (with only operation), HS group (duplicating HS model, and divided into shock 1 h and shock 2 h subgroups). The thoracic duct rings (n=48 in each group) were prepared for assaying the lymphatic reactivity to NE and calcium sensitivity by lymphatic tension measurement technique in vitro with isolated perfusion system. Meanwhile, the effects of angiotensin Ⅱ (Ang Ⅱ) and insulin (Ins) on lymphatic reactivity were also observed. RESULTS: Compared with sham group, the NE concentration-response curves in HS 1 h and HS 2 h groups, and calcium concentration-response curves in HS 2 h group were obviously shifted to right. The lymphatic reactivity to NE, contraction to calcium, maximum effect(E_max)and avidity index (pD₂) were markedly reduced. In HS group, after incubating with calcium sensitizer Ang Ⅱ, the lymphatic reactivity to NE and calcium sensitivity were significantly increased but reduced in sham group. However, calcium sensitivity inhibitor Ins decreased the lymphatic contractile response to NE and Ca²⁺. CONCLUSION: The lymphatic hypo-reactivity in hemorrhagic shock rats is related to calcium desensitization, indicating a mechanism of lymphatic hypo-contraction.     

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.     

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.     

Substance P enhances pump function of isolated lymphatics from hemorrhagic shock rats

AIM: To establish a technique of lymphatic perfusion in vitro and to determine the effect of substance P (SP) on lymphatic contractility during the process of hemorrhagic shock (HS). METHODS: Male Wistar rats were randomly divided into control group (surgical procedure only) and HS group . Thoracic ducts were isolated from HS rats at the corresponding time points. The segment of thoracic duct was prepared, perfused in vitro at the transmural pressure of 3 cmH₂O and stimulated with gradient concentrations of SP to measure the lymphatic contractile activity. The end-systolic diameter, end-diastolic diameter, contraction frequency (CF) and passive diameter of isolated lymphatics were measured. The contraction amplitude (CA), tonic index (TI) and fractional pump flow (FPF) were also calculated.RESULTS: SP increased lymphatic CF, TI and FPF, and the effects were enhanced with the increase in the concentration of SP. The CF, TI and FPF of 2 h- and 3 h- shocked lymphatics were elevated to/over the value of baseline levels before the experiment by SP at the concentration starting from 3×10^-8 mol/L. At the same concentration of SP, the CA of lymphatics showed no significant statistical difference among the groups. However, with the increase in SP concentration, the lymphatic CA had a downward trend in all groups.CONCLUSION: SP enhances the pump function of lymphatics not only under physiological condition, but also in shock during different stages.     

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.     

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.     

Protective effects and mechanism of heat shock response on cardiovascular system in rats after heat exposure

AIM:To study protective effects and mechanism of heat shock response (HSR) on cardiovascular system in rats after heat exposure.METHODS:The study was divided into 2 experiments:①Protective effects of HSR on cardiovascular system in rats after heat exposure.SD rats randomly allocated into 2 groups:heat shock group (HS group), sham control group(SC group).HS group were treated with heat shock, but SC group weren't.After re-covering for 20 h at room temperature, two groups exposed to death in thermal environment, and blood pressure and elec-trocardiogram were measured continuously.Through Chart software mean arterial pressure(MAP), existent time etc were acquired.②SD male rats randomly allocated into 3 groups:HS group, SC group and normal temperature control group(NC group).NC group weren't treated.The treatment in HS and SC group was identical with in the first experiment, but it would be terminated at 73 min after heat exposure, meanwhile content of MDA of myocardium were measured.RESULTS:① Existent time in HS group was longer than that in SC group and shock arrived later; ② During earlier period after heat exposure MAP had no significant changes between HS and SC group, but after 60 mins MAP in HS group were higher than that in SC group; ③ Compared with NC group, content of MDA in myocardium in SC group was higher significantly at 73 min after heat exposure. Howerer, content of MDA in HS group was lower than in SC group, and had no significant changes with NC group.CONCLUSION:Through decreasing production of MDA in myocardium, HSR has a protective effect on cardiovascular system in rats after heat exposure.     

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 mesenteric lymph reperfusion on multiple organs injury in superior mesenteric artery occlusion shock rats

