Cyanidin-3-O-glucoside Protective Effect On LPS-induced Endothelial Cells As Well As Therapeutic Action Of Acute Lung Injury

Acute respiratory distress syndrome (ARDS) is the more severe form of acute lung injury (ALI). ARDS is a complex devastating clinical syndrome characterized by increased production of pro-inflammatory cytokines, neutrophil accumulation, disruption of pulmonary endothelial and epithelial cell capillary barriers, and leakage of proteins into alveolar space. Diverse predisposing factors participate in ARDS development Such factors include sepsis, shock, and pneumonia Pathogenesis of ARDS/ALI may be related to a variety of factors, such as inflammatory mediators, products of oxidative stress, endothelial and alveolar epithelial injury. Inflammatory mediators and cytokines leading to the increasing of pulmonary microvascular permeability, causing lung compliance decreased lung surface tension increases, the alveoli collapse, reducing the effective lung ventilation area, resulting in V/Q serious imbalance, thereby forming progressive hypoxemia. Recent studies have found that timely and effective anti-inflammatory, antioxidant and other measures to improve the capacity of the lung tissue oxygenation, to improve lung function, correct hypoxemia has important significance.As a cell wall component of gram-negative bacteria, lipopolysaccharide (LPS) is an important pathogen leading to ARDS. Experimental administration of LPS has also been used to induce ARDS in animal models. LPS can bind to the cell membrane receptor of the monocytes/macrophages, which play an important role in the development of most ARDS cases, increase endothelial permeability and tissue damage, enter the bloodstream, and elicit inflammatory response with excessive production of inflammatory cytokines and edema in the lungs.Activation of novel therapeutic targets, namely, nuclear factor-kappa B (NF-?B) and mitogen-activated protein kinase (MAPK), is an important step in ARDS development. Upon exposure to LPS, NF-?B will be activated and release numerous cytokines, including tumor necrosis factor-a (TNF-a), interleukin (IL)-1?, and IL-6, as well as reactive oxygen species (ROS), which play a critical role in ARDS. thus, MAPK pathways, such as p38 MAPK, c-Jun NH2-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), also participate in ARDS developmentCyanidin-3-O-glucoside (C3G), an anthocyanin belonging to the flavonoid family, commonly present in food and vegetables in human diet, such as blueberries and purple sweet potato. Previous studies found that cyanidin-3-O-glucoside enables the cell membrane with anti-lipid peroxidation and oxygen free radical scavenging effect, and can be related to the role of endothelial cells by inflammation and oxidative stress cell signaling pathways, thus prevent the progression of endothelial dysfunction.Our main design assessed the protection afforded by C3G in an ARDS model in vivo, as well as in human umbilical vein endothelial cells (HUVECs) in vitro, to further investigate the possible protective mechanisms related to the suppression of NF-?B and MAPK pathways.This study aimed to investigate the protective ability of C3G against inflammatory and oxidative injuries, as well as to clarify the possible mechanism in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) in vitro and acute respiratory distress syndrome mouse model in vivo. Provides a new way of thinking and approach to the prevention and treatment of acute respiratory distress syndrome.METHODSIn vitro studyHUVEC Cells were sub-cultured at confluence and used between the third and eighth passages for all experiments. All the endothelial cells were incubated with or without various concentrations of C3G, which were always added 1 h prior to LPS (1 ?g/mL) stimulation. Control cells were not exposed to LPS. TNF-?, IL-1?, and IL-6 concentrations in the culture supernatant of HUVECs were measured using the ELISAkits.In vivo studyAdult male Kunming mice (age 8 to 10 weeks, weighing 18 g to 20 g). Experimental animals were randomly allocated into three groups, namely, the control group, LPS group, and C3G+LPS group. C3G (12.5 mg/kg,25mg/kg,50mg/kg) was intraperitoneally injected to animals. After 1 h, the LPS and C3G+LPS groups were intraperitoneally injected with LPS (20 mg/kg) to induce lung injury. At 6 h after the mice were induced with LPS, Measuring bronchoalveolar lavage fluid (BALF) cell count (total cells, macrophages, neutrophils), enzyme-linked immunosorbent assay measured the levels of inflammatory cytokines (TNF-a, IL-6 and IL-1?), And myeloperoxidase (MPO), superoxide dismutase (SOD) and malondialdehyde (MDA) levels in lung tissue homogenates. Measurement of lung wet/dry ratio, HE staining of lung tissue. Using relevant Western blot technology to detect LPS induced ARDS activation status of key regulatory protein (p65/p-p65, I?B/p-I?B, MEK/p-MEK, JNK/p-INK, P38/p-P38, ERK /p-ERK) level of NF-?B and MAPK signaling pathways.RESULTSHUVECs or male Kunming mice were pretreated with C3G 1 h before LPS stimulation. C3G significantly inhibited the production of pro-inflammatory cytokines (TNF-a, IL-6, and IL-1?) in cell supematants and bronchoalveolar lavage fluid (BALF) as determined by ELISA. Histopathologic examination with hematoxylin and eosinstaining showed that C3G pretreatment substantially suppressed inflammatory cell infiltration, alveolar wall thickening, and interstitial edemain lung tissues. C3G markedly prevented LPS-induced elevation of malondialdehyde and myeloperoxidase levels in lung tissue homogenates, wet/dry ratio of lung tissues, total cells, and inflammatory cells (neutrophils and macrophages) in BALF. Moreover, C3G reduced superoxide dismutase activity in the lung tissue homogenates. Western blot assay also showed that C3G pretreatment significantly suppressed LPS-induced activation of nuclear factor-kappaB (NF-?B) and mitogen-activated protein kinase (MAPK) signaling pathways by blocking the phosphorylation of I?B?, NF-?B/P65, ERK, p38, and JNK in the lung tissues. In summary, C3G may ameliorate LPS-induced injury, which results from inflammation and oxidation, by inhibiting NF-?B and MAPK pathways and playing important anti-inflammatory and anti-oxidative roles.