Effects Of Tert-butylhydroquinone On The Lung Injury Of Chicken Induced By Fine Particulate Matter From Chicken Houses

The fine particulate matter(PM_2.5)from chicken houses is seriously polluted,with complex components and high toxicity.PM_2.5 will easily induce inflammation in the respiratory tract and is becoming a huge health risk in chicken production.Previous studies in our laboratory found that PM_2.5 in chicken houses showed various characteristics,such as high concentration,rich in a couple of heavy metal ions and endotoxin,and carrying a variety of harmful bacteria and fungi.In addition,PM_2.5 could induce an inflammatory reaction,and upregulate the expression of genes involved in oxidative stress and apoptosis in alveolar epithelial cells.Tert-butylhydroquinone（TBHQ）,a feed additive,has the capacity for antioxidant and anti-inflammation.However,the studies on the effects of TBHQ on PM_2.5-induced lung injury in chicken is rare.Consequently,we chose chicken primary AECⅡcells and chick embryos to establish models of cell injury and lung damage induced by PM_2.5 from chicken houses,exploring the mitigation and mechanism of TBHQ.This study can provide a theoretical basis and countermeasures for preventing or alleviating chicken respiratory health problems caused by PM_2.5 from chicken houses.The main research results are as follows:1.Isolation,culture and identification of chicken primary alveolar typeⅡepithelial cellsThe aim of the study was to establish a stable technique for isolation,purification and culture of chicken typeⅡalveolar epithelial cells（AECⅡ）,and provide a research model to investigate the effects of air pollutants and other environmental stress factors on chicken alveolar function.The lung tissue of 16-day-old chick embryos was digested with type I collagenase.A combination of differential centrifugation and differential adherent methods was used to separate the cells,and their purification was conducted using chicken immunoglobulin G（Ig G）immunosorbent assay.Identification of the cells was performed by immunofluorescence and alkaline phosphatase in the final.The results showed that the chicken AECⅡcells obtained from each chick embryo by this method could reach（1.75±0.25）×10⁷.The cell purity is（94.3±1.5）%,and the cell viability is（92.0±1.2）%.When the cells were cultured for about 18～24 h,they stretched and sticked to the wall.They were polygonal or rectangular in shape,closely connected with each other,and grew like islands with lots of fine particles inside them.After 24～72 h,the cells have proliferated and metabolized vigorously,the nucleus is obvious,the cytoplasm is rich,and the cells gradually connect to form a monolayer.After 72 h,the intracellular particulate matter decreased,and the cell morphology changed.Immunofluorescence showed the expression of epithelial cell marker cytokeratin-19（CK19）.Alkaline phosphatase staining showed diffuse red or red-brown particles in the cytoplasm.This method is an effective technique for the isolation,purification and culture of chicken alveolar epithelial cells with high cell yield and purity,which can satisfy general research in vitro.2.Effects of tert-butylhydroquinone on chicken primary alveolar typeⅡepithelial cells damage induced by fine particulate matter from chicken housesThis experiment aimed to investigate the protective effect of TBHQ on chicken primary AECⅡcell injury induced by PM_2.5 from chicken houses.Firstly,the previous experiment method was used to extract chicken primary AECⅡcells in this study.Then,a model of chicken primary AECⅡcell damage induced PM_2.5 was established.Subsequently,the cells were treated with TBHQ or PM_2.5 to detect cell integrity,intracellular ROS levels,and the expression of genes involved in pyroptosis and necroptosis,studying the effect of TBHQ.Our results showed that（1）after chicken primary AECⅡcells were exposed to different concentrations of PM_2.5(0,3.125,6.25,12.5,25,50,100μg·m L^-1)for a certain period of time（6,12,24 h）,the cell viability showed a concentration-and time-dependent decline.With increasing concentrations of PM_2.5,chicken AECII swelled,ruptured and even generated fragments.The greater the concentration of PM_2.5,the greater the degree of cell disruption.The release of lactate dehydrogenase（LDH）and the number of propidium iodide（PI）-positive cells were both observably increased in chicken AECII treated with PM_2.5（50 or 100μg/m L）compared with those in the control group（P<0.05,P<0.01）.（2）The addition of TBHQ significantly increased cell viability（P<0.01）and decreased LDH release levels（P<0.01）compared to the PM_2.5 group.Immunofluorescence assay results showed that the fluorescence density of PI-positive cells and intracellular reactive oxygen species（ROS）levels in the TBHQ and PM_2.5 co-treatment group were significantly lower compared to the PM_2.5 group（P<0.05,P<0.01）.RT-PCR results showed that the expression levels of pyroptosis-related genes（NLRP3,IL-18,IL-1β）and necroptosis-related genes（RIPK1 and RIPK3）were significantly higher in the PM_2.5 group compared with the control group（P<0.01）.However,compared with the PM_2.5 group,the pyroptosis-related genes（NLRP3,Caspase-1,IL-18,IL-1β）and necroptosis-related genes（RIPK3）in the TBHQ and PM_2.5 co-treatment group were significantly reduced（P<0.05,P<0.01）.These results indicated that TBHQ could reduce the upregulation of genes involved in pyroptosis and necroptosis,cell membrane fragmentation and LDH release caused by PM_2.5from chicken houses.Besides,TBHQ improved cell viability and alleviated cell damage by inhibiting ROS production in chicken primary AECII cells.3.Effects of tert-butylhydroquinone on lung injury of chick embryos induced by fine particulate matter from chicken housesChick embryos,a model of oxidative stress,are closer to the body’s internal environment than cells,and more accurately grasp the exposure dose.Therefore,to further clarify the mitigation effect of TBHQ on lung injury induced by PM_2.5 from chicken houses,chick embryos were selected as the research model in this study.Firstly,a model of lung tissue damage induced by PM_2.5 from chicken houses was established（experiment 1）,and then the optimal TBHQ concentration was screened（experiment 2）.Finally,the alleviating effect of TBHQ on lung injury of chick embryos was studied（experiment 3）.The experiments were respectively treated with PM_2.5 or TBHQ,and lasted for 5 days.After 5 d,the survival of chick embryos were recorded,lung tissues were collected.Eggs,embryos and lung tissues were weighted.The lungs of chick embryos were observed histologically,the activities of MDA,T-SOD,and T-AOC in lung were detected.The expressions of proptosis related genes（NLRP3、Caspase-1、IL-18、IL-1β）and necroptosis related genes（RIPK1、RIPK3、MLKL）were also detected.The results suggested that（1）in the experiment 1,different concentrations of PM_2.5(0,25,50,100μg·m L^-1)from chicken houses did not affect the survival of chick embryos,but the lung tissue of chick embryos in the 0.25 mg·m L^-1 PM_2.5 group showed inflammatory cell infiltration.The lung tissues of chick embryos in 0.5 mg·m L^-1 and 1 mg·m L^-1 PM_2.5 groups showed pulmonary edema.（2）In the experiment 2,0～10μg·m L^-1 TBHQ had no effect on the survival rate of chick embryos.In contrast,chick embryos in the 100μg·m L^-1 TBHQ group showed death and inflammatory cell infiltration in lung tissue.0～100μg·m L^-1 TBHQ also had no effect on embryo weight,lung tissue weight and related indices of chick embryos.MDA levels in chick embryo lung tissue increased with increasing TBHQ concentrations.However,T-SOD and T-AOC levels decreased with increasing TBHQ concentrations.Based on these results,the concentration of 0.1μg·m L^-1 of TBHQ was selected for the follow-up study.（3）In the experiment 3,there was no effect on embryo weight,lung tissue weight and related indices in all groups.Compared with the PM_2.5 group,inflammatory cell infiltration and MDA levels were reduced in the lung tissue of chick embryos co-treated with TBHQ and PM_2.5.RT-PCR results showed that the expression of the Caspase-1 gene in the TBHQ group,the expression of IL-1β（P<0.01）and RIPK1（P<0.01）genes in the PM_2.5 group were significantly increased compared with the control group.Compared with the PM_2.5group,the expression of IL-18（P<0.01）,IL-1β（P<0.01）,RIPK1（P<0.01）,and MLKL（P<0.01）genes was significantly downregulated in the TBHQ and PM_2.5 co-treatment groups,and Caspase-1（P<0.01）,RIPK3（P<0.05）genes were significantly increased.These results indicated that TBHQ could alleviate inflammatory damage in chick embryonic lung tissues by inhibiting lipid peroxidation reactions and reducing the expression of pyroptosis and necroptosis related genes induced by PM_2.5 from chicken houses.In summary,TBHQ can reduce the expression of genes related to pyroptosis and necroptosis induced by PM_2.5 from chicken houses to alleviate lung damage.