AIM: To explore the effect of mesenteric lymph reperfusion (MLR) on blood pressure, survival rate and organ injury in superior mesenteric artery occlusion (SMAO) shock rats.METHODS: Male Wistar rats were randomly divided into 4 groups: in sham group, only anesthetized and operation; in MLR group, performed 1 h occlusion of mesenteric lymphatics (ML) followed by 2 h of reperfusion; in SMAO group, performed 1 h occlusion of superior mesenteric artery (SMA) followed by 2 h of reperfusion; in MLR+SMAO group, performed 1 h occlusion of SMA and ML followed by 2 h of reperfusion. Histopathological and function changes of the lung, liver, kidney and myocardium in rats were assessed after 3 h of mean arterial pressure (MAP) was observed continuously in rats. The survival rate of 24 h was recorded.RESULTS: ① The 24 h survival rates of rats in sham, MLR, SMAO group (100%, 83.3%, 66.7%, respectively) were significant higher than those in MLR+SMAO group (0%). ② The changes of MAP in 4 groups were not statistically different before occlusion. The MAP in SMAO group was higher than that in MLR+SMAO and sham groups at multi-time points after clamping. After reperfusion, the change of MAP in MLR and sham groups was not obvious, and the MAP in SMAO group at multi-time points was decreased than that in MLR and sham groups. MAP in MLR+SMAO group was decreased compared with SMAO, MLR, sham group at all time points after reperfusion. ③ The levels of AST, ALT, BUN, Cre, LDH-1 and CK-MB in MLR+SMAO group in serum were higher than those in sham, MLR and SMAO groups, and these indexes in SMAO group were higher than those in sham and MLR groups. The morphologic observation showed that the structures in kidney, lung, liver and myocardium were normal in sham and MLR groups, however, inflammation, hemorrhage, congestion and necrosis were found in MLR+SMAO group, but only mild histopathological injury was found in SMAO group.CONCLUSION: Our data suggest that the MLR aggravates the multiple organs injury in SMAO shock, therefore, intestinal lymph pathway may play an important role in the pathogenesis of SMAO shock.     

Effect of hypertonic saline solution on the geometry of erythrocyte in rats subjected to hemorrhagic shock

AIM:To study the effect of hypertonic saline solution on the geometry of erythrocyte in hemorrhage-shocked rats,using micropippette aspiration technique. METHODS:Wistar rats were randomized into three groups of 0.9% NaCl(NS),7.5% NaCl(HS) and 5% NaCl-3.5% NaAc(HSA). The animals were bled to reach a mean arterial pressure of 5.3 kPa in 10 minutes and maintained in shock for 90 minutes. 4 mL/kg NS,HS or HSA was given intravenously and respectively in 5 minutes following hemorrhagic shock. The blood was collected to determine the geometry of erythrocyte at baseline,after shock and treatment. RESULTS:There were no significant difference in surface area/volume ratio and spherical index between baseline and shock.The diameter of erythrocyte were decreased obviously following hemorrhagic shock. The surface area/volume ratio was significantly lower in HS group than that in NS and HSA group after treatment. HS and HSA raised spherical index of erythrocyte remarkbly compared with NS.Otherwise,the spherical index of erythrocyte was reduced significantly in HSA group compared to HS group. Both HS and HSA decreased diameter of erythrocyte notably compared with NS. CONCLUSION:Both HS and HSA have deleterious effects on the geometry of erythrocyte in rats subjected to hemorrhagic shock.Whereas, HSA could decrease the side effect compared with HS.     

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…     

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.     

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.     

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.     

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.     

Effects of astragaloside IV on autophagy in rats with cerebral ischemia/reperfusion injury

AIM: To investigate the effects of astragaloside IV (AS-IV) on autophagy in rats with cerebral ischemia/reperfusion (I/R) injury. METHODS: The focal cerebral ischemia/reperfusion of rat left middle cerebral artery occlusion (MCAO) was induced by suture method. Male SD rats (n=70) were randomly divided into sham operation group, I/R group, solvent control group, AS-IV group, AS-IV+autophagy inhibitor (3-methyladenine, 3-MA) group, 3-MA group and autophagy activator (rapamycin, Rapa) group. Except for sham operation group, the rats in other groups were subjected to ischemia for 2 h and reperfusion for 24 h. The rats with successful modeling were selected according to Zea Longa scoring criteria. The volume of cerebral infarction was measured by TTC staining. The morphological changes of nerve cells in the rats were observed with Nissl staining. The phenomenon of autophagy was observed under transmission electron microscope. The protein expression of beclin-1 and LC3-Ⅱ was determined by Western blot. RESULTS: No neurological deficit in sham operation group was observed, and the cerebral infarction was not found. Compared with sham operation group, obvious cerebral infarction was observed, the Nissl bodies were small in size and number and stained light, typical autophagosomes were observed, and the protein expression of beclin-1 and LC3-Ⅱ was increased in I/R group (P<0.05). Compared with I/R group, the volume of cerebral infarction was decreased obviously, neurological deficit restored significantly, and the number of autophagosomes and the protein expression of beclin-1 and LC3-Ⅱ were increased in AS-IV group and Rapa group (P<0.05). However, no significant difference between solvent control group and I/R group was observed (P>0.05). Compared with AS-IV group, the neurological deficit was serious, the volume of cerebral infarction and the number of autophagosomes were increased, while the expression of beclin-1 and LC3-Ⅱ was decreased in AS-IV+3-MA group and 3-MA group (P<0.05). CONCLUSION: Astragaloside IV may play an important role in atte-nuating cerebral ischemia/reperfusion injury by activating autophagy.     

