Study On The Mechanism Of Vanillin's Preprotective Effect On LPS Induced Acute Lung Injury

Background:Acute lung injury(ALI)/ acute respiratory distress syndrome(ARDS)is a severe respiratory disease that causes pulmonary inflammation,destroys alveolar capillary membrane cells,and leads to severe acute respiratory failure with a high mortality rate.The latest outbreak of COVID-19 is also an acute lung injury caused by a viral infection.At present,there is no specific molecular marker in clinical practice,which provides a certain difficulty for its diagnosis and treatment.Mechanical ventilation is usually used for treatment,but it is only supportive treatment and is prone to iatrogenic complications such as barotrauma and circulatory disorders.The treatment should focus on the root cause of the disease.There is no effective drug used in clinical,for drug research of acute lung injury(ALI)has become hot spot,but the application of drugs such as corticosteroids,albuterol doesn't get the support of clinical trials,and there are some side effects,so looking for effective,economic and slight side effects drug for treatment of acute ALI/ARDS is imminent.When ARDS occurs,the lungs often exhibit a strong inflammatory response accompanied by a large amount of inflammatory cells infiltration,leading to the release of pro-inflammatory factors such as tumor necrosis factor?(TNF-?),interleukin-1?(il-1?),and IL-6.Lipopolysaccharides(LPS)are known irritants that trigger an inflammatory response in various organs,such as the intestine,nerves,and mammary glands.At present,the technique of creating airway inflammation through LPS stimulation to build ALI animal models has been widely accepted by researchers.Previous studies have shown that lipopolysaccharide stimulates a large number of inflammatory cells and neutrophils to infiltrate bilateral lungs,releasing relevant pro-inflammatory mediators and affecting a variety of inflammatory pathways.Redox homeostasis is maintained by the balance of oxidants and free radicals,especially the scavenging capacity of reactive oxygen species(ROS)and endogenous antioxidant systems.However,after exposure to harmful stimuli,the production of excessive reactive oxygen species may impair antioxidant capacity and produce toxicity to cellular components,including DNA,lipids and proteins,increasing the oxidative burden and thereby leading to oxidative stress.There is increasing evidence that oxidative stress is involved in the pathogenesis of various diseases,such as atherosclerosis,diabetes,cancer,and airway disorders.Potential antioxidant pathways can be mediated by endogenous or exogenous compounds that may be therapeutic targets to address ALI.Vanillin is a kind of natural product,widely exists in nature,often in vanilla bean,citronella,lilac bud,beetroot,sulfonate and other natural substances exist,often used in fragrances of spices,cosmetics,or food.Caraway beans have been used to obtain natural vanillin for nearly 500 years.We were surprised to find that relevant studies showed that vanillin had anti-inflammatory,antioxidant,anti-mutagenesis and anti-tumor pharmacological effects,which provided a breakthrough for us to develop clinical ALI/ARDS drugs.Therefore,vanillin was selected as the research object to explore the molecular mechanism of its pre-protective effect on acute lung injury via inflammatory response and oxidative stress.We will investigate in vivo and in vitro the role of vanillin in the regulation of ERK,p38,AKT,and NF-?B during acute lung injury and the relationship between vanillin and inflammatory factors.The oxidative damage caused by acute lung injury was simulated by bronchial epithelial cells,and the specific molecular mechanism of vanillin on oxidative factors and ROS regulation was verified,providing scientific basis for the application of vanillin in the treatment of ALI/ARDS,also providing clues to the pathogenesis and early intervention for treatment of COVID-19.Methods:1.Study on the anti-inflammatory effect of vanillin in acute lung injury(1)Establish an animal model of acute lung injury,and use HE staining to evaluate the degree of lung injury in mice;MPO was used to detect the degree of neutrophilic infiltration,and ELISA was used to evaluate the protein expression levels of inflammatory factors IL-6,IL-1?,and TNF-?.Expression of ERK,p38,AKT and NF-?B was determined by western blot.(2)In vitro experiments were conducted to verify the molecular mechanism of vanillin's pre-protective effect.CCK8 experiment selected the appropriate concentration of cell drug administration,and ELISA method was used to evaluate the protein expression levels of inflammatory cytokines IL-6 and TNF-?.Expression of ERK,p38,AKT and NF-?B was determined by western blot.2.Study on the antioxidant effect of vanillin in acute lung injury(1)In vitro,the protein expression levels of FRAP,ROS,SOD,GSH,CAT and MDA in the lung tissues of mice were detected by ELISA.Real-time fluorescence quantitative PCR was used to detect the expression levels of HO-1,GCLM,GCLC and NQO1 genes in related cell protective genes,and western blot was used to detect the expression levels of Nrf2,N-Nrf2,p-AMPK,H3K9/14 and HO-1 proteins.