Study On The Correlation Between Inflammatory Reaction And Bronchopulmonary Dysplasia And The Preventive And Therapeutic Effects Of Oridonin

Part Ⅰ:Study on the correlation between bronchopulmonary dysplasia and early inflammatory cytokines expression in premature infantsBackgroundInflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia(BPD).Various pathogenic factors such as mechanical ventilation,oxygen therapy,and infection all cause damage to the airway endothelium and pulmonary interstitium through inflammatory reactions.Numerous studies have shown that the expression of inflammatory cytokines in bronchoalveolar lavage fluid(BALF)and serum in children with BPD are significantly increased,including IL-1,IL-6,IL-8,IL-18,and TNF-α and so on.The release of inflammatory cytokines can activate innate immune responses,generate cascade reactions,which further regulate the secretion of various cytokines,chemokines and growth factors,leading to the imbalance between the damage and repair of alveolar type Ⅱ epithelial cells,and finally resulting the formation of BPD.Research has confirmed that systemic inflammatory reactions occur in the early neonatal period,prior to the clinical symptoms of children with BPD,which provides a window period for the prevention and treatment of BPD and supports early inflammatory intervention in BPD.This study conducted a meta-analysis by collecting published studies on the correlation between BPD and early cytokine levels in premature infants at home and abroad,providing evidence-based basis for the direction and timing of clinical intervention in inflammatory response to BPD.ObjectiveThe method of meta-analysis was used to analyze the level of cytokines in premature infants within 24 hours,to explore whether there is a correlation between the occurrence of BPD and the level of early cytokines,and to provide evidence-based basis for seeking new therapeutic targets for BPD.Methods1.China National Knowledge Infrastructure,China Biomedical Literature Database,Wanfang Medical Database,VIP database,PubMed and EMBase were searched by computer,and the published literatures on the correlation between cytokine levels and bronchopulmonary dysplasia in premature infants were screened.2.Newcastle-Ottawa Scale(NOS)was applied to evaluate the quality of the included literatures,extract the required data,and use Stata16.0 for data analysis.3.Comparison of early cytokine levels between BPD group and non-BPD group was measured in Standardized mean difference(SMD)and 95%Confidence Interval(95%CI).Q test and I2 test were used to measure the heterogeneity among various studies,and funnel plot combined with Egger’s test were used to detect whether publication bias existed.ResultsA total of 27 related literatures were included,with a total of 2393 premature infants,involving seven cytokines including IL-1(5),IL-6(7),IL-8(7),IL-10(5),IL-33(4),TGF-β(8)and TNF-α(8),and the results showed that IL-1(SMD=1.061,95%CI:0.800～1.322,p<0.001),IL-6(SMD=1.171,95%CI:0.989～1.354,p<0.001),TGF-β(SMD=0.949,95%CI:0.735～1.146,p<0.001),TNF-α(SMD=1.805,95%CI:1.554～2.056,p<0.001),indicating that the level of cytokines IL-1,IL-6,and TGF-β、TNF-α in BPD group within 24 hours after birth was higher then non-BPD group,with a statistically significant difference.The funnel plot combined with Egger’s test showed no publication bias.Statistical analysis of IL-8,IL-10,and IL-33 showed strong heterogeneity among the literature.Repeated search for the cause of the heterogeneity failed to improve,and the correlation analysis between the three cytokines and BPD was abandoned.ConclusionThe expression levels of IL-1,IL-6,TGF-β and TNF-α within 24 hours after birth in premature infants in BPD group were significantly higher than those non-BPD group,suggesting that the occurrence of BPD is correlated with the increase of the above 4 cytokines.Early attention to the levels of inflammatory cytokines and intervention can be an important target for prevention and treatment of BPD.Part Ⅱ:Study on the therapeutic effect and mechanism of oridonin on experimental bronchopulmonary dysplasia by inhibiting inflammatory reactionBackgroundBronchopulmonary dysplasia is a chronic lung disease caused by a combination of multiple factors on the basis of genetic susceptibility and premature lung immaturity.A large number of studies have shown that inflammatory reactions play a key role in the pathogenesis of BPD,and the expression of