Role of deacetylase sirtuins in sepsis: beneficial or harmful? / 中华危重病急救医学

Sepsis,life-threatening organ dysfunction caused by a dysregulated host response to infection,is a major public health concern.To date,the mechanism of sepsis is not completely understood,which is still a huge task ahead of numerous clinical and laboratory researchers.Recently,increasing evidences show that deacetylase sirtuins play an important role in sepsis and the function of sirtuins are varied in different stages of sepsis.More importantly,the mechanism of sirutins is not fully understood.The sirtuins family is composed by sirtuin 1-7 members.Among them,sirtuin 1 is widely reported.In addition to sirtuin 1,other members of sirtuins are also involved in the regulation of inflammation or metabolism signaling following sepsis.Of note,the sirtuins may interact with each other and form a precious control mechanism.Herein,we tried to summarize the recent paper from PubMed,to explain the possible mechanism of distinct role of sirtuin 1/2,to generalize the downstream effects of sirtuin 3 action,and to describe the interactions among sirtuins members on sepsis,which might be helpful for our future research and potential clinical applications.

Changes of platelet mitochondria in rats with severe hemorrhagic shock and intervention effect of polydatin / 中华创伤杂志

Objective To investigate relationship between the variation degree of platelet mitochondria in rats with severe hemorrhagic shock and the degree of shock.Methods Thirty-six Wistar rats were divided into sham group,shock 30,60,and 120 minutes groups,shock 120 minutes + normal saline (NS) + blood reinfusion group (NS group) and shock 120 minutes + polydatin (PD) + blood reinfusion group (PD group) according to random number table,with six rats per group.Content of ATP in platelets was detected by fluorescein-luciferase assay kit; structure of platelet mitochondria by electron microscope; state of mitochondrial permeability transition pore by Calcein-AM and CoCl2 ; change of mitochondrial membrane potential (△Ψm) by JC-1 mitochondrial membrane potential kit; lipid hydroperoxide (LPO) in platelets by LPO assay kit; stability of platelet lysosomes by acridine orange (AO).Results ATP released from platelets was reduced significantly in shock 60 minutes group (P ＜0.01) and with the prolong of shock period,further reduction was observed,particularly in NS group [(50.75 ± 9.15)％ of normal value].Mitochondrial swelling with poorly defined crista structure,declined △Ψ and low lysosome stability (pale cells were increased) were observed in shock 30 minutes group.Calcein fluorescence in mitochondria was faded in shock 60 minutes group (P ＜ 0.01).Whereas in PD group,all the above indices presented some recovery with ATP level returned to nearly (79.57 ± 8.48) ％ of normal value in particular.Conclusions Platelet mitochondrial dysfunction takes place at 30-60 minutes following severe shock.Hence,it may be served as an non-invasive index for the diagnose and treatment of severe shock.

Effects of basic fibroblast growth factor on expressions of collagen and fibronectin in normal skin and hypertrophic scar fibroblasts / 中国组织工程研究

BACKGROUND:Basic fibroblast growth factor(bFGF)can promote production of collagen,fibronectin and matrix enzyme in healing wounds.However,dysregulation of this process,such as the abnormal coordination of cell proliferation,extracellular.matrix and neovasculadzation formation,or remodeling of the wound matrix will lead to excess accumulation of scar tissues.OBJECTIVE:To investigate effects of bFGF on normal skin wound healing and hypertrophic scar formation.METHODS:Normal and hypertrophic scar fibroblasts from tissue biopsies from 5 patients who underwent plastic surgery for repairing hypertrophic scars were isolated and cultured.The expressions of collagen,fibronectin and protein synthesis were detected by RT-PCR and ELISA.The mitochonddal membrane potential changes were measured using JC-1 staining and flow cytometry.Simultaneously,adenosine tdphosphate(ATP)levels were determined by chemiluminescence method.The effects of bFGF on these indexes of normal and hypertrophic scar fibroblasts were observed.RESULTS AND CONCLUSION:Hypertrophic scar fibroblasts become slower after being exposed to bFGF,which selectively inhibited type Ⅰ collagen production in hypertrophic scar fibroblasts(P<0.05).Although bFGF inhibited type]collagen production,it had no effect on type Ⅲ collagen expression in both normal and hypertrophic scar fibroblasts.However,fibronectin expression in the normal fibroblasts was up-reguleted after bFGF treatment(P<0.05).In addition,the mitochonddal membrane potential tended to depolarization,although no statistical difference,in hypertrophic scar fibroblasts treated with bFGF(10 or 100 μg/L).bFGF treatment increased the cellular ATP levels in the normal fibroblasts,while there were no significant alterations in the hypertrophic scar fibroblasts over a treatment of bFGF(10 or 100 μg/L,P<0.05).The results suggest that there are differential effects and mechanisms on the skin fibroblasts with bFGF treatment in normal wound healing and hypertrophic scar formation.

