Study On LncGRN/miR-6615-5p Regulating Thymocyte Apoptosis Through SMAD7 In Chickens Exposured To Ammonia

Ammonia is one of the polluted gases with irritating odor in intensive farms.Poultry is more sensitive than ruminants.Ammonia reduces the performance of chickens and even leads to the death of chickens,which seriously threatens the health of poultry and breeding personnel.Ammonia produced in livestock production process is also an important source of air pollutants.The environmental and health risks caused by ammonia in the atmosphere have become one of the main indicators of environmental and health risk assessment.Therefore,it is urgent to study the mechanism of ammonia poisoning in chickens,deepen the understanding of the biological mechanism of ammonia exposure,and provide theoretical basis for the prevention and treatment of ammonia poisoning in chickens.In this study,the effects of chicken exposure to ammonia on apoptosis of immune organs were studied on thymus tissue in vivo and thymus lymphocyte and spleen lymphocyte in vitro.In vivo,80 one-day-old broilers were randomly divided into two groups,40 broilers in each group,the control group(low NH3 concentration,5 mg/m3 for 0-6 weeks)and the ammonia treatment group(high NH3 concentration,20 mg/m3 for 0-3 weeks,45 mg/m3 for 4-6 weeks).The ammonia concentration was set according to the national environmental quality standard for livestock and poultry farms(NY/T388-1999).The ammonia exposure concentration of the treated group was the ring of livestock and poultry farms.The exposure concentration of ammonia in adult chickens was twice as high as that in domestic livestock and poultry farms.After 42 days of ammonia exposure,thymus tissues were isolated for microscopic and ultrastructural observation,TUNEL assay for cell apoptosis,total transcriptome sequencing,proteomic quantitative analysis,and subsequent RNA and protein analysis.Double luciferase reporter gene detection system validated the targeting binding of mi R-6615-5p(micro RNA-6615-5p)with SMAD7,lnc GRN and mi R-6615-5p;In vitro,chicken thymocytes and spleen lymphocytes were cultured in primary culture.Ammonium chloride was used as a substitute for ammonia to simulate the exposure environment.The concentration of ammonium chloride was determined by CCK-8 assay.Apoptosis was observed by AO/EB staining,lymphocyte apoptosis was analyzed by flow cytometry,mitochondrial membrane potential was observed by fluorescence microscopy,and calcium ion in mitochondria was detected by fluorescence probe.Changes of reactive oxygen species were detected by ROS fluorescence probe.Antioxidant enzymes(GPx and GST4),inflammatory cytokines(HO-1,NF-κB,COX-2,i NOS,TNF-α,TGF-β,IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10 and IL-12,apoptosis-related genes(BCL-2,BAX,BAK,Cytc,APAF1,Caspase-9 and Caspase-3)were verified by real-time quantitative PCR and immunoblotting.Changes of expression levels,over-expression and knock-down transfection were used to detect the effect of mi R-6615-5p on apoptotic pathway.The results show that:(1)Ammonia exposure leads to changes in thymus histomorphology,including inflammatory injury,proliferation of vascular endothelial cells,infiltration of inflammatory cells,increase of apoptotic bodies,nuclear condensation,chromatin condensation,swelling and vacuolation of mitochondria,and apoptosis.Thymic lymphocytes and splenic lymphocytes were cultured in vitro.The apoptotic rate of lymphocytes increased with the increase of ammonia exposure,and the apoptotic level increased with the increase of concentration.TUNEL assay showed that the apoptotic rate was 13.46% in ammonia treatment group compared with 3.20% in control group(P < 0.05).