Hydrogen-rich Lactated Ringer's Solution Attenuates Lung Injury On Post-burn Delayed Resuscitation

BackgroundBurns mainly refers to the damage of skin, mucous membranes and even deep tissue caused by heat, chemicals, electricity, radiation, etc. An extensive burn injury, as a serious trauma makes the body suffer a heavy strike, is extremely complex pathophysiological process, which not only leads to local damage of the burned area, but also induces distant visceral organ injury away from burn, leading to a series of disorders of various systems and organs in the structure and function. Thus delayed resuscitation after burn injuries is the secondary hit to the body, often resulting in multiple organ failure, in which the lung is the most apt to be affected. Previous data indicated that an extensive burned patients with delayed resuscitation-induced acute lung injury (ALI) is considered relevance with the high mortality rate of burns. Recent studies have confirmed that a large number of oxygen radicals and pro-inflammatory cytokine-induced systemic inflammatory play a key role in the pathogenesis of ALI induced by extensive burn with delayed resuscitation. The damage of vessel endothelial barrier composed of endothelial cells plays a critical role in the process of ALI formation. Recent reports showed that hydrogen molecule could selectively combine and eliminate hydroxyl radical ('OH), the most toxic one of ROS, and showed anti-inflammatory and anti-apoptotic effect in some disease models. The purpose of this study was to observe the effects of Hydrogen-rich Lactated Ringer's solution (HRS) on acute lung injury caused by extensive burns with delay resuscitation, and to provide a more effective method for the treatment of burns. The study consists of three parts. Part OneThe Anti-oxidative and Anti-inflammatory Effects of Hydrogen-rich Lactated Ringer's Solution on Lung of Rats with Post-burns Delayed ResuscitationObjective:To investigate the anti-oxidative and anti-inflammatory effects of HRS on the lung tissue of rats in the delayed resuscitation in severe burns.Methods:After preparation of HRS, according to random number table,36SD rats were randomly divided into3groups (n=12):sham burns group (S), burns plus normal Lactated Ringer's solution (NRS) treatment group (BR) and burns plus HRS treatment group (BH). The group S rats underwent a mimic process of burn injury using37℃water bath without fluid replacement. The group BR and group BH rats were subjected to30%total body surface area (TBSA) full-thickness scald. NRS and HRS were administered1/2,1/4, and1/4of the total calculated resuscitation volume according to Parkland formula at7hours,9hours and17hours post-burn respectively. All rats were administered with1%Even's blue via sublingual vein and measured blood pressure30minutes before they were sacrificed. Arterial blood was collected for blood gas analysis. Left lung was fixed for lung vascular permeability measurement and HE staining which was double-blind scored, Right lung was collected for detecting the oxidation index (including MDA levels and SOD activity), and the content of proinflammatory cytokines IL-1β and TNF-a.Results:Blood pressure in group BR was lower than that in group S and group BH (P<0.05), thus the blood pressure in the BH group was significantly higher than it in group BR and close to it in the group S. Blood gas analysis showed that PaO2in group BR was lower than that in group S. There was no significant differences between the PaO2in group BH and that in the group S, but both were higher than that in the group BR (P<0.05). Pathological scores of the group BR was higher than that in group BH (P<0.01). Evans blue concentration in the group BR in the lung was significantly increased (P<0.01vs. BH group) at24hours after injury. The lung tissue content change of MDA, SOD at24hours after the burn, and lung MDA content of the group BH is lower than those in the group BR (P<0.05); The level of lung tissue SOD in the group BH was between that in the group S and BR and was significantly higher than that in the group BR (P<0.01). IL-1B and TNF-a content in lung tissue of group BH was between that in the group S and in group BR, higher than that in the group S, but significantly lower than that in the group BR (P<0.01).Conclusion:the protective role of HRS to lung tissues in extensive burn with delayed resuscitation was showed in three aspects:(1) hydrogen reduced lung oxidative stress damage and resumed the activity of enzymatic ROS scavenger in vivo;(2) hydrogen suppressed the over-production and release of proinflammatory cytokines, IL-1B and TNF-a, and played an important role in inhibiting the amplification of inflammatory response;(3) hydrogen alleviated the pathological change and improved lung function. All these demonstrated that HRS could partly replace and resume cellular protective role of superoxide dismutase as a ROS scavenger for lung tissues, HRS is more suitable for fluid replacement than NRS in rats with post-burn delayed resuscitation.Part TwoThe Effects of Hydrogen-rich Lactated Ringer's Solution on Apoptosis at Lung of Rat at Post-burn Delayed ResuscitationObjective:To explore the inhibition effects of HRS on apoptosis of the lung tissue due to delayed resuscitation in burned rats, as well as the impact of HRS on several apoptotic protein.Methods:Paraffin sections of lung tissue made from the Part One of this study were tested by TUNEL, and apoptosis was observed in situ. Lung tissue powder from the Part One of this study were used to observe the expression changes of apoptotic protein Bcl-2, Bax and Caspase-3by western blotting.Results:The group S had few apoptotic cells, or only a small number of apoptotic cells. The number of apoptotic cells in the Group BR and the group BH increased, they were higher than that in the group S (P<0.01), and the number of apoptotic cells in the group BH is less than it in BR the group. The level of cleaved Caspases-3protein in the group S had no significant activation, the cleaved Caspases-3protein in the group BR was significantly increased, while it was higher than that in the S group, but lower than that in the group BR (P<0.05). The pro-apoptotic protein Bax expression in the group BR was significantly higher than that in the S group and group BH (P<0.01). Anti-apoptotic Bcl-2protein expression of lung tissue in the S group and group BH was significantly higher than that in the group BR(P<0.01).Conclusion:The expression of pro-apoptotic proteins, Bax and Caspase-3protein, were downregulated in delayed resuscitation of burned rats by HRS. Conversely, the expression of anti-apoptotic protein Bcl-2was upregulated by HRS. These results confirmed the consequences of apoptosis in rat lung tissue by TUNEL assay. The results show that the protective role of HRS on lung tissue in rat with delayed resuscitation post-burn through regulating of the expression of apoptotic protein and reducing apoptosis.Part Three The Effects of Hydrogen-rich Medium on Shape and Apoptosis of HUVEC Induced by Burn SerumObjective:Using human umbilical vein endothelial cells (HUVEC) model cultured in vitro, to observe the influence of hydrogen-rich medium on endothelial cell contraction and apoptosis under burn serum, to further understand the effects of the hydrogen on vascular permeability, and provide a new thought for the mode of severe burns injury treatment.Methods:After preparation of hydrogen-rich medium, human umbilical vein endothelial cells (HUVEC) cultured in6-well plates were divided into normal serum control group (C), burn serum plus normal medium group (B), burn serum plus hydrogen-rich medium group (H). The cells were incubated for8h and the cytoskeleton was stained using the rhodamine phalloidin. The apoptosis was detected by flow cytometry after Cells were incubated in same group for24h.Results:In the group C, cell filamentous actin were distributed both in the centre and periphery of cells, and they connected to each other between cells and cells, the formation of peripheral actin ribbon. Recombinant of filamentous actin of endothelial cells in group B, the F-actin microfilaments are shorter, thicker, and appeared fuzzy, focus on the central portion of cell. Cell morphology in the group H was roughly circular. The changing extent of intracellular filamentous actin in the group H was between those in group B and group H where the rate of cell apoptosis was increased induced by burn serum,. The rate of apoptosis in the group B was significantly higher than that in the group C, P<0.01. The rate of apoptosis in group H was lower than that in group B, P<0.01.Conclusion:Burn serum can lead to morphological changes and apoptosis of HUVEC in vitro. Hydrogen-rich medium can relieve contraction of HUVEC influenced by burn serum, and reduce the occurrence of HUVEC apoptosis. These results provide circumstantial evidence for the reduction of the permeability of the lung tissue in burned rats with delayed resuscitation by using HRS. For the treatment of burns, our finding provides new ideas and means for burns treatment according to the causality hypothesis that we can interfere with vascular endothelial cell barrier using HRS so as to achieve exudation and reduce fluid volume.