| Hydrogen sulfide（H2S）is one of the main air pollutants in poultry farming environment,which is mainly caused by the degradation of sulfur-containing organic matter in poultry excrement.In livestock and poultry houses,H2S concentrations in the houses are often excession due to improper air ventilation and manure disposal.Long-term H2S exposure can cause a variety of stress response in animals,such as weakening the physique of livestocks and poultries,weakening anti-stress capacity,reducing disease resistance and many other adverse effects.At the same time,it is also an occupational hazardous gas,which can damage the respiratory,digestive,cardiovascular,and immune systems to human and animals at long-term exposure.H2S caused immune damage greatlyr educes the fungus-or virus-resisting ablity of the chickens.However,the mechanism of H2S induced immune damage is still unclear.Therefore,it is necessary to further explain the mechanism of immunotoxicology caused by H2S.In the northern of China,the weather is cold in winter,and the contradiction between heat preservation and ventilation in poultry houses is more obvious than elsewhere.In this study,the H2S contents in a certain city in northern of China was investigated in chicken houses,and the H2S exposure model was established with an environmental control warehouse.We measured the broiler production performance and immune function related indicators,and the results confirmed that H2S could reduce chicken production performance and damage immune function;On the 42 days of age,the thymic tissues were isolated to observe the histomorphology and ultrastructural changes,measure the cytokines and apoptosis.After,the transcriptomics sequencing analysis was performed and the ciRNA-mi RNA-m RNA negative regulated networks was constructed,which explain the non-coding RNAs are involved in H2S caused inflammatory response and apoptosis initially;Then an in vitro H2S exposure model was established using chicken macrophages（HD11）.We detected effects of H2S on cell activity and proliferation,apoptosis and cell cycle;Furthermore,a mi R-15a and ciR-PTPN23 knockdown/overexpression cell model was constructed respectively,and the targeting relationship between mi R-15a and E2F3 or ciR-PTPN23 was verified by the dual luciferase reporter system;We also measured the m RNA and protein expressions of apoptosis and cell cycle-related genes,which were the downstream of ciR-PTPN23/mi R-15a/E2F3 axis,to preliminarily explain that H2S caused thymic apoptosis through this axis.This study further clarified H2S-mediated immunotoxicity,and provided a new theoretical for researching the environmental toxicological in livestock and poultry.The main results of the study were showed as follows:（1）H2S contents survey results showed that among the 17 chicken houses surveyed in a certain city in northern of China from December 2017 to January 2018,the average H2S concentration in most of chicken houses was lower than the national limit except 2 houses;The highest H2S concentration were exceeded the national limit in 5 chicken houses.This results indicating that the H2S content in some chicken houses were overlimit,and the concentration fluctuates greatly.Further analysis found that the H2S concentration in the flat-fed house was significantly higher than that in the cage,and it was lower in the brooding house than that in the rearing chicken house.（2）The growth production performance evaluation showed that H2S exposure significantly reduced the body weight,average daily weight gain,and abdominal fat rate of broilers,but had no significant effect on average daily feed intake,feed conversion rate,slaughter rate,and eviscerate percentage.The muscle quality revealed that H2S exposure increased the yellowness,reduced the p H and tenderness of the pectoral muscle,but had little effect on water loss.（3）The immune performance-related indicators results showed as following:H2S reduced the serum antibody titers of NDV,the relative weight of the thymus,bursa of Fabricius and spleen in broiler;It also increased the expression of thymus IL-4 and IL-10 levels,while reducing the IL-12and IFN-γlevels;The results also found H2S reduced the Ig A and Ig M levels,the number of CD4+and CD8+T lymphocytes,and the decreased trend ratio of CD4+/CD8+T lymphocytes in thymus.（4）Histological morphology and ultrastructural observations showed that thymus tissues exhibited severity structural alterations after H2S exposure,with blurred boundary of cortex and medulla,and inflammatory infiltration;Ultrastructural observations revealed that,after H2S exposure,the thymus showed a number of cells displayed markedly swollen mitochondria with the degeneration or loss of cristae.The nuclear membrane appared to lose the structural integrity and shrinkage,and the autophagosome were increased.The results of apoptosis detection also showed that H2S caused abnormal apoptosis of thymus.（5）Transcriptomics analysis showed that H2S exposure caused 9 differential expression ciRNAs,17 differential expression mi RNAs,and 172 differential expression m RNAs;Bioinformatics analysis found that these differentially expressed m RNAs were mainly enriched in the cell cycle,antigen processing and presentation,cytotoxic and inflammatory response biological processes;Heat map analysis found that some differential expression m RNAs involved in inflammatory response and apoptosis;Combined analysis revealed multiple ciRNA-mi RNA-m RNA interaction networks.q RT-PCR and Western blot verificated that the ciR-PTPN23-mi R-15a-E2F3regulatory network plays an important role in apoptosis and cell cycle.Then we measured the apoptosis（Bcl-2,Bax,Cyt-C,Caspase-9,Caspase-3,etc.）and cell cycle（Cyclin D1,Cyclin E1,CDK2 and CDK6,etc.）related genes,and the results found that H2S significantly up-regulate pro-apoptosis genes and down-regulate anti-apoptosis genes,and the cell cycle-related genes;H2S exposure model in vitro showed that H2S significantly inhibited HD11 cell proliferation,cause cell apoptosis and G1/S phase arrest.（6）Double luciferase report anaysis results confirmed the targeting relationship between mi R-15a and E2F3.mi R-15a overexpression reduced the E2F3 expression,inhibited HD11 cell proliferation,causing apoptosis and G1/S phase arrest.It also up-regulated expression of pro-apoptotic genes such as Bcl-2,Bax,Cyt-C,Caspase-9 and Caspase-3,while down-regulated anti-apoptotic gene Bcl-2 and the cell cycle-related genes Cyclin D1,Cyclin E1,CDK2 and CDK6.However,knockdown mi R-15a showed the opposite results compared to the mi R-15a overexpression.（7）Fluorescence in situ hybridization results showed that both the ciR-PTPN23 and mi R-15a are localized in the cytoplasm,providing a basis for the ce RNA theory.Dual luciferase report system confirmed the taegetinng relationship between ciR-PTPN23 and mi R-15a.Knockdown ciR-PTPN23increased the expression of mi R-15a and decreased the expression of E2F3,inhibited HD11proliferation,caused apoptosis and G1/S phase arrest.Moreover,it also caused pro-apoptosis-related genes up-regulated,while down-regulated anti-apoptotic gene and the cell cycle-related genes.However,overexpression ciR-PTPN23 showed the opposite results compared to the ciR-PTPN23 overexpression.In summary,the H2S contents in some winter chicken houses in a certain city appared excessived due to improper ventilation;Long-term H2S exposure reduced the production performance,damaged the immune function of broiler;It also damaged thymic histological and ultrastructural,causing tissue inflammation and apoptosis,affecting the thymic T lymphocytes maturation.Further study revealed that H2S could cause thymic apoptosis via ciR-PTPN23/mi R-15a/E2F3 axis.This study This experiment enriches H2S immunotoxicity research,provides practical guidance for prevention and control of H2S pollution scientifically in poultry houses,and also provides a theoretical for development of H2S special-effect rescue drugs. |
