High dosage lipopolysaccharide-induced duodenal, cecal, hepatic, and cardiac inflammation, programmed cell death, permeability in chicken embryos models

Lipopolysaccharide (LPS) from gram-negative bacteria initially induces the pro-inflammatory cytokines storm and causes inflammatory cascade responses. However, the LPS with higher dosage induced duodenal, cecal, hepatic, and cardiac inflammation remains elusive. Specific pathogen-free chicken embryos (n = 72) were allocated to the control, LPS groups (10 μg, 24 μg, 50 μg, 100 μg, 170 μg/egg, respectively). Fifteen day old embryonated eggs were injected abovementioned solutions via the allantoic cavity by disposable syringes. On embryonic day 19, the tissues of the embryos were collected for histopathology, RNA extraction, real-time PCR, and immunohistochemistry investigation. The results demonstrated that there was inflammatory responses (heterophils infiltration or macrophages accumulation) presented in the duodena, ceca, livers, and hearts after LPS induction. The duodenal mRNA expressions of inflammatory-associated mediators (TLR4, IFNγ, IL-1β, IL-6, IL-8, MMP9, MMP3, p38, or NF-κB1) were significantly upregulated after LPS induction (10 μg, 24 μg, 50 μg, 100 μg, or 170 μg /egg) when compared with the control group, respectively. Duodenal immunopositivity of TLR4, MMP9, and MMP3 significantly increased following LPS induction (24 μg or 50 μg) compared to the control group. Meanwhile, the hepatic mRNA expressions of inflammatory-associated factors (IFNγ, MMP3, IL-1β, IL-10, TNFα, IL-8, or NF-κB1) significantly increased after LPS induction (10 μg, 24 μg, 50 μg, 100 μg, or 170 μg /egg) when compared with the control group, respectively. Additionally, cardiac mRNA expression of TLR4, IFNγ, IL-1β, IL-8, IL-10, MMP3, MMP9, and TNFα was significantly increased in all five LPS groups compared to the control group. Cardiac protein expressions of TLR4 or IFNγ significantly increased when compared 100 μg LPS group with the control group. Duodenal and cecal mRNA expressions of programmed cell death-related factors presented irregular. The mRNA expression of hepatic pyroptosis-associated gene AMPKα2, Beclin-1, Bcl-2, CASP1, or CASP12 after LPS induction (10 μg, 24 μg, or 50 μg/egg) increased when compared with the control group. Furthermore, the cardiac mRNA expressions of pyroptosis-related gene CASP1 and CASP12 in five LPS groups increased when compared with the control group. Cardiac autophagy-related gene Bcl-2, ATG5, or LC3B enhanced in LPS groups (10 μg, 50 μg, or 100 μg/egg) when compared with the control group, whereas LC3A, CASP1, or Drp1 mRNA expression in five LPS groups reduced when compared with the control group, respectively. The mRNA expressions of duodenal mucosal barrier function-associated mediators Claudin 1 and PEPT1 were upregulated after LPS induction (10 μg or 50 μg/egg) when compared five LPS groups with the control group, respectively; nevertheless, duodenal Mucin 2 and SGLT1 mRNA expression reduced in four groups (24 μg, 50 μg, 100 μg, or 170μg /egg) when compared with the control group, as well as cecal mRNA expressions of Mucin 2, occludin, SGLT1.The mRNA expressions of liver permeability-related gene (claudin 1 and occludin) increased in the five groups when compared with the control group, as well as cardiac permeability and energy metabolism-related gene (AMPKα2, APOA4, PPARα, SGLT, and claudin1). In conclusion, LPS can induce duodenal, hepatic and cardiac inflammation, initiate energy deficiency, autophagy, programmed cell death, enhanced intestinal mucous barrier function, tight junction, and permeability in chicken embryos.