(2)Total transcriptome analysis showed that circ RNA,lnc RNA,micro RNA and RNA were involved in the stress response to ammonia exposure.Bioinformatics analysis revealed that differentially expressed genes were involved in oxidative stress,inflammation,and apoptotic processes.The lnc GRN-mi R-6615-5p-SMAD7 regulatory network was identified by ce RNA analysis.The pathway was verified by q RT-PCR and Western blot in thymus tissues in vivo and spleen lymphocytes in vitro.Transcriptional genome thermogram showed differential expression of oxidative stress,cytokines and apoptotic genes after ammonia exposure.Proteomic analysis showed that the functions of differential proteins were closely related to oxidative stress,inflammation,immune response and apoptotic process.The expression of differentially expressed proteins Q5F3P4,E1BXY6,E1C765 and Q5ZMR6 is closely related to apoptosis-related genes BCL-2,Caspase-9 and Caspase-3.The differentially expressed proteins play an important role in regulating mitochondrial pathway apoptosis.(3)Detection of double luciferase reporter gene confirmed that there was a targeted binding relationship between mi R-6615-5p and SMAD7,lnc GRN and mi R-6615-5p.In primary cultured splenic lymphocytes,the expression of mi R-6615-5p was overexpressed and knocked down.The results showed that the expression of mi R-6615-5p was significantly increased in overexpressed lymphocytes,the expression of target gene SMAD7 and protein was decreased,and the expression of target gene SMAD7 and protein in knocked-down lymphocytes was increased.(4)The results showed that ammonia exposure inhibited the expression of GPx and GST in thymus tissue and the expression of GPx protein,increased ROS production in lymphocyte exposed to ammonia in vitro,inhibited the expression of GPx and GST,and inhibited the expression of GPx protein.(5)By detecting the changes of inflammatory cytokines,it was found that ammonia exposure induced the expression of inflammatory cytokines HO-1,NF-κB,COX-2 and i NOS in thymus tissue,inhibited the expression of TGF-beta,induced the expression of IL-1β,IL-2,IL-6,IL-8 and IL-12,inhibited the expression of IL-10,and induced the expression of HO-1,NF-κB and i NOS.Ammonia exposure in vitro induced the expression of cytokines HO-1,NF--κB,COX-2 and TGF-β in splenic lymphocytes,inhibited the expression of i NOS,induced the expression of HO-1 and NF-κB,and inhibited the expression of i NOS.(6)After ammonia exposure,mitochondrial membrane potential of thymic lymphocytes and splenic lymphocytes decreased,mitochondrial calcium content increased,ROS activity increased,and the effect became more obvious with the increase of concentration.By detecting the changes of apoptosis-related genes,we found that the expression of BCL-2 was inhibited in thymus tissues exposed to ammonia,and the expression of BAX,BAK,Cytc,APAF1,Caspase-8,Caspase-9 and Caspase-3 was induced.The expression of BAX,Cytc,Caspase-8,Caspase-9 and Caspase-3 was also induced.The expression of BCL-2 was inhibited by splenic lymphatic ammonia exposure in vitro;BAX,BAK,Cytc,APAF1,Caspase-8,Caspase-9 and Caspase-3 were induced;the expression of BAX,Cytc,Caspase-8,Caspase-9 and Caspase-3 were induced.mi R-6615-5p overexpression transfection induces lymphocyte apoptosis.CONCLUSION: The differential expression genes in thymus tissues of chickens exposed to ammonia were screened and identified by full transcription histology and proteomics analysis.Bioinformatics analysis revealed that the differential genes and proteins participated in many physiological and pathological processes,such as oxidative stress,inflammation,and apoptosis.Luciferase assay confirmed that there was a targeted binding relationship between mi RNA-6615-5p and SMAD7,lnc GRN and mi R-6615-5p.As ce RNA,lnc GRN regulates the expression of SMAD7 through mi R-6615-5p.Ammonia exposure induces apoptosis of chicken thymocytes by regulating SMAD7 through lnc GRN/mi R-6615-5p.In vivo and in vitro experiments confirmed that ammonia exposure induced oxidative stress,inflammatory reaction and cell apoptosis in chicken thymus and lymphocyte,and ammonia exposure induced apoptosis through mitochondrial pathway.It enriched the understanding of the biological mechanism of ammonia exposure and provided reference for the prevention and control of ammonia poisoning in chickens.