Investigation Of Hydrogen Sulfide Content In A Chicken House In Winter And Its Effect On Tracheal Mucosal Pyroptosis

Hydrogen sulfide（H₂S）is a colorless and toxic air pollutant with pungent odor.It can cause tracheal injury.Content of H₂S in large closed livestock and poultry houses is often too high due to poor heat preservation and ventilation,which leads to mucosal damage of livestock and poultry in winter in northern China.Pyroptosis is a kind of programmed death caused by oxidative stress.Some transcripts called competitive endogenous RNAs（ce RNAs）chelate with micro RNAs（miRNAs）and reduce their inhibitory effect on other transcripts,thus affecting oxidative stress and pyroptotic death.Noncoding RNAs such as circular RNAs（circRNAs）are often involved in the ce RNA mechanism as molecular sponges of miRNAs.However,there were few studies on the transcription profile changes of circRNAs and circRNA-related ce RNA after H₂S exposure.Moreover,the regulatory mechanism on ROS and pyroptosis of circRNA-related ce RNA in chicken tracheal mucosa needs to be further explored.Therefore,this study investigated the content of H₂S in chicken houses in a city of northern China in winter and replicated the model of exposure to H₂S in environmental bins.Transcriptome sequencing on the trachea of chickens exposed to H₂S for 42 days was performed to obtain the differential expression profile of circRNAs and their regulatory ce RNA network.Pyroptosis in trachea of 42-day-old chickens exposed to H₂S was detected by immunohistochemistry,Western blot,and real-time quantitative PCR.The effect of H₂S exposure on pyroptotic death was detected by establishing a model of chicken liver cancer（LMH）cells exposed to H₂S in vitro.In this study,miR-6631-5p knockdown or overexpression and Dual specificity protein phosphatase 6（DUSP6）knockdown LMH cell models were constructed respectively.The targeting relationship between miR-6631-5p and circRNA-IGLL1-17828（circRNA-17828）or DUSP6 was verified by dual luciferase gene reporting system.Subsequently,the effects of miR-6631-5p knockdown or overexpression and DUSP6 knockdown cells on ROS level,the morphological changes of pyroptotic cells and the expression of related genes of NLR family pyrin domain containing 3（NLRP3）inflammasome in pyroptotic pathway were detected.The results are as follows:（1）The results of H₂S content survey showed that the average concentration of H₂S exceeded the national limit standard in two houses which seeded 0nd-21nd broilers,the exceeding rate was30.36%,and the highest concentration was 2.79 mg/m³in December 2020.The average concentration of H₂S exceeded the national limit standard in four houses which seeded 21nd-42nd broilers,the exceeding rate was 46.43%,and the highest concentration was 14.65 mg/m³.Average concentration of H₂S in the other chicken houses were lower than the national limit standard.（2）RNA-seq showed that 16 circRNAs were differentially expressed in the trachea of chickens exposed to H₂S.Among them,11 circRNAs were up-regulated and 5 circRNAs were down-regulated.Bioinformatics analysis showed that differentially expressed genes were involved in NOD like receptor signaling pathway and cell-cycle regulation pathway.Interleukin-1β（IL-1β）and interleukin-18（IL-18）,the key inflammatory mediators in NOD like receptor signaling pathway,were involved in immune response and immune system processes.These results suggested that some indicators of NOD like receptor signaling pathway may exert in H₂S-induced injury.（3）Transcriptomic analysis and bioinformatics prediction showed that miR-6631-5p had complementary pair base with circRNA-17828 and DUSP6,respectively.After H₂S exposure in vivo and in vitro,the expressions of circRNA-17828 and DUSP6 were down-regulated but miR-6631-5p was up-regulated.Dual luciferase reporter gene system confirmed that miR-6631-5p targeted with circRNA-17828 or DUSP6,respectively.After miR-6631-5p knockdown or overexpression,the expression trends of DUSP6 and circRNA-17828 were negatively correlated with miR-6631-5p.After DUSP6 knockdown,the expression trend of circRNA-17828 was positively correlated with DUSP6,and the expression trend of miR-6631-5p was negatively correlated with DUSP6.This further confirmed the existence of circRNA-18728/miR-6631-5p/DUSP6 regulatory axis.（4）H₂S exposure up-regulated pyroptosis indicators including NLRP3,apoptosis-associated speck-like protein（ASC）,Caspase 1（Casp-1）,IL-1βand IL-18.Hoechst/PI/Annexin V staining results showed that pyroptotic cells showed co-staining of Hoechst,PI and Annexin V,cell swelling or rupture even with characteristic large bubble after exposure to H₂S.Knockdown of miR-6631-5p could inhibit the morphological changes of pyroptosis induced by Na HS（H₂S exposure in vitro）.Compared with Na HS treatment group,knockdown of miR-6631-5p significantly up-regulated the expression levels of circRNA-18728 and DUSP6,and down-regulated the expression of pyroptotic indicators.These results indicated that H₂S induced pyroptotic death via circRNA-17828/miR-6631-5p signaling.（5）Overexpression of miR-6631-5p or knockdown of DUSP6 induced the up-regulation of pyroptosis indicators,morphological characteristics of pyroptotic death and ROS accumulation.But high pyroptotic rate,up-regulation of pyroptotic indicators and ROS accumulation induced by miR-6631-5p overexpression were significantly decreased after treatment with N-acetyl-L-cysteine（NAC）.Similarly,pyroptotic activation and ROS accumulation induced by DUSP6 knockdown were inhibited after miR-6631-5p knockdown.This suggested that miR-6631-5p/DUSP6 induced pyroptotic death through ROS accumulation.In conclusion,content of H₂S in many chicken houses exceeded the standard in 16 chicken houses of a large chicken farm in northern China.Further research found that H₂S regulated ROS level through circRNA-18728/miR-6631-5p/DUSP6 axis to mediate pyroptosis of chicken trachea mucosal cells.This study was the first to reveal the mechanism of ROS-related ce RNA mediated pyroptosis in tracheal cells exposed to H₂S.It enriched the toxicity study of H₂S,provided important guidance for respiratory diseases research and scientific prevention and control of harmful gases in poultry houses.