Preconditioning with 3-nitropropionic acid induces rat cerebral ischemic tolerance and increases expression of glucose transporter 1 and glucose transporter 3

AIM: To explore the mechanism of 3-nitropropionic acid (3-NPA) preconditioning that induces cerebral ischemic tolerance in rats by affecting the expression of brain-type glucose transporters (GLUT1 and GLUT3) at mRNA and protein levels in cerebral tissues.METHODS: The male SD rats were used in the experiments and divided randomly into sham operation group (sham group, n=4), control group of 3-NPA preconditioning (3-NPA group, n=4), cerebral ischemia group (M group, n=16) and 3-NPA preconditioning group (IPC group, n=16). M group and IPC group were further divided into 4 subgroups according to the different reperfusion time(4 h, 12 h, 24 h and 48 h). All rats were killed at the corresponding time points. The cerebral tissues in the ischemic side (left) and coronal intermediate 1/3 of cortex were collected. The protein levels and mRNA expression of GLUT1 and GLUT3 were determined by Western blotting and RT-PCR. RESULTS: Compared with M group, the ischemic reperfusion and 3-NPA preconditioning induced the upregulation of GLUT1 and GLUT3 at protein levels with significant differences (F=5.848, P<0.05 and F=6.295, P<0.05, respectively), especially after ischemia-reperfusion for 48 h. The mRNA expression of GLUT1 in IPC group began to increase at 4 h, peaked at 48 h after reperfusion, with significant difference as compared to M group at the corresponding reperfusion time points in each group or sham group. In contrast, the mRNA expression of GLUT3 in IPC group increased at 24 h, and was the highest at 48 h as compared to cerebral ischemia group at the corresponding reperfusion time points or sham group.CONCLUSION: 3-NPA preconditioning increases the expression of GLUT1 and GLUT3 at protein and mRNA levels to maintain the energy supply in brain tissues, indicating a cerebral protective mechanism.     

Establishment and evaluation of rat heart ischemia-reperfusion injury model in vivo

AIM: To establish and evaluate a rat model of heart ischemia-reperfusion injury in vivo. METHODS: Seventy-two male Sprague-Dawley rats weighing(250±50)g were randomly divided into sham operation group(sham), ischemia-reperfusion group(I/R) and normal group. The animals were anesthetized and heparinized. Myocardial ischemia-reperfusion was induced by ligating the left anterior descending coronary artery with "U-shape tube" for 35 min followed by 120 min or 240 min reperfusion in vivo. The heart infarct size was measured by triphenyltetrazolium chloride(TTC) staining. The myocardial cell apoptotic index was determined by the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling(TUNEL). Immunohistochemical method was used to detect the expression of Bcl-2 and Bax in rat ischemia myocardium. The blood level of MB isoenzyme of creatine kinase(CK-MB),cardiac troponin I(cTnI),nitric oxide(NO),malondialdehyde(MDA), total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px) were detected after reperfusion for 2 h and 4 h. RESULTS: Compared with normal group and sham group, there were obvious changes of ST-T segment and Q wave in the electrocardiogram of I/R group. The blood level of CK-MB, cTnI, NO, MDA and GSH-Px in I/R group increased(P<0.05,P<0.01) after reperfusion for 2 h and 4 h, and the blood level of T-SOD in I/R group after reperfusion for 2 h and 4 h also increased(P<0.05). The heart infarct size in I/R group was the largest as compared to other groups. Among these groups, the apoptotic index of I/R group was the highest and the Bcl-2/Bax ratio in I/R group decreased(P<0.01).CONCLUSION: The rat model of heart ischemia-reperfusion injury in vivo can be successfully established with the "U-shape tube". There are obviously changes of heart infarct size, blood level of CK-MB, cTnI, NO, MDA, T-SOD and GSH-Px, myocardial apoptotic index and Bcl-2/Bax ratio between I/R rats and control animals.     

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.     

Dual effects of antithrombin Ⅲ on inflammatory factor and bloo d coagulatory factor in rats with hemorrhagic shock

AIM:To observe the changes of inflammatory factors and blood coagulatory factors and effects of antithrombin Ⅲ (ATⅢ) on activated inflammatory factors and blood coagulatory factors in rats with hemorrhagic sho ck .METHODS:The rat model of hemorrhagic shock was set up.40 SD rats were randomized into four groups:sham operation,shock,routine dose ATⅢ and high dose ATⅢ groups,each group was composed of 10 SD rats.Shock group w as administered common resuscitation fluid,routine dose ATⅢ group was administ ered ATⅢ 20 U/kg,high dose ATⅢ group was administered ATⅢ 100 U/kg everyday for successive three days.Plasma NF-κB,6-Keto-prostaglandinF1α,E-selecti n,sICAM-1,thrombin-ATⅢ complexes,thrombinogen fragment F1+2 (PF F1+2),D-dimer and TMD levels were detected.RESULTS:Plasma NF-κB,sICAM-1,E -selectin levels were signifi cantly lower in high dose ATⅢ group than those in shock group and routine dose ATⅢ group (P<0.01).No significant difference between shock group and rou tine dose ATⅢ group was observed.Plasma 6-Keto-prostaglandinF1α levels were h igher in routine dose ATⅢ and high dose ATⅢ groups than that in shock group.P lasma thrombinogen F1+2,D-dimer,TMD,TAT levels were higher in shock rats ( P<0.01).CONCLUSION:Hemorrhagic shock activates coagulatory and inflamma tory reactions.High dose ATⅢ inhibits inflammatory reactions.