ST is one kind of intestinal pathogens and its infection will cause gastrