Study On The Function Of TLR4 Signaling Pathway In Lipopolysaccharide-induced Spleen Injury In Young Chicks

The spleen is the largest peripheral immune organ in the chicken.It is easily damaged during microbial infection,which in turn leads to immunosuppression.The initial period of hatching is a critical stage for the survival of chickens.At this time,the acquired immunity of the chicks is not fully developed,and innate immunity plays an important role.Toll-like receptors(TLRs),as the most important class of pattern recognition receptors in innate immunity,can specifically identify pathogen-associated molecular patterns(PAMPs)and thus trigger a series of signal transduction and regulate the body’s immune response by releasing inflammatory mediators.Among them,TLR4 can recognize lipopolysaccharide(LPS),a cell wall component of gram-negative bacteria,and represents the first line of defense against natural immunity.TLR4 is widely expressed in a variety of cells of the spleen and is involved in mediating tissue damage in the body.However,most of these studies have focused on humans and mice.Studies on the relationship between TLR4 signaling pathway and spleen damage in chicks have rarely been reported.Therefore,in this study,one-day-old Cobb broiler chickens were used to investigate the changes of spleen structure,cell apoptosis and proliferation,inflammatory factors under Salmonella typhimurium derived from LPS stimulation by using HE,and immunohistochemical stainings,fluorescent quantitative PCR and Western Blot techniques.The dynamic changes in the expression of TLR4 signaling pathway-associated molecules were investigated as well.The main research contents and results are as follows: 1.LPS-induced spleen injury in chicksLPS derived from Salmonella typhimurium was intraperitoneally injected into 1-day-old Cobb broilers.At 12 h,36h and 72 h after LPS stimulation,the body weight of the chicks was significantly lower than that of the control group(p<0.01),and the spleen weight showed a downward trend at 36 h and 72 h after LPS stimulation.The ellipsoidal cells exhibited nuclear degeneration and nuclear lysis at 12 h after LPS stimulation.The lesions deepened at 36 h,the nuclear degeneration and nuclear lysis disappeared after 72 h,but the ellipsoid structure was not obvious.The above results indicated that LPS stimulation induced acute spleen damage in chicks.2.Effects of LPS stimulation on apoptosis and cell proliferation in spleen of chicksImmunohistochemistry was used to investigate the expression of ssDNA and proliferating cell nuclear antigen(PCNA)in the chicken spleen;The expression of P53,BCL2,BAK,BAX,caspase-3,and caspase-8 were detected by quantitative PCR at mRNA levels.The expression of caspase-3 was detected by Western Blot at protein level.Apoptosis significantly increased at 12 h and 36 h after LPS stimulation(p<0.05),the expressions of apoptosis-related genes P53,BCL2,BAK and BAX were significantly up-regulated at 12 h and 72 h after LPS stimulation(p<0.05).There was no significant difference in the expression of caspase-3 and caspase-8 at 36 h.Cell proliferation was significantly reduced(p<0.05)at 12 h and 36 h post LPS stimulation,and there was no significant difference between the LPS and control groups at 72 h.The above results showed that LPS stimulation promoted spleen cell apoptosis and inhibit spleen cell proliferation.Interestingly,spleen cell apoptosis is mainly mediated through the activation of apoptosis-related genes P53,BCL2,BAK,BAX.3.The effect of LPS stimulation on the expression of inflammatory factors in the spleen of chickensImmunohistochemistry was used to study the expression of IL-6 at protein level;Q-PCR was used to detect the expression of IL-6 and TNF-a at mRNA level.Following LPS stimulation,the expression of inflammatory cytokines IL-6 and TNF-a was significantly higher than the control group(p <0.01)at 12 h and 72 h,but down-regulated at 36 h.The above results show that LPS stimulation can lead to overexpression of inflammatory factors in the spleen,which in turn leads to spleen damage in the young chicks.4.Effects of LPS stimulation on the expression of LEP100 in the spleen of chickensThe expression of LEP100 was detected by immunohistochemical staining.After LPS stimulation,the expression of LEP100 was upregulated and significantly higher at 12 h than the control group(p <0.01),continued to increase at 36 h,while down-regulated at 72 h.The above results indicated that LPS stimulation induced overexpression of LEP100 in the spleen,causing spleen damage in chicks.5.Effect of LPS stimulation on the expression of TLR4 signaling pathway-related molecules in the spleen of chickensThe expression pattern of TLR4 protein was detected by immunohistochemistry and Western Blot,and mRNA expression of TLR4 and its down-stream signaling molecules including myeloid differentiation factor 88(MyD88)and nuclear factor-κB(NF-κB)were detected by Q-PCR.The results of immunohistochemistry showed that the expression of TLR4 was significantly increased(p<0.05)at 36 h after LPS stimulation,and gradually decreased at 72h;the Western Blot result was similar to that of immunohistochemical staining at 36 h after LPS stimulation.Q-PCR results showed that TLR4 expression was down-regulated at 12 h,and was up-regulated at 36 h and 72 h after LPS stimulation.The expression of MyD88 and NF-KB showed a wave change after LPS stimulation in the chicken spleen.The expression of MyD88 and NF-KB increased at 12 h and was higher than that of the control group.It decreased at 36 h and increased at 72 h.These results showed that: LPS stimulation activated the TLR4 signaling pathway.Taken together,the above results indicated that LPS derived from Salmonella typhimurium activated TLR4 signaling pathway,inducing overexpression of inflammatory cells and inflammatory factors,apoptosis and hence causing splenic injury.