Carbon Monoxide Protection Of Neonatal Acute Lung Injury By Interfering With Cx43-ERK Signaling Pathway

Background and objective Acute lung injury(ALI)caused by various causes is still the most common clinical critical illness causing disability and even death of newborns.The acute respiratory distress syndrome(ARDS)which is associated with ALI can cause a lot of damage to the lung tissue structures and seriously affect the lung function.It is easy to develop into the refractory neonatal respiratory failure and death.At present,the study of neonatal ALI/ARDS is still at early stage at home and abroad,and there is a lack of large sample data in clinical research.Carbon monoxide(CO)is an important endogenous gas signal molecule,which is usually formed by the decomposition of heme by heme oxygenase.As a byproduc t of heme catabolism,it has many biological effects in vivo and in vitro,such as anti-inflammatory,anti-apoptotic,anti-oxidation,anti-proliferation,vasodilation and so on.More and more studies have found that the increase of CO level in a certain extent has a protective effect on the necrosis of a variety of cells.Previous studies of our group shown that low concentration of CO can not only reduce ALI by inhibiting the apoptosis of alveolar epithelial cells(AEC),but also reducing AEC necrosis.Unfortunately,the molecular pathway through which CO reduces AEC necrosis and protects ALI has not been fully revealed.Recently,it is believed that cell necrosis can be regulated by signaling molecules,and there are more complex signaling pathways,i.e.necroptosis,just like apoptosis.However,it is not clear what role procedural necrosis plays in the protection of ALI by CO.Further research is needed.Persistent pulmonary hypertension of newborn(PPHN)is a common serious disease in Neonatal Intensive Care Unit(NICU).It is a serious threat to the health of newborns and also leads to the death of newborns.At present,the treatment of PPHN at home and abroadis limited,so it is urgent to find new treatment methods to reduce the mortality and disability rates.CO has the biological effects of vasodilating and inhibiting the proliferation of smooth muscle cells.Studies have shown that both pulmonary vascular smooth muscle cells and endothelial cells have the expression of heme oxygenase(HO)protein whi ch is one of the important sites for the production and release of the endogenous CO.The endogenous HO/CO system may be involved with the formation of oxygen-induced pulmonary hypertension in low levels.To further study the signal transduction pathway of CO inhibiting AEC programmed necrosis,and to provide a new theoretical basis and new ideas for the development of new drugs for the prevention and treatment of neonatal respiratory critical illness by exogenous inhalation of low concentration CO.Methods 1.The animal model of ALI in neonatal rats was established by intratracheal injection of lipopolysaccharide(LPS)through endotracheal intubation.2.Carbon monoxide releasing molecule 3(CORM3)or inactive carbon monoxide releasing molecule 3(i CORM3)was intraperitoneally injected into ALI animal model of neonatal rats.Lung tissue samples were collected for histological examination,and the total number of cells and protein content in bronchoalveolar lavage fluid(balf)were determined.Detection of expression of Cx43 and necrosis-related markers were quantified real-time quantitative PCR and Western blot.3.A549 cells were used to complete cell recovery and passage.Cell transfection was used to study the protective effect of CO on LPS-induced ALI by down-regulating Cx43.4.Neonates with mechanical ventilation PPHN were randomly given inhalation of nitric oxide or CO.Clinical data were collected and related indicators were detected.Results 1.Both intratracheal instillation of LPS or intraperitoneal instillation of LPS can establish animal models of ALI in neonatal rats,manifested as lung wet/dry weight ratio(w/d)increases,and the total cell number and protein concentration in bronchoalveolar lavage fluid(BALF)increases.2.LPS-induced lung injury was significantly improved by CORM3 administration,but i CORM3 administration had no significant effect.3.CORM3 can improve the expression of Cx43 induced by LPS,and overexpression of Cx43 can weaken the protective effect of CORM3 on lung.4.LPS can increase the expression of necrosis-related markers MLKL,RIP1,RIP3 and FADD,and CORM3 can significantly inhibit the expression of these markers.5.CORM3 can attenuate the activation of extracellular signal-regulated kinases(ERK)induced by LPS,and ERK inhibitor U0126 can attenuate its protective effect on necroptosis.6.Inhalation of nitric oxide can reduce pulmonary artery pressure(n = 8,P < 0.05)and inhalation of carbon monoxide can also reduce pulmonary artery pressure to a certain extent(n = 5,P < 0.05).7.Inhalation of carbon monoxide can reduce the levels of inflammatory markers such as interleukin-1-beta,tumor necrosis factor-alpha,interleukin-8 and interleukin-6(n=5,P<0.05).Conclusion: 1.Animal models of ALI in neonatal rats can be established by intratracheal instillation of LPS or intraperitoneal injection of LPS,which provides a better animal model for the study of ALI.2.Carbon monoxide reduces LPS-induced ALI AEC necroptosis.3.Carbon monoxide reduces LPS-induced ALI AEC necroptosis by down-regulating Cx43.4.Carbon monoxide protects LPS-induced ALI by down-regulating the ERK signaling pathway activated by Cx43.5.Carbon monoxide has anti-inflammatory effect and plays a certain role in reducing pulmonary artery pressure.