Effect Of Sulfated Polysaccharides From Seaweed On Stress-induced Inflammatory Responsein Chicken Spleen

The inflammatory response caused by stress is a major problem in poultry farming today.Continuous inflammatory irritation is one of the major causes of reduced immunity and increased mortality in poultry,which causes huge economic losses to the poultry farming industry.We conducted a study on the effect of glucocorticoid-induced stress on immune organ gene expression in chickens by transcriptome sequencing and found that the MyD88 gene has a potential role in regulating immune organ function in poultry under stress through the TLR4/MyD88 signalling pathway.Therefore,whether the MyD88 gene is involved in the inflammatory response caused by stress-induced spleen damage in poultry during production,which in turn affects the immune function of the organism,deserves further investigation.Sulfated Polysaccharides from Seaweed(SPS)has good anti-inflammatory,anti-viral,anti-tumour activity and modulation of the body’s immunity.At present,there are few studies on the application of SPS in poultry as a natural immunomodulatory agent,and the molecular mechanism of SPS regulating inflammatory response is still unclear.Exploring the molecular mechanism of SPS regulating inflammatory response in poultry immune organs has important guiding significance for poultry production.In this study,the stress and inflammation model of beak trimming stress and macrophage(HD11)of chicken were constructed.ELISA,CCK-8,LDH,q RT-PCR,Ed U and flow cytometry were used to detect the effects of SPS on the expression of MyD88 gene and related genes in TLR4/MyD88/NF-κB signaling pathway,proliferation and apoptosis of HD11 cells in the spleen of chickens.To explore the molecular regulatory mechanism of SPS regulating stress inflammatory response in poultry spleen.The main research elements and results are as follows.Part 1 Effect of SPS on the inflammatory response of the spleen in chicks induced by beak trimming stress.Two hundred Gushi roosters of similar weight and health at 1 day of age were selected and randomly divided into five groups A,B,C,D and E.Group A was the control group,fed the basal diet without SPS;groups B,C,D and E were the beak trimming stress group,with 0 mg/kg,80 mg/kg,160 mg/kg and 240 mg/kg of SPS added to the basal diet respectively.Acclimatised to 10 days of age,slaughtered and samples collected after the 1st,3rd,5th and 7th d after beak trimming.Fully automated blood biochemistry analyser,ELISA and q RT-PCR were used to detect changes in immune-related indicators.The results showed that the serum levels of CORT,LDH,TC,IL-6,TNF-α,IL-1β and NF-κB were significantly higher(P<0.05)and Ig M,Ig G,CD3 and CD4 were significantly lower(P<0.05)in group B chicks at different time points after beak trimming compared to group A,indicating that the chick beak trimming stress inflammation model was successfully constructed.MyD88 gene expression was significantly higher in spleen tissue in group B compared to group A(P<0.05).Compared with group B,the addition of different concentrations of SPS all improved the immune organ index of chicks,reduced the level of inflammatory factors in serum,down-regulated the relative expression of MyD88 and pro-inflammatory genes in the spleen,alleviated the inflammatory response caused by beak trimming stress and improved the immune status of the organism.The addition of 160 mg/kg SPS had the most significant effect(P<0.05).Part 2 Effect of MyD88 on the immune function of HD11 cells.In a chick beak-trimming stress-induced inflammation model,MyD88 gene was significantly overexpressed in the spleen,and it was hypothesized that MyD88 gene might play a key role in regulating stress-induced inflammatory response in the chicken spleen.Therefore,we aim to explore the effect of MyD88 gene on the immune function of HD11 cells at the cellular level by constructing MyD88 overexpression vectors and interfering fragments and transfecting them into HD11 cells.The results showed that MyD88 overexpression significantly upregulated the relative expression of pro-inflammatory factors IL-1β,TNF-α,IL-8,NF-κB and TLR4genes(P<0.05),significantly inhibited the activity and proliferation of HD11 cells(P<0.05)and promoted apoptosis(P<0.05);the interference results showed an opposite trend to the overexpression results.This result indicates that the MyD88 gene has a pro-inflammatory effect,Promoting the expression of inflammation-related genes,inhibits cell proliferation,promotes apoptosis and regulates the immune function of HD11 cells.Part 3 Effect of SPS on the inflammatory response of HD11 cells induced by LPS.A pro-inflammatory model of HD11 cells was constructed by adding LPS.The optimal addition concentration of SPS in HD11 was screened by CCK-8 and LDH assay,and whether SPS regulates the inflammatory response of HD11 cells through MyD88 gene by transfecting MyD88 overexpression plasmid and interfering fragment with LPS and by co-treating SPS with LPS and detecting the expression level of inflammatory cytokines by q RT-PCR.The results showed that the pro-inflammatory model of HD11 cells was successfully constructed by stimulating the cells with 2μg/m L of LPS for 9 h.50 μg/m L SPS significantly increased the survival rate of HD11 cells(P<0.05)and had no significant toxic effect on the cells.MyD88 had a synergistic effect in combination with LPS,further promoting the expression of inflammatory genes such as IL-1β,TNF-α,IL-8,NF-κB and TLR4(P<0.05)and exacerbating the cellular inflammatory response induced by LPS.Co-treatment of SPS with LPS significantly down-regulated the expression levels of genes involved in the TLR4/MyD88/NF-κB inflammatory signalling pathway(P<0.05)and alleviated the inflammatory response of cells stimulated by LPS.In summary,SPS may alleviate the beak trimming stress-induced inflammatory response in chicks and the LPS-induced inflammatory response in HD11 cells by down-regulating the TLR4/MyD88/NF-κB inflammatory signalling pathway mediated by the MyD88 gene.