Effect of limited volume resuscitation on hemodynamic changes in pregnant rabbit with hemorrhagic shock / 中华妇产科杂志

Objective To determine the effects of two fluid resuscitation strategies on the changes of hemodynamic variables,serum concentration of tumor necrosis factor-alpha(TNF-α)and interleukin-6 (IL-6)in a clinically relevant model of uncontrolled hemorrhagic shock in pregnant rabbits.Methods Hemorrhagic shock was induced by bleeding via carotied artery,followed by transection of a medium vessel in gestational sac.Experimental design consisted of three phases,shock phase(0-30 min),prehospital phase(30-90 min)and hospital phase(90-180 min).Twenty pregnant rabbits were randomly divided into two groups(n=10/group),aggressive fluid resuscitation group(PNL group)and limited volume resuscitation group(PLH group).In the shock phase,animals were hemorrhaged by blood withdrawal to mean arterial pressure(MAP)of 40-45 mm Hg(1 mm Hg=0.133 kPa)via carotid artery.In the prehospital phase,a medium vessel in the gestational sac was transected,then the animals in the PNL group and PLH group were resuscitated with 0.9% normal saline(NS)and shed blood to MAP of 80,60 mm Hg respectively.In the hospital phase,bleeding was controlled by surgical intervention and all the animals were reinfused with shed blood and NS to MAP 80 mm Hg.Hemodvnamic variables and respiration rate were monitored and blood samples were collected for TNF-α and IL-6 measurement.and finally subsequent volume resuscitation and survival rate were recorded.Results (1)At 120 min,the respiration rate and heart rate in the animals assigned to PLH group was(66±16)bpm,(235±41)bpm respectively,which were significantly lower than those in PNL group(P＜0.01),while MAP and central venous pressure in the PLH group was(80.4±7.2)mm Hg,(8.0±4.4)cm H2O,respectively,which were significantly higher than those in PNL group(P＜0.01);(2)The serum concentration of TNF-α,IL-6 of all the animals were markedly increased after hemorrhagic shock.and peak at 24 min.The serum concentration of TNF-α,IL-6 in animals assigned to PLH group were(105±67)ng/L,(118±51)ng/L respectively,which were significantly lower than those in PNL group(P＜0.01).The serum concentration of TNF-α,IL-6 in the animals assigned to PLH group were decreased to normal at 480 min;(3)The subsequent blood transfusion volume and NS resuscitation volume in PLH group in prehospital phase were(16.0±2.2)ml,(39.0±5.5)ml respectively,while those in hospital phase were(28.0±6.7)ml,(90.0±7.1)ml respectively,which were significantly lower than those in PNL group(P＜0.05);(4)The 24 and 72 hours survival rate in the animals assigned to PLH group were 100%,90% respectively;which were significantly higher than those in PNL group(P＜0.01).Conclusion Limited volume resuscitation improves thermodynamic changes of pregnant rabbit,attenuates the increase of serum concentration of TNF-α,IL-6,and results in higher survival rate.Limited volume resuscitation is an ideal means for hemorrhagic shock resuscitation in pregnant rabbit.

