| Transforming growth factor-beta(TGF-β) is an epidermal growth factor which can improve the mucosal repair capacity, and plays an important role in the repair of intestinal injury. This experiment was designed to investigate the expression of the key factos of TGF-β/Smads signaling pathway in the chicken’s injury intestinal mucosal, and the role they played in intestinal mucosal injury.In this experiment, egg-type cockerels(Roman) were used to study. The acute and chronic intestinal injury were induced by injecting chicks E.coli and feeding chicks Eimeria tenella oocysts. First, the immune organ index and inflammatory cytokines in serum were measured, the expression of inflammatory cytokines and tight junction proteins in the injury gut were also detected; we detected the expression of the key factors of TGF-β/Smads signalinging pathway in the injury gut, spleen and liver, and part of factors’ proteins were detected by Western Blot. We would like to reveal the expression and funcation of TGF-β/Smads signaling pathway in different organs when chicken gets an intestinal mucosal injury, and prepare to make a futther and deeper study of TGF-β/Smads signaling pathway in intestinal mucosal injury.Experiment 1: The role of TGF-β/Smads signaling pathway in chicken acute mucosal injury induced by E.coli72 6-day-old chicks were randomly divided into control group and E.coli group,each group had 6 replicates with 6 chicks. Each chick of E.coli group was injected1 m L E.coli bacterial suspension with a concentration of 6×109 cfu /m L; the control group were injected with an equal volume sterile LB broth.6 hours after injection, 1chick from each replicates were chosen to be killed, the jejunum was collected, then the total RNA was extracted, after reverse transcription, we detected the gene expression of the inflammatory cytokines IL-1β, IL-6, TNF-α,tight junction proteins ZO-1,2,the key factors of TGF-β/Smads signalinging pathway TGF-β2,3,4,TβRⅠ,Ⅱ, Smad2,3,7. The experiment result showed that IL-6 gene expression was suppressed in early inflammation, IL-1β gene expression significantly increase(P<0.05). ZO-1, 2 gene expression didn’t change significantly(P>0.05), which meand the permeability was not changed. TGF-β4’s gene expression increased significantly(P<0.05), accompanied by a significant decrease(P<0.05) of TGF-β2,3. TβR Ⅰ, Ⅱand Smad 3 had no significant change, but the gene expression of Smad2,3 decreased significantly(P<0.05).Experiment 2: The role of TGF-β/Smads signaling pathway in chicken chronic mucosal injury induced by E.tenellaPart 1:The expression of inflammatory cytokines and tight junction proteins and the content of inflammatory cytokines in serum120 16-day-old chicks were randomly divided into control group and E.tenella group, each group had 6 replicates with 10 chicks. Each chick of E.tenella group was fed 105 oocysts, the control group was fed with normal saline. In 6h, 72 h, 5th day, 6th day and 8th day after infection, 1 chick from each replicates were chosen to be killed,spleen、 bursa were collected and record immune organs index. Cecal was collected,and total RNA was extracted, after reverse transcriptase, we detected the expression of the inflammatory cytokines IL-1β, IL-6, TNF-α, TLR4 and tight junction proteins ZO-1,2.The result showed that the spleen’s weight was increased significantly(P<0.05) on 5th and 6th day after infection. TLR4 and IL-6 gene expression decreased significantly(P<0.05) in 6h after infection; TNF-α and IL-1βgene expression increased significantly(P<0.05) on 5th day after infection; TNF-α and IL-6 gene expression increased significantly(P<0.05) on 8th day after infection. ZO-1 gene expression decreased significantly(P<0.05) in 6h and 5th 、 6th 、 8thday, ZO-2 geneexpression decreased significantly(P<0.05) in 6h, but increased significantly(P<0.05)on 5th,6th,8th day after infection. We collected chichen’s serum on 6th and 8th day after injection, and used Elisa kit to check IFN-γ,IL-1β,IL-6,IL-12 p40’s contents, but we found no significant difference between control group and E. tenella group(P>0.05).Part 2:The expression of the key factors of TGF-β/Smads signaling pathway in the injury gut and other organs, and the content of some factors’ proteins:In 6h, 72 h, 5th day, 6th day and 8th day after infection, 12 chicks of each group were killed. Spleen, liver, cecum were collected, and total RNA was extracted, after reverse transcriptase, we detected the expression of TGF-β2,3,4, TβR Ⅰ, Ⅱ,Smad2,3,7 in these organs. And TGF-β2, 3, Smad2’s proteins’ content were detected by Wstern Blot. The result showed that TGF-β2 gene expression in cecum decreased significantly(P<0.05) in 6h,5th,6th,8th day after infection, TGF-β3 decreased significantly(P<0.05) in 6h,6th day, TGF-β4 decreased significantly(P<0.05) in 72 h,8th day, so did TβRⅠ,Ⅱ in 6h,5th,6th day(P<0.05). TGF-β2 gene expression in spleen decreased significantly(P<0.05) in 6h, 6th day, TGF-β3 decreased significantly(P<0.05) in 5th, 6th day, and increased significantly(P<0.05) in 8th day, TGF- β4 gene expression decreased significantly(P<0.05) in 72 h, and increased significantly(P<0.05) on 5th day, Smad2, 3 gene expression increased at first and decreased then,returned to normal level finally. TGF- β2,3, TβRⅠ,Ⅱ, Smad2,7 gene expression in liver increased significantly(P<0.05) on 8th day, before this time, the gene expression of TGF-β2 had a significantly increased in 72 h after infection, and a significantly decreased on 6th day(P<0.05). The gene expression of TβR Ⅰ had a significantly increased in 72 h and 6th day( P<0.05). The gene expression of Smad3 had a significantly increased on 6th day( P<0.05). The content of TGF-β2, 3, Smad2’s proteins had no significantly change in 6h,72 h,6th and 8th day after infection(P>0.05). |
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