Abstract Background: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. Methods: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatiotemporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. Results: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). Conclusions: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

An in vitro experiment was conducted to study the effects of chitosan on the secretion of cytokines and expression of inducible nitric oxide synthase mRNA in peritoneal macrophages of broiler chicken. In the experiment, peritoneal macrophages were incubated for 24 h in culture medium supplemented with 0 (control), 40, 80, 160 and 320 µg/mL chitosan. The results showed that chitosan tended to increase quadratically the levels of interleukin-1 (P = 0.093) and interleukin-2 (P = 0.106) in the culture fluid of peritoneal macrophage. Chitosan also significantly enhanced inducible nitric oxide synthase mRNA expression of peritoneal macrophage in a quadratic dose-dependent manner (P < 0.05) and tended to promote quadratically the secretion of nitric oxide (P = 0.053) and inducible nitric oxide synthase (P = 0.157) in peritoneal macrophages. This result implied that one of the mechanisms by which chitosan modulated immune functions in chickens might be chitosan activating expression of inducible nitric oxide synthase and then improving the secretion of nitric oxide.