Regulation of P38 and MKK6 kinase on heat-induced Raw264.7 cell apoptosis / 基础医学与临床

Objective To elucidate the role of P38 signaling pathway on heat-induced apoptosis in monocytic cell line Raw264.7.Methods Raw cells were transfected with constitutively active mutant MKK6b(E)and dominant negative mutant P38(AF),or the empty cloning vector pcDNA3 and apoptosis was detected by flow cytometric analysis.Results The ectopic expression of P38 mutant was confirmed by immunostaining with the antibody against the Flag-epitope tag.Expression of MKK6b(E)led to a marked increase in P38 kinase activity in transfected cells and induced a 4-fold increase in the number of apoptotic cells as compared to that in cultures of control transfected cells.Meanwhile the expression of MKK6b(E)increased the apoptotic rate of Raw cells induced by heat.In contrast,the dominant-negative mutant P38(AF)inhibited Raw cells apoptosis induced by heat.Conclusion The activation of the MKK6-P38 MAP kinase signaling pathway is required for heat-induced apoptosis in Raw264.7 cells.

The effect of microelement powder on membrane potential of vascular endothelial and smooth muscle cells of rats / 中国病理生理杂志

AIM: To study the effect of microelement powder (MP) on membrane potential of vascular endothelial and smooth muscle cells of rats in order to elucidate the mechanism of microcirculation improvement by MP. METHODS: Cultured pulmonary vascular endothelial cells (EC) and aortic smooth muscle cells (SMC) of rats and detecting the changes of cellular membrane potentials by using potential-sensitive fluorescent probe and laser jet confocal microscope. RESULTS: MP hyperpolarized SMCs significantly. Glybenclamide (2 μmol/L), a blocker of KATP channel, which had no effect on membrane potential of SMCs, reversed the hyperpolarization of MP completely; MP hyperpolarized ECs slightly, but the effect was unaffected by glybenclamide. CONCLUSION: MP hyperpolarizes SMCs by activating KATP channels and leads to dilation of microvessels and improvement of microcirculation.

The ATP-sensitive K(+) channel and membrane potential in the pathogenesis of vascular hyporeactivity in severe hemorrhagic shock / 中华创伤杂志(英文版)

OBJECTIVE: To elucidate the mechanism of vascular hyporeactivity following severe hemorrhagic shock (HS) by studying the changes of ATP-sensitive potassium channels' (K(ATP)) properties and membrane potential of mesenteric arteriolar smooth muscle cells. METHODS: Single channel currents were studied on cell-attached and inside-out patches of enzymatically isolated mesenteric arteriolar smooth muscle cells (ASMCs). Membrane potentials of arteriolar strips and ASMCs were recorded by intracellular membrane potential recording method and confocal microscopy, respectively. RESULTS: K(ATP) channels in ASMCs were activated, which induced smooth muscle hyperpolarization following vascular hyporeactivity in HS. CONCLUSIONS: Hyperpolarizing effect of K(ATP) channel activation plays an important role in low vasoreactivity during severe hemorrhagic shock.

Rho kinase inhibitor Y-27632 prevents partially burn serum-induced endothelial barrier dysfunction / 中国病理生理杂志

AIM: To investigate whether small GTPase RhoA's downstream effector Rho kinase mediates burn serum-induced endothelial hyperpermeability. METHODS: Primary cultured rat dermal microvascular endothelial cells (DMECs) were exposed to serum isolated from burned or sham burn rats for 6 hours and 8 hours, respectively, and did or didn't pretreated or post-treated with Y-27632 (30 ?mol/L), a specific inhibitor of Rho kinase. ECs were then prepared for routine scanning electron microscopy observation, or stained with rhodamine-phalloidin for F-actin visualization. Permeability to FITC-albumin was evaluated using EC monolayers. RESULTS: Stimulation with 15% burn serum for 6 h changed the ultrastructure on cellular surface of DMECs with appearance of ripple marks instead of microvillus. The small protuberances at cellular lateral were shorten and the gaps were seen between adjacent cells. Post-treatment of Y-27632 reversed the changes of ultrastructure on the cellular surface. Burn serum induced a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity of the peripheral filament bands and formation of the long and thick stress fibers, lamellipodia and filopodia. The stress fibers were diminished by pretreatment or post-treatment of Y-27632. But lamellipodia and filopodia were not influenced by pretreatment or post-treatment of Y-27632. Pre-treatment of Y-27632 also attenuated significantly the increase in EC monolayer permeability stimulated by burn serum for 6 h. However, post-treatment of Y-27632 could not attenuated burn serum-induced endothelial hyperpermeability response although their Pa values were lower than simple burn serum group's. CONCLUSION: These findings indicate that Rho kinase is involved in the mediation of burn serum-induced endothelial actin cytoskeleton reorganization and early stage of barrier dysfunction. [

