Huang Ys

Serial experimental and clinical studies on the pathogenesis of multiple organ dysfunction syndrome (MODS) in severe burns

These serial clinical and experimental studies were designed to clarify the pathogenesis of postburn MODS. Both animal and clinical studies were performed. In animal experiments, 46 male cross-bred dogs were cannulated with Swan-Ganz catheters and 39 of them were inflicted with 50% TBSA third degree burns (7 were used as controls). The burned dogs were randomly divided into 4 groups: immediate infusion, delayed infusion, delayed fast infusion and delayed fast infusion combined with ginsenosides. All dogs were kept under constant barbiturate sedation during the whole study period. Hemodynamics, visceral MDA, mitochondrial respiratory control rate (RCR) and ADP/O ratio, ATP, succinic dehydrogenase (SDH), organ water content as well as light and electron microscopy of visceral tissues were determined. In the clinical study, 61 patients with extensive deep burns were chosen, of which 16 sustained MODS. Plasma TXB2/6-keto-PGF1alpha ratio, TNF, SOD, MDA, circulatory platelet aggregate ratio (CPAR), PGE2, interleukin-1, total organ water content and pathological observations of visceral tissues from patients who died of MODS were carried out. Results demonstrated that ischemic-reperfusion damage due to severe shock, sepsis and inhalation injury are three main causes of postburn death. All inflammatory mediators increased markedly in both animals and patients who sustained organ damage or MODS. SDH, RCR, ADP/O and ATP decreased significantly. These findings suggested that ischemic damage and systemic inflammatory response syndrome (SIRS) initiated by mediators or cytokines might be important in the pathogenesis of postburn MODS.

Improvement of early postburn cardiac function by use of Panax notoginseng and immediate total eschar excision in one operation

Cardiac dysfunction development in the early stage postburn has been an important problem in burn treatment. However, no effective therapies are available for use in clinical practice. In this study, we sought to determine whether early total eschar excision (EEE) in one operation and the traditional Chinese herb Panax notoginseng (PNS) would be helpful in improving early postburn cardiac function. 160 Wistar rats were randomly divided into burn (burn group, n = 50), burn treated with EEE (EEE group, n = 50), burn treated with PNS (PNS group, n = 50) groups and normal controls (n = 10). All rats except the normal control were given a 30% TBSA full skin thickness burn and resuscitated with Ringers lactate. EEE was performed immediately after the burn group received the first intraperitoneal injection of Ringers lactate. The wound was covered with homoskin from normal rats. In the PNS group, two doses of PNS (200 mg/kg for each dose) were given intraperitoneally immediately and 4 h postburn. Cardiac contractile function and cardiac troponin T (TnT) were determined at 1, 3, 6, 12 and 24 h postburn. Results showed that cardiac contractile parameters including AOSP, AODP, LVSP and +dp/dt(max) all declined and were still significantly lower than the control values at 24 h postburn. Cardiac TnT was elevated markedly and reached a level 25 times higher than control at 12 h postburn. In EEE and PNS groups, the reduction of cardiac contractile function was limited as compared with that in the burn group. Levels of TnT in both EEE and PNS groups were significantly lower than in the burn group 6 h postburn later. The findings of this study demonstrated that both EEE and PNS were effective in improving early postburn cardiac function.

The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

In both cardiomyocytes and HeLa cells, hypoxia (1% O2) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.

Roles of ischemia and hypoxia and the molecular pathogenesis of post-burn cardiac shock.

OBJECTIVE To evaluate the roles of ischemia and hypoxia in the development of post-burn cardiac shock and its molecular pathogenesis. METHODS One hundred and fifty healthy adult Wistar rats were divided into the control group and burn group inflicted with 30% total body surface area third degree burn. Groups were processed at 1, 3, 6, 12 and 24h post-burn. Myocardial contractile function, myocardial microvascular permeability, volume of regional myocardial blood flow, levels of myocardial myosin light chain 1 (CM-LC1), myocardial NF-kappaB (nuclear factor kappa B) activity, MPO (myeloperoxidase), TNFalpha (tumor necrosis factor alpha) mRNA expression and levels of myocardial TNFalpha were measured. MAIN RESULTS Myocardial microvascular permeability began to rise at 1h post-burn and was still rising at 24h (2.1 times as high as that of the control group); the volume of regional myocardial blood flow fell significantly and remained at a level markedly lower than that in the control group; CM-LC1 also rose significantly and reached a level 18.6 times as high as that in the control group; myocardial NF-kappaB activity and TNFalpha mRNA expression were significantly promoted; elevation of levels of myocardial TNFalpha and MPO activity occurred; cardiac mechanic parameters including LVSP, +/-dp/dt max significantly decreased while LVEDP increased. CONCLUSION The findings of the present study suggest severe myocardial damage due to ischemia and hypoxia following burns; promotion of myocardial NF-kappaB activity and TNFalpha mRNA expression in myocardium may be an important factor in the development of post-burn cardiac shock.

