The Protective Effect Of Caffeine Via Down-regulation Of NLRP3 Through NF-?B In BPD Lung

Objective:Bronchopulmonary dysplasia?BPD?is the most common respiratory disease in premature infants,and the pathogenesis is not yet clear.The main pathological changes of the new BPD are alveolar development arrest and pulmonary vascular maldevelopment.Researches have shown that the imbalance between damage and repair of type?alveolar epithelial cell is the key point to the development of BPD.Genetic susceptibility accompanied with a variety of environmental factors such as inflammation,oxidative stress,mechanical ventilation,preterm and other environmental factors can eventually lead to the damage of type?alveolar epithelial cell.The damaged type?alveolar epithelium appears dysregulation of proliferation/apoptosis,transformation disorders,abnormal transformation even necrosis,which seriously affect the immature lung development and ultimately lead to the occurrence of BPD.Therefore,it is of great scientific and clinical significance to explore the possible pathogenesis and clinical therapy of BPD.Inflammation plays an important role in the early development and progression of BPD.A large number of studies have shown the expression of inflammatory factors in serum and BALF?Bronchoalveolar lavage fluid?of BPD children increased significantly,including IL-1,IL-6,IL-8,IL-18 and TNF-?.The release of inflammatory factors can activate the innate immune response,produce cascade reactions,further regulate the secretion of various cytokines,chemokines and growth factors,and ultimately lead to the imbalance of damage and repair of type?alveolar epithelial cell,and eventually form BPD.The relationship between the maturation and secretion of IL-1 beta and IL-18 is closely related to the inflammatory body.Inflammasome is a complex that consist of multi protein which is assembled by cytoplasmic pattern recognition receptors?PRRs?,and take an important part in innate immunity.They can feel and recognize pathogen associated molecular patterns?PAMPs?and the danger signal molecule model or host derived?DAMPs?,then recruitment and activation of protease activated caspase-1 to produce mature IL-1?and IL-18,finally resulting in inflammation.NLRP3 is the most famous member of inflammasome,and take part in lots of chronic inflammatory disease,such as COPD,lung fibrosis and asthma.NLRP3 is activated by direct ROS exposure and can by regulated in several ways,such as transcription,post-transcription and post-translational modification.NF-?B seems to be an effective regulator of NLRP3 in the transcriptional way,supported by several researches.It has been reported that the lung development of transgenic mice with NLRP3 knockout is significantly better than that of the widetype under hyperoxia,suggesting that NLRP3 is one of the key factors that influence the development of lung.Caffeine as the methylxanthine drugs,because of better efficacy,high safety and long half-life,is previously used for prevention and treatment of apnea.while a large number of subsequent studies found that the incidence of BPD in caffeine group was significantly lower than that without caffeine.This clarified the preventive effect of caffeine in lung development,and has been applied in the clinical routine application in premature infants.Some studies also tested the protective effects of caffeine on immature lungs in the BPD animal model.However,it is not clear how caffeine protects the immature lungs.Previous studies showed that the inflammatory factors such as IL-1,IL-6 and IL-8 in the serum and BALF of the caffeine group were significantly lower than those in the control group,suggesting caffeine has a good anti-inflammatory effect.Nuclear factor kappa B?NF-?B?is an important transcription factor in inflammation,the present studies had confirmed it can promote the transcription of IL-1?,IL-6 and other inflammatory factors,and participate in the immune response,which is closely related to the occurrence of BPD.Therefore,based on the previous BPD neonatal rat model,we trying to clarify the protective effect of caffeine on BPD lung,and confirm whether caffeine can inhibit the expression and activity of NLRP3 by NF-?BMethods:newborn rats were divided into 4 groups:N group:air+NS,NC group:air+caffeine,H group:model+NS,HC group:model+caffeine,caffeine was given intraperitoneal injection of 20mg/kg per day to from the first day after birth until the 21th day of life,the N group and