Relationship between nitric oxide and prognosis of rats with severe burns / 中国病理生理杂志

AIM: To investigate the variation of nitric oxide(NO) and NO synthase(NOS) in rats during the early stage of severe burn and their possible relation with prognosis of severe burns.METHODS: Levels of NO - 2/NO - 3, the metabolic products of NO, nNOS and iNOS protein in brain, lung and duodenum of rats were measured before and after burns. Survival times of rats in each group were also measured.RESULTS: Levels of NO - 2/NO - 3 in rats after burn increased remarkably, selective inducible NOS( iNOS) inhibitor aminoguanidine (AG), and nonselective NOS inhibitor L-NAME can inhibit this increasing. Levels of neuronal NOS(nNOS) protein in normal rats were low, and iNOS could not be detected. Levels of nNOS protein increased mildly in all observed tissues and the levels of iNOS protein increased remarkably after burn. Administration of L-NAME and AG made the increase of nNOS more apparently but could not affect the level of iNOS. Survival time of rats decreased in L-NAME group and increased in AG group compared to control group.CONCLUSION: Symptoms such as vascular ralaxation and hypotension in burn shock are connected mainly with over-increased iNOS. [

Change of ICAM-1 expression in intestine tissue of mice induced by LPS and role of p38 MAPK in its expression / 中国病理生理杂志

AIM: To study the change of intercellular adhesion molecule-1(ICAM-1) expression in intestine tissues of mice induced by LPS and regulatory effect of p38 mitogen-activated protein kinase (p38 MAPK) on ICAM-1 expression. METHODS: Protein and mRNA of ICAM-1 were measured using Western blotting and RT-PCR respectively in intestine tissue of BALB/c mice treated by lipopolysaccharide(LPS) or LPS plus SB203580, a specific inhibitor of p38 MAPK. RESULTS: Compared with control group, the expression of ICAM-1 protein and mRNA was increased significantly by LPS stimulation in dose- and time-dependent manner. ICAM-1 expression reached peak value at 12-36 h after LPS stimulation. 20.0 mg/kg of LPS could induce the maximum of ICAM-1 expression. Pretreatment of mice with SB203580 for 30 min could inhibit significantly LPS-induced expression of ICAM-1 protein and mRNA expression in mouse intestine tissues. CONCLUSIONS: These data highlight that LPS could up-regulate ICAM-1 protein and mRNA expression in intestine tissue of mice in dose- and time-dependent manner, and p38 MAPK signal pathway plays an important role in ICAM-1 expression induced by LPS. It suggests that inhibition of p38 MAPK might be a useful principle for the prevention and treatment of intestine damage of endotoxic shock.

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells / 中国病理生理杂志

AIM and METHODS: To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION: The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression

Signal transduction pathway and blocking strategies of NF-?B / 中国病理生理杂志

NF-?B is thought of as a genetic switch to control expressions of many target genes and directly participates in pathogenesis of infection, inflammation, stress, immunoresponse, cellular apoptosis, toxic shock and tumor as well as cell-cycle regulation and cell differentiation. The overactivation of NF-?B is intimately involved in many human diseases. Various therapeutic strategies against NF-?B, to date, include anti-inflammatory drugs, antioxidants, immunosuppressive agents, inhibitors of protease and proteasome, prostaglandings, nitric oxide, IL-10, microbial products, synthetic inhibitors, antisense oligonucleotides and decoy deoxyoligonucleotides. Studies are underway to develop NF-?B member-specific and cell type-specific drugs that can inhibit the activation of NF-?B only in target cells and that may become a novel way to treat the human diseases.

Burn rat serum suppresses transient outward potassium current (I_(to)) / 中国病理生理杂志

AIM: To study I_ to channel function in severe burn and its contribution to cardiac dysfunction induced by severe burn. METHODS: CHO-K1 cells were transfected with human Kv4.3, the major subunit of human I_ to channel. The expressed Kv4.3 channels were recorded by whole-cell patch clamp and the effects of rat burn serum on Kv4.3 current densities and kinetics were observed. RESULTS: Kv4.3 channels expressed in CHO-K1 cells were endowed with the characterization of fast activation and inactivation, which was quite similar to that of native I_ to channels in cardiomyocytes. Rat's burn serum at the concentration of 2% decreased the current density significantly. At +40 mV, the current density in control group was (67.6?15.1) pA/pF, in contrast to (32.3?9.7)pA/pF in burn serum-treated group (P

Effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured endothelial cells / 中国病理生理杂志

AIM: This experiment is to investigate the effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured human umbilical endothelial cells.METHODS: The human umbilical vein endothelial cell lines ECV-304 were incubated with LPS at different concentrations for 30 min. VE-cadherin was detected by immunofluorescence with primary mAb of VE-cadherin and FITC-conjugated secondary antibody. F-actin was detected with fluorescence staining with rodamine-phalloidin. RESULTS: At high concentration, LPS could induce reorganization of VE-cadherin with the formation of serrata cellular border and enlargement of intercellular gaps, which were apparently different from that in normal conditions with the high fluorescence intensity at cell-cell junction. F-actin depolymerization could also be induced by LPS at high concentration with the formation of stress fiber and filopodia. CONCLUSION: LPS(300 ?g/L) could induce reorganization of VE-cadherin and F-actin in human umbilical vein endothelial cells.

Construction and expression of red fluorescent protein vectors containing different regions of human eNOS promoter / 中国病理生理杂志

AIM: To construct the plasmid vectors containing different regions of human eNOS promoter coupled to a red fluorescent protein reporter gene, which may express in mammalian cells. METHODS: Different regions of human eNOS promoter were subcloned respectively into a red fluorescent protein vector, pDsRed1-1. These recombinant vectors, pDsF1033Red, pDsF494Red and pDsF166Red, were then transfected into NIH3T3 cell lines, followed by the observation under a fluorescent microscope. RESULTS: After identified to be right by double restriction enzyme digestion, PCR and sequencing, the vectors might be effectively expressed in NIH3T3 cells. 95 % of the red fluorescent emitted by a red fluorescent protein dispersed all over the cells, appearing at 48-60 h after transfection, reaching peak at 96-144 h, becoming the strongest in light at 144 h, gradually disappearing after 168 h and remaining little red fluorescent in 21 days. The quantity and intensity in expressions of red fluorescent protein drived by different regions of human eNOS promoter were clearly lower than by a strong promoter, p CMVIE . CONCLUSION: The red fluorescent protein reporter gene vectors containing different regions of human eNOS promoter are successfully constructed and may efficaciously express in mammalian cells, appearing not strong transcriptional activities, which provide practical and feasible tools to study functions of different regions of human eNOS promoter and roles of cis-elements in it. [

Effects of nitric oxide inhibitors on NO mRNA expression and burn shock in rats with thermal injury / 中国病理生理杂志

AIM: To identify the effects of nitric oxide synthase (NOS) inhibitor on NO production, expression of NOS and mean artery pressure (MAP) in rats with severe burns. METHODS: After administration of non-selective NOS inhibitor, L-NAME, and selective inducible NOS (iNOS) inhibitor, aminoguanidine (AG), to rats with severe burns, levels of NO - 2/NO - 3 in blood, mRNA expression of nerve NOS (nNOS) in lung and duodenum, MAP in each group were calculated. RESULTS: Levels of NO - 2/NO - 3 in blood of rats increased significantly post burn, which could be inhibited by L-NAME and AG, especially by L-NAME. Expression of nNOS mRNA in lung and duodenum of rats increased post burn, which could be enhanced by AG and L-NAME. MAP of rats decreased gradually post burn and administration of AG could slow down this process significantly. CONCLUSION: cNOS and iNOS could play different roles in the pathophysiology of burn shock. Over-expression of iNOS could be closely related to the pathogenesis of burn shock.

The regulation of polydatin on [Ca~(2+)]i of myocardial cells in the rat / 中国病理生理杂志

AIM: To elucidate the mechanism of polydatin(PD) in increasing the contractility of myocardial cells by observing the cytosolic free calcium concentration ([Ca 2+ ]i) of myocardial cells of rats. METHODS: The cells were labelled with fluo-3-AM, and [Ca 2+ ]i was determined by use of confocal microscopy (ACAS 570). RESULTS: In the study, we found that [Ca 2+ ]i of myocytes was elevated 10min after adding PD (0.6 mmol/L), but [Ca 2+ ]i of some cells increased to 111.80%?2.22% vs baseline, the others to 224.00%?24.33%. The effect of PD was inhibited remarkably by pretreated with EGTA(2mmol/L), verapamil (50 ?mol/L), a kind of L-calcium channel antagnist, and tetrodotoxin ( 1 ?mol/L), a kind of sodium channel blocker 10 min before PD, the fluorescence value were decreased to 53.00%?9.02% , 52.00%?7.07% and 72.67%?12.70% respectively vs baseline (P

The effect of intercellular adhesion molecule-1 monoclonal antibody on microcirculation of burn shock in rats / 中国病理生理杂志

AIM: To detect the effect of intercellular adhesion molecule-1(ICAM-1) monoclonal antibody on microcirculation disorder in burn shock of Wistar rats. METHODS: The blood flow velocity and diameter of venule were measured with RBC tracking correlator and IV550 model video microscaler in burn shock models of rats. The number of leukocytes adhered on venule wall was calculated under microscope. The animal survival time was observed. RESULTS: ICAM-1monoclonal antibody could attenuate the falling of mean arterial pressure, significantly reduce the number of leukocytes adhered on venule wall, and obviously prolong the animal mean survival time, but less than 24h. CONCLUSION: ICAM-1 monoclonal antibody can decrease the number of adhered leukocytes to endothelial cells, attenuate the tether of leukocytes to venule and improve microcirculation and protect tissue cells in burn shock of rats. However, a comprehensive therapy should be taken in severe shock.

Carbon monoxide, another gaseous messenger of the body / 中国病理生理杂志

Endogenous carbon monoxide (CO) is proposed to be an intracellular signaling molecule. As a new gaseous messenger, CO shares with nitric oxide (NO) the ability of modulating neuroendocrine function and inducing vasorelaxation, mainly by stimulating soluble guanylate cyclase and regulating the level of cGMP. This review emphasized the endogenous source of CO and the interaction of CO- and NO-generating systems. It also discussed the biological effects of CO and the underlying mechanisms.

Hypertonic NaCl-NaAc improves the microcirculation in rats subjected to hemorrhagic shock / 中国病理生理杂志

AIM: To study the effects of hypertonic NaCl-NaAc on microcirculation in hemorrhage-shocked rats.METHODS: SD rats were randomized into three groups of 7 5% NaCl(hypertonic saline, HS),5% NaCl-3 5% NaAc(hypertonic sodium acetate, HSA) and 0 9% NaCl(normal saline, NS) 4 mL/kg HS,HSA or NS was given intravenously following hemorrhagic shock The microcirculation of spinotrapezius muscle was observed RESULTS: HS increased mean aortic pressure more significant than HSA Variables including arteriolar and venular diameter,velocity and volumetric flow rate and open capillaries were increased and erythrocyte aggregation was decreased in 5 min after resuscitation with both HS and HSA solutions 5 min later,variables were deteriorated in HS group After local treatment, arteriolar and venular diameters were dilated significantly in HSA group CONCLUSION:HSA had superior effects to HS in improving microcirculation of hemorrhage-shocked rats