cytokines in bronchoalveolar lavage fluid(BALF)and serum in children with BPD is significantly increased.The meta-analysis in the first part also confirmed that the occurrence of BPD was correlated with the early elevation of some inflammatory cytokines.Previous studies have mostly focused on the expression of cytokines,but the maturation and secretion of inflammatory cytokines such as IL-1β and IL-18 are closely related to inflammatory bodies.It is reported that the lung development of transgenic mice under hyperoxia which NLRP3 inflammatory body was knockout is significantly superior to wild type mice,indicating that NLRP3 is one of the key factors affecting the lung development of BPD.Therefore,downregulation of inflammatory response and NLRP3 expression through drug intervention may provide new thinking and methods for clinical treatment of BPD.NF-κB can involve in inflammation and immune response of the body,whose activation is related to the production of TNF-α,IL-1β,IL-6,IL-8,and IL-10.Hyperoxy-induced lung injury is partly mediated by the ERK pathway,and the activation of ERK can mediate the generation of inflammatory cytokines and recruitment of inflammatory cells,and participate in the pathogenesis of BPD.Oridonin is a diterpenoid extract of Rabdosia rubescens.It has been widely used in anti-tumor therapy in Asian countries for many years,and has various pharmacological activities such as anti-inflammatory and antioxidant activities.A large number of studies have shown that Ori alleviates lung inflammation in rats by inhibiting the release of pro-inflammatory cytokines.Meanwhile,studies have confirmed that Ori can induce nuclear transcription of nuclear factor E2 related factor 2(Nrf2)to exert anti-inflammatory and oxidative effects.However,whether Ori has an inhibitory effect on the inflammatory response induced by BPD has not been reported.Therefore,this study aims to observe the effect of Ori on improving lung injury in BPD through in vitro and in vivo studies,explore the mechanism of Ori inhibiting inflammatory response,downregulating inflammatory bodies,and inhibiting their function,and providing experimental and theoretical basis for clinical anti-inflammatory treatment of BPD with traditional Chinese medicine.Objectives1.To observe the protective effect of Ori on neonatal rats with BPD induced by hyperoxia,and observe the therapeutic effect of Ori on neonatal rats from the aspects of reaction,weight gain,pathological changes of lung tissue,production of pro-inflammatory cytokines and NLRP3,and recruitment of inflammatory cells.To observe the protective effect of Ori on AECII injuried by H2O2 in vitro.2.Detection the activation of proinflammatory signaling pathway NF-κB and ERK in vitro and vivo,and the activation of antioxidant signaling pathway Nrf2 in lung tissue,and the expression of antioxidant heme oxygenase-1(HO-1)and NADPH quinone oxidoreductase(NQO-1).The production of active oxygen species(ROS)and mitochondrial membrane potential of AECII were measured in vitro.Explore the mechanism of Ori action.Methods1 Animal test1.1 Animal modeling and administrationA total of 83 newborn Wistar rats were randomly divided into 4 groups within 24 hours after birth:a control group(Ctrl)of 20 rats,a BPD model group(BPD),an Ori low-dose group(Ori 10mg/kg),and an Ori high-dose group(Ori 20mg/kg)of 21 rats each.After birth,the normoxic control group was fully fed in normoxic state,while the other three groups were placed in an 80%-85%hyperoxia box 24 hours after birth,fed in the same manner as the control group.Different interventions were given since hyperoxia induction respectively:The Ori low dose group was given Ori 10 mg/kg,the Ori high dose group was given Ori 20 mg/kg,the normoxic control group and the BPD group were given equal amounts of physiological saline,which were injected intraperitoneally,once every other day,until all samples were collected until day 21,and alveolar lavage fluid and lung tissue were taken respectively.1.2 Observe the protective effects of Ori1)Observe the reaction and weight gain of rats in each group;2)Lung tissue:HE staining was used to observe the morphological changes of the lung induced by hyperoxia,and the radial alveolar count(RAC)and chord length were counted;Masson staining was used to observe pulmonary fibrosis;Western blot was used to detect the expression of NRLP3,IL-1,and IL-6 in lung tissue;3)Bronchoalveolar lavage fluid:Inflammatory