Prospective clinical and experimental studies on the cardioprotective effect of ulinastatin following severe burns

OBJECTIVE To investigate the preventive effect of ulinastatin on shock in the heart after burn. METHODS In an open prospective clinical study 34 adults with burns >50% total body surface area were randomly divided into control (B) and ulinastatin-treated (U) groups. All underwent routine treatment, and group U received 100,000U ulinastatin intravenously three times a day for 1 week. In an animal experiment, 72 healthy rats underwent equivalent burn, similar division into groups B and U, and resuscitation according to Parklands formula. Rats in group U received ulinastatin (40,000U/kg) immediately after burn. Myocardial pathomorphology, plasma cTnI, CK-MB and PMNE, myocardial MDA, TNF-alpha, IL-10 and caspase-3 activity and cardiocyte apoptosis were determined. RESULTS Plasma cTnI, CK-MB, and PMNE were higher in clinical group B than group U. In the animal experiment, plasma cTnI, CK-MB, myocardial MDA, TNF-alpha, IL-10 and caspase-3 activity, and apoptotic index and myocardial pathomorphological changes were significantly less in group U than in group B, save IL-10. CONCLUSION The clinical and experimental data showed that ulinastatin relieved myocardial damage from severe burn. The mechanism might involve modulation of the anti- and pro-inflammatory balance and lipid peroxidation, and decreased myocardiocyte apoptosis.

Calcium induced the damage of myocardial mitochondrial respiratory function in the early stage after severe burns.

OBJECTIVE To explore the role of Ca(2+) in the damage to myocardial mitochondrial respiratory function in the early stage after severe burns. METHODS An experimental model of 30%TBSA full-thickness skin scalding was reproduced in rats. Myocardial mitochondria were isolated from control and burned rats in the 1st, 3rd, 6th, 12th and 24th hour post-burn. The mitochondrial respiratory function, contents of mitochondrial calcium ([Ca(2+)](m)), activities of mtPLA(2), mtNOS, F(0)F(1)-ATPase and cytochrome c oxidase were determined. RESULTS (1) At the 1st hour post-burn, [Ca(2+)](m) was increased significantly and the myocardial mitochondrial respiratory function was significantly reinforced. At the same time, mitochondrial respiratory control rate (RCR) was elevated and positively correlated with [Ca(2+)](m) (r=0.8415, P<0.01). At the 3rd, 6th, 12th and 24th hour post-burn, [Ca(2+)](m) increased further to a higher level, however, the mitochondrial respiratory function was decreased from the peak value at 6h, and RCR was negatively correlated with [Ca(2+)](m). (2) The activities of mtNOS and mtPLA(2) were higher significantly at the 3rd, 6th, 12th and 24th hour post-burn than that of the control. After severe burns, mtNOS and mtPLA(2) activities were both positively correlated with [Ca(2+)](m) (r=0.8945, P<0.05; r=0.9271, P<0.01, respectively). (3) The F(0)F(1)-ATPase synthetic activity increased at the 1st hour post-burn, but it decreased to 51.4, 44.9, 77.6 and 87.4% of that of the control at the 3rd, 6th, 12th and 24th hour post-burn respectively. The F(0)F(1)-ATPase hydrolytic activity decreased at the 1st hour post-burn and increased at the 3rd, however, it decreased again at the 6th, 12th and 24th hour post-burn. The activity of cytochrome c oxidase at the 3rd, 6th, 12th and 24th hour was low compared to the control. CONCLUSIONS The changes of [Ca(2+)](m) were involved in damage to or regulation of mitochondrial respiratory function after severe burns. Appropriate increase of [Ca(2+)](m) reinforced the mitochondrial respiration at 1st hour after of burn injury, but Ca(2+) severe overload impairing F(0)F(1)-ATPase and cytochrome c oxidase directly, or, indirectly by activation of mtPLA(2) and mtNOS, might play an important role in damage to myocardial mitochondrial respiratory function at later stages after severe burns.

