3,5,4’-tri-O-acetylresveratrol Attenuates Seawater Aspiration-induced Lung Injury In Rat Models

Background and ObjectiveDrowning is a major but often neglected public health problem and about500,000people over world would die from drowning each year. The lung is the most vulnerableorgans when during near-drowning. As well demonstrated, Acute Lung Injury (ALI)resulted from seawater inhalation is much more severe than that of caused by fresh water.Pathological changes of Seawater Inhalation Induced Acute Lung Injury (SWI-ALI)dependents on both the composition and quantity of water aspirated and the mechanismsare very complicate. What confirmed is that pulmonary inflammation and lung edema arethe most frequently observed pathological damages. Seawater Inhalation Induced AcuteRespiratory Distress Syndrome (SWI-ARDS) would be the outcome of SWI-ALI If nottreated or treated inappropriately. Although， nuermous research has been done onmechanisms of SWI-ALI/ARDS and how to treat this injury, there is no solid theory orreasonable method to cure this disease. Nuclear factor (NF-κB) is a transcription factor expressed in almost all cell types,which facilitates the expression of many genes and plays a critical role in cell proliferation,differentiation, apoptosis and cancer. In most cells, NF-κB complexes are inactive,locating in the cytoplasm and binding to inhibitory proteins named IκB family. NF-kBactivators, such as LPS, TNF-α and IL-1, can cause almost complete degradation of IkBswithin minutes, and then, the freed NF-κB homodimers or heterodimers may translocateinto the nucleus and recognize κB sites on their target genes for DNA interaction andtranscriptional activation. Numerous research show that NF-κB regulates multipledownstream molecular expressions, Hypoxia Induced Factor-1α (HIF-1α) and InducedNitric Oxide Synthase (iNOS) are involved. Based on that knowledge, the expression ofNF-κB and its related signaling molecules became the research focus of this project.Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic compound which is aphytoalexin synthesized by a wide variety of plant species. t has broad biological andpharmacological functions, such as cardio-protection, liver protection, radiation protection,anti-cancer, anti-ageing, anti-oxidation and neuro-protection. Therefore, the interest of thescientific community in resveratrol has substantially increased over the last years.However, because of its poor pharmacokinetic, bio-availability properties and shorthalf-life (8-14min), resveratrol cannot be used as an injury protective drug.3,5,4′-tri-O-acetylresveratrol, product of resveratrol with three hydroxyls replaced byacetyls, is a kind of prodrug. Evidence showed that3,5,4′-tri-O-acetylresveratrol wouldbecome resveratrol in body and exhibited an anti-γ-irradiation effect. The objective of thisresearch was to investigate the effect of3,5,4′-tri-O-acetylresveratrol on SWI-ALI/ARDSin vivo and in vitro, and explore possible mechanisms.MethodsPart Ⅰ48healthy SD rats were at random divided into6groups: A: Control group; B：Seawater drowning group; C:3,5,4′-tri-O-acetylresveratrol (50mg/kg)+Seawaterdrowning group; D:3,5,4′-tri-O-acetylresveratrol (150mg/kg)+Seawater drowning group;E:3,5,4′-tri-O-acetylresveratrol (450mg/kg)+Seawater drowning group; F: PDTC (100mg/kg)+Seawater drowning group. Rats from C, D and E groups were pretreated with 3,5,4′-tri-O-acetylresveratrol (50,150or450mg/kg body weight) for7days beforemodeling; and rats from F group were pretreated with PDTC (100mg/kg body weight)for7days before modeling. Seawater (4ml/kg) was instilled at a steady speed within4mininto both lungs. All rats were sacrificed at4h after seawater instillation. Histologicalchanges were assessed to study lung injuries; cytokines in lung samples were monitoredby ELISA to reflect inflammation; T-SOD and MDA activity were detected to examineoxidative stress in lung tissues. Besides, we also tested the expression of NF-κB, iNOSand HIF-1α to probe the possible protecting mechanism of3,5,4′-tri-O-acetylresveratrolon AWD-ALI.Part2A549cells during exponential growth phase were divided into4groups: A: Normal;B: Cells stimulated by seawater (0.25ml per1ml total volume); C: Cells pretreated byresveratrol (200μmol/L)+seawater (0.25ml per1ml total volume); and D: Cellspretreated by PDTC (200μmol/L)+seawater (0.25ml per1ml total volume). After4hstimulation Immunofluorescence was carried out in order to investigate the expression ofNF-κB, iNOS and HIF-1α in theA549cells.ResultsPart1Compared with the control, Four hours after seawater instillation induced pulmonaryedema, infiltration of inflammatory cells in the lung tissues and alveoli, and alveolardamage. The expression of TNF-α、IL-1β、MDA and NO were significantly increased(p<0.05) while T-SOD activity was decreased (p<0.05); meanwhile, seawater inhalationseverely induced the expression of NF-κB、HIF-1α and iNOS (p<0.05). On the other hand,pre-treatment of3,5,4′-tri-O-acetylresveratrol markedly reduced the expression of TNF-α、IL-1β、MDA and NO (p<0.05), more impotently, the expression of NF-κB、HIF-1α andiNOS were also significantly decreased.(p<0.05).Part2Data from Immunofluorescence showed that the expressions of NF-κB, iNOS andHIF-1α were up-regulated in A549cells4hours after seawater exposure. In addition,treatment of resveratrol and PDTC significantly inhibited the up-regulation of NF-κB, iNOS and HIF-1α induced by seawater. Besides, there was no significant differentbetween the effects of resveratrol and PDTC.ConclusionNF-κB and its related signaling molecules took part in the on step and development ofseawater induced acute lung injury, and3,5,4′-tri-O-acetylresveratrol may ameliorateSWI-ALI by interfering those signaling pathway.