(2)In vivo experiments: the optimal concentration of vanillin for pre-protection of bronchial epithelial cells was selected by CCK8 method.Cellular immunofluorescence assay was used to analyze the effect of vanillin on Nrf2 nucleation and intracellular H3K9/14 acetylation.The effects of vanillin on Nrf2,N-Nrf2 and p-AMPK protein expression in bronchial epithelial cells were determined by western blot.The expression levels of related oxidases HO-1,GCLM,GCLC and NQO1 were detected by real-time fluorescence quantitative PCR.Double luciferase reporter gene was used to detect the binding of Nrf2 to ARE promoter.The expression levels of related oxidases HO-1,GCLM,GCLC and NQO1 were detected by real-time fluorescence quantitative PCR.Protein expressions of Nrf2,N-Nrf2 and p-AMPK were detected by western blot after using AMPK pathway blocker CC.The expression levels of related oxidases HO-1,GCLM,GCLC and NQO1 were detected by real-time quantitative PCR after using histone acetylation inhibitor AA.To simulate the environment of acute lung injury,CCK8 was used to measure the cell activity and select the most appropriate stimulus ratio.Protein expression levels of FRAP,ROS,SOD,GSH,CAT and MDA in mouse lung tissues were detected by ELISA after Nrf2 inhibitor was used.Expressions of Nrf2,p-AMPK and H3K9/14 proteins were detected by western blot.The expression levels of related oxidases HO-1,GCLM,GCLC and NQO1 were detected by real-time fluorescence quantitative PCR.Results:1.The mouse ALI model was successfully constructed,and vanillin could inhibit the degree of lung tissue damage induced by LPS in acute lung injury;Compared with the LPS group,the MPO level of vanillin+LPS group was significantly lower,and the protein expression levels of IL-6,IL-1? and TNF-?were also significantly lower.Vanillin up-regulated ERK,p38,AKT and NF-?B in lung tissues of mice.2.Vanillin can down-regulate the expression of inflammatory factors IL-6and TNF-? secreted by macrophages,and promote the protein expression of ERK,p38,AKT and NF-?B in macrophages.3.Compared with the LPS group,the expression levels of FRAP,SOD,GSH and CAT proteins in the lung tissues of the vanillin+LPS group were significantly increased,the expression levels of ROS and MDA were decreased,the transcription levels of the relevant cell protective genes HO-1,GCLM,GCLC and NQO1 were increased,Nrf2,N-Nrf2,p-AMPK,H3K9/14 and HO-1 proteins were increased.4.Vanillin can increase Nrf2 nuclear accumulation and H3K9/14 acetylation level in bronchial epithelial cells and increase with time.Vanillin can promote the expression of Nrf2,N-Nrf2 and p-AMPK proteins in bronchial epithelial cells,and promote the binding of Nrf2 and ARE promoters,thus leading to the transcription of downstream cell protective genes HO-1,GCLM,GCLC and NQO1.AMPK inhibitors inhibited the vanillin-induced up-regulation of p-AMPK,Nrf2 and N-Nrf2.The vanillin-induced up-regulated transcriptional levels of HO-1,GCLM,GCLC and NQO1 were inhibited after the histone acetylation inhibitor AA was used.Compared with cells in the oxidative damage group,the expression levels of FRAP,SOD,GSH and CAT proteins were significantly increased after stimulating cell injury with pre-protection of vanillin,while the expression levels of ROS and MDA were decreased.The Nrf2 inhibitor group weakened the effect of vanillin on FRAP,SOD,GSH,CAT,ROS and MDA.Compared with oxidation-damaged cells,transcriptional levels of HO-1,GCLM,GCLC and NQO1 were increased in bronchial epithelial cells after vanillin was added,and expression levels of protein HO-1 were increased in Nrf2,N-Nrf2,p-AMPK,H3K9/14.Conclusion:1.Vanillin can play a pre-protective role in acute lung injury and reduce inflammation through the regulation of ERK,p38,AKT and NF-?B to reduce the expression levels of inflammatory cytokines.2.Vanillin can enhance the total antioxidant capacity of lung tissues and cells,enhance the protein expression of related antioxidant enzymes,reduce the expression of ROS,and up-regulate the transcription levels of relevant cell protective genes so as to play their antioxidant role.3.Vanillin can activate Nrf2 by AMPK pathway,at the same time promote the combination of Nrf2 with ARE promoter,increasing transcription levels of cell protection genes,and ultimately enhance the protein expressions of antioxidant enzymes,reduce the secretion of ROS and MDA to play its antioxidant role.To sum up,our experimental results have confirmed the pre-protective effect of vanillin on acute lung injury,which can be achieved by anti-inflammatory and antioxidant functions.The interaction between inflammatory response and oxidative stress remains to be further explored.Inflammatory response and oxidative stress,as two important mechanisms of acute lung injury,may be a new direction in the treatment of ALI/ARDS,and we provide a scientific theoretical basis for vanillin as a candidate for clinical treatment of ALI.