Activation of the prolyl‐hydroxylase oxygen‐sensing signal cascade leads to AMPK activation in cardiomyocytes

The proline hydroxylase domain‐containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP‐activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2‐specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2‐RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK‐α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin‐dependent kinase kinase (CaMKK) with STO‐609, or RNAi‐mediated down‐regulation of CaMKK α inhibited PHI‐induced AMPK activation significantly. In contrast, down‐regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI‐induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca2+/CaMKK‐dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.

HSP70 protects intestinal epithelial cells from hypoxia/reoxygenation injury via a mechanism that involves the mitochondrial pathways

Though recent studies have reported the importance of several endogenous cytoprotective factors including heat shock protein 70 (HSP70) that protect intestinal epithelial cells (IECs) from the effects of stress and injury, the exact mechanism of HSP70 underlying cytoprotection against hypoxia/reoxygenation induced IEC injury remains unclear. The present study was designed to investigate the possible mechanisms by which HSP70 protected IECs against hypoxia/reoxygenation injury and focused on the effects of HSP70 on IEC apoptosis induced by hypoxia/reoxygenation injury. Recombinant adenoviruses (Ad-HSP70) were transfected into the intestinal epithelial cell line in vitro and then suffered from 90 min of hypoxia followed by 60 min of reoxygenation. The LDH leaking, apoptosis, and mitochondrial membrane potential (Psi(m)) were evaluated after hypoxia/reoxygenation. The expression of HSP70, cytochrome c and Bcl-2 protein was determined by Western blot or immunofluorescence analysis. The results show that HSP70 protein was highly expressed in the IECs at 48h following Ad-HSP70 transfection. HSP70 overexpression could reduce LDH leakage and cell apoptosis in IECs following hypoxia/reoxygenation injury. Furthermore, the overexpression of HSP70 significantly reversed the decrease of mitochondrial membrane potential and the release of mitochondrial cytochrome c in IECs during hypoxia/reoxygenation. HSP70 overexpression was also associated with the increasing expression of Bcl-2 protein in IECs during hypoxia/reoxygenation. We conclude that HSP70 protects IECs against hypoxia/reoxygenation induced apoptosis through increasing Bcl-2 expression, which in turn could inhibit the mitochondria-related apoptotic pathway that involves the disruption of the Psi(m) and release of cytochrome c from mitochondria.

Inhalation injury in southwest China—The evolution of care

AIM This study aims to review the changes in management of inhalation injury and the associated reduction in mortality over the past 2 decades. METHODS The records of burn patients with inhalation injury hospitalised in our institute from 1986 to 2005 were retrospectively analysed. The incidence of inhalation injury and the associated mortality were analysed. Meanwhile, the relationship of inhalation injury with age, total burn area, tracheostomy intubation and mechanical ventilation were studied. RESULTS The incidence of inhalation injury was 8.01% in the total 10 608 hospitalised burn patients during the 20 years surveyed. Inhalation injury was always associated with large-sized burn and was more common in adults. The incidence of tracheostomy and mechanical ventilation increased from 39.46 and 30.28% in the period from 1986 to 1995 to 70.12 and 39.74% from 1996 to 2005, respectively. The overall mortality of inhalation-injured burn patients was 15.88% compared with 0.82% of the non-inhalation group. The mortality of the burn patients with inhalation injury dropped from 25.29% during the first 10 years to 11.71% during the second decade (p<0.01). Mortality secondary to inhalation injury as the lead cause decreased from 14.56 to 6.29% (p<0.01). CONCLUSION The care of inhalation injury has made significant progress over the past 2 decades. The early diagnosis of inhalation injury, early airway control and pulmonary function assistance with mechanical ventilation contribute to the reduction of mortality.

MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1

Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O2) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization.