H₂S Exposure-induced Oxidative Stress And Energy Metabolism Disturbance Promoted LPS-mediated Apoptosis And Autophagy In Broiler Hepatocytes Through The PI3K/AKT Signaling Pathway

Hydrogen sulfide（H₂S）is an atmospheric pollutant,which has a severe impact on the natural environment and human health.H₂S is also a common harmful gas in poultry houses.Excessive H 2S exposure can reduce immunity and laying rate,and increase the mortality of livestock.Apoptosis and autophagy are vital mechanisms of the in maintaining homeostasis and balance in the organism.To investigate the molecular mechanism of H₂S exposure inducing apoptosis and autophagy in chicken liver cells and the effect of H₂S exposure on liver injury caused by lipopolysaccharide（LPS）.180 1-day-old Ross 308 male broilers were randomly assigned into two groups:The control group and H₂S group.The H₂S gas exposure model was constructed through the H₂S environmental control cabin.In the H₂S group,the H₂S concentration was 4.0±0.5 mg/m³（0-21 days old）and20.0±0.5 mg/m³（22-42 days old）.The control group was not treated with additional H₂S gas,and the concentration of H₂S was controlled below 2.0mg/m³.At 42 days old,30 broilers were randomly selected from the control group and the H₂S group,and named the LPS group and the H₂S-LPS group.The LPS group and the H₂S-LPS group were intraperitoneally injected with LPS（200μg/kg）and euthanized 5 hours later.Liver tissues were collected,ultrastructure observation,oxidative stress kit detection（T-AOC,CAT,SOD,GSH,GSH-Px and MDA）,energy metabolism（HK1,HK2,LDHA,LDHB,PFK,PK,SDHB and ACO2）,PI3K/AKT pathway（p-PI3K/PI3K and p-AKT/AKT）,apoptosis（caspase3,BCL-2 and bax）and autophagy（Beclin1,ATG5 and LC3-II/LC3-I）related gene expression levels were detected by real-time fluorescence quantitative PCR（q RT-PCR）,Western blot immunoblotting.The results are as follows:（1）The observation of histopathological structure showed that clear intracellular structure,complete organelle structure and normal ultrastructure were observed in the control group.Nuclear agglutination,chromatin condensation and marginalization,mitochondrial swelling,spina bifida and vacuolation,apoptotic bodies and autophagosomes were observed in both LPS group and H₂S group.Compared with the LPS group and the H₂S group,the characteristic lesion of cell apoptosis in the H₂S-LPS group was more serious,and the number of autophagic vesicles was also larger,and the damage of mitochondria was more significant.These results indicated that H₂S caused apoptosis and autophagy damage to the liver tissues of chickens,LPS caused apoptosis and autophagy damage to the liver tissues of chickens,and H₂S aggravated liver damage caused by LPS.（2）The TUNEL examination of liver tissue indicated that H₂S could induce liver cell apoptosis,and it could aggravate apoptosis of liver cell induced by LPS.（3）The results of oxidative stress detection showed that,compared with the control group,the activity of SOD,CAT,T-AOC and GSH-Px in LPS group and H₂S group were significantly reduced,the level of GSH was significantly reduced,the MDA was significantly increased.Compared with the LPS group and the H₂S group,the GSH level of the H₂S-LPS group decreased,SOD,GSH-Px,T-AOC and CAT activities decreased,and the MDA level increased.These indicated that the exogenous H₂S caused oxidative stress in the liver tissues of chickens,and H₂S aggravated the oxidative stress caused by LPS.（4）The detection results of energy metabolism related genes at the m RNA and protein levels indicated that both H₂S and LPS caused the energy metabolism disorder of chicken liver and H₂S aggravated the energy metabolism disorder of chicken liver tissue caused by LPS.（5）q RT-PCR analysis and Western blot results of PI3K/AKT pathway indicated that exogenous H₂S gas exposure aggravates the effect of LPS on PI3K/AKT pathway in chicken liver cells.（6）Detection results of apoptosis-related genes（caspase3,bax and BCL-2）showed that H₂S can induce hepatocyte apoptosis through the mitochondrial pathway and aggravate the hepatocyte apoptosis induced by LPS.（7）Detection results of autophagy-related genes（Beclin1,ATG5 and LC3-II/LC3-I）showed that H₂S exposure causes autophagy in chicken liver cells,and exacerbated autophagy damage in chicken liver caused by LPS.In summary,H₂S exposure can induce energy metabolism disorders and oxidative stress in the liver of broilers,and activate PI3K/AKT pathway to trigger apoptosis through mitochondrial pathway and autophagy.In addition,H₂S can promote LPS-mediated hepatocyte apoptosis and autophagy through the PI3K/AKT pathway.Our study elucidated the mechanism of H₂S exposure to induce apoptosis and autophagy in broilers,and H₂S exposure can cause liver damage in broilers and aggravate LPS induced liver damage.