cells were counted with a blood cell counter.The expression of proinflammatory cytokines(IL-1,IL-6)in the bronchoalveolar lavage fluid was detected by ELISA,to evaluate the lung injury induced by hyperoxia in rats and the protective effect of Ori.1.3 Explore the mechanism of OriWestern blot was used to detect the activation of proinflammatory signaling pathway NF-κB and ERK in lung tissue and alveolar lavage fluid,and the activation of antioxidant signaling pathway Nrf2,the expression of HO-1 and NQO-1 in lung tissue were detected to explore the mechanism of Ori.2.Cell test2.1 Cell model construction and groupingAECII was treated with different concentrations of H2O2 and Ori,and CCK8 was used to determine the cell survival rate,determine the safe drug concentration,and construct an AECII oxidative damage model.They were divided into control group(Control),H2O2 injury group(H2O2),Ori 2um+H2O2,Ori 5um+H2O2,Ori 10um+H2O2.In the control group,AECII was cultured under normal conditions,while in the H2O2 injured group,AECII was stimulated with 100um/L H2O2 for 3 hours.In the three groups pretreated with Ori at different doses,AECII was pretreated with 2um/L,5um/L,and 10um/L Ori for 3 hours,and then stimulated with 100um/L H2O2 for 3 hours.2.2 CCK8 was used to detect the survival rate of cells in each group,and Western blot was used to detect NLRP3 and IL-1β in each group.To observe the effect of hyperoxia on the inflammatory expression of AECII and the protective effect of Ori.2.3 Western blot was used to detect the activation of signaling pathways NF-κB and ERK,to explore the mechanism of anti-inflammatory effect of Ori.The production of reactive oxygen species(ROS)and mitochondrial membrane potential were detected.Results1.Animal test1.1 The protective effect of Ori on BPD rats:compared with the normal oxygen control group,the weight gain was limited of BPD group,lung development was impaired,the number of alveoli was reduced,the alveolar cavity was enlarged,and pulmonary fibrosis was significant.The pulmonary inflammatory reaction was significant.Neutrophils(Neu)count,myeloperoxidase(MPO)and the contents of pro-inflammatory cytokines IL-1β and IL-6 increased in alveolar lavage fluid,and the expressions of IL-1β,IL-18 and NLRP3 increased in lung tissue.After Ori intervention,the growth and development of rats and lung tissue injury were improved,and the flow of inflammatory cells,NLRP3 inflammatory bodies and the production of cytokines IL-1 β and IL-18 were reduced.1.2 The mechanism of action of Ori:Compared with BPD group,Ori inhibited the activition of pro-inflammatory signaling pathways of NF-kB and ERK,reduced the lung tissue collagen deposition,improved high oxygen induced pulmonary fibrosis,activated the Nrf2 pathway,increased the level of antioxidant NQO-1 and HO-1.It is suggested that Ori can reduce inflammation by inhibiting the activation of pro-inflammatory signaling pathway,promote the activation of antioxidant signaling pathway,enhance antioxidant activity,and play a protective role in neonatal rats with hyperoxic BPD.2 Cell test1.1 Ori’s protective effect on AECII injuried by H2O2:Compared with the control group,Ori improves cell survival rate and reduces the expression of pro-inflammatory cytokines induced by oxidative damage in a dose-dependent manner within the safe range:After different concentration of Ori pretreatment,the expressions of IL-1β,IL-18 and NLRP3 decreased compared with the non-intervention group;1.2 Mechanism of Ori:Compared with the control group,Ori inhibits the activation of the proinflammatory signaling pathway NF-κB and ERK,reduces the production of proinflammatory cytokines and plays an anti-inflammatory role;Ori reduces ROS production,inhibits mitochondrial damage induced by hyperoxia,and plays an antioxidant role.Conclusion1.Ori has an effective protective effect on neonatal rats with hyperoxia induced BPD,improving their weight gain,lung appearance,and pathological changes in lung tissue;It has a protective effect on AECII in vitro,significantly improving the survival rate of cells;Both in vivo and vitro,it reduces the production of proinflammatory cytokines and the recruitment of inflammatory cells;2.Ori inhibits the activation of proinflammatory signaling pathway NF-κB and ERK,plays an anti-inflammatory role;Activates the Nrf2 pathway,increases the production of antioxidants HO-1 and NQO-1,reduces ROS production,and exerts antioxidant effects.