H group received the same volume of normal saline;animals in H and HC groups were put in a closed bin with oxygen concentration of 80-85%,while animals in control groups lived in the air with oxygen concentration of 21%.On the 14^th days,10 rats in each group were sacrificed to get lungs.The upper lobe of the right lung was used to test the ratio of wet/dry weight of lung,and the middle lobe of the right lung was fixed in 4%paramaldehyde,subjected to conventional paraffin embedding for hematoxylin and eosin?H&E?and immunohistochemistry staining.The remaining lung tissue was stored at-80?for western blot analysis and mRNA detection.After 14days of oxygen inhalation,animals in the H group and HC group were transferred to air and all the animals were killed at day 21 and collected lung tissue in the same way of day14.We calculated the RAC value and MLI from HE staining,and observe elastin deposition by Weigert staining.The growth factors TGF-?1 and PDGF level,together with the cleaved caspase-3 and PCNA were tested by Western blot in lung tissue.We analyzed the mRNA and protein level of NLRP3,caspase-1 p10,IL-1?and IL-18,also test the correlation analysis between NLRP3 protein expression and IL-1??IL-18 protein expression.The protein expression of pNF-?B and pI?B?also tested by western blot,we also did a Pearson test between the protein expression level of pNF-?B and that of NLRP3.All data were summarized by Mean+SD,two groups were compared by student's t test,and multi groups were compared by the one-way ANOVA statistical analysis using SPSS20.0 software?SPSS Inc,IL,USA?.The p value less than0.05?p<0.05?is considered statistically significant.Results:1.Ratio of wet/dry weight of lung is higher in H group than other 3 groups on both 14^th day and 21^th day,that of HC group is also higher than N and NC groups;2.Compared with H group,the RAC value in HC group is higher and the MLI value is less,but it is still significant different from that of N group and NC group;3.Normal elastic fiber is stained in N group and NC group is located in the top of the second septa in alveolar and wall of bronchus,but it is less in secondary septa in H group and most of them stain on the mesenchyme;4.The vascular density tested by CD31immunochemistry stain show a significant decrease in H group compared with other 3groups;the stain of HC group is better than H group but still lower than N group and NC group;5.The protein expression of cleaved caspase-3 is much more in H group than other3 groups,and the protein expression of PCNA is lower compared with other group,there was no different among N group,NC group and HC group;6.protein expression of growth factor TGF-?1 and PDGF were higher than other 3 groups,the protein level of TGF-?1 is still higher in HC group than N group and NC group,but there is no significance of PDGF expression among HC group,N group and NC group;7.Immunohistochemistry of NLRP3 show the cytoplasmic and perinuclear space deposition of NLRP3 in alveolar epithelium and macrophages,the expression level increased in H group than other three groups,there were no difference among HC group,N group and NC group;8.Western blot show the protein level of NLRP3,caspase-1 p10,IL-1?,IL-18 significantly increased in H group than other three groups,there was no significant difference among NLRP3 expression of HC group,N group and NC group;9.mRNA of NLRP3,IL-1?,IL-18 and caspase-1 increased in H group than other groups,the IL-1?and IL-18 mRNA levels in HC group are higher than N and NC group,also with the caspase-1 mRNA level decreased significantly with the N and NC group;10.Pearson test show the positive correlation between NLRP3 protein level and IL-1?,IL-18protein level;11.Western blot showed the protein expression level of pNF-?B in HC group is less than H group,the deposition which in cytoplasma in N group,NC group and HC group,but aberrantly nuclear expression in H group,also do the p I?B;12.Pearson correlation analysis showed that there is a positive correlation between pNF-?B protein level and NLRP3 protein level.Conclusion:1.Based on the animal model we confirm the protective effect of caffeine used early and sustainedly in BPD,which is in accordance with that in preterm infant clinically;2.Caffeine can inhibit the expression and function of NLRP3 in lung tissue,also alleviated the inflammation reaction.3.NF-?B activity can suppressed by caffeine and the pNF-?B protein expression is positive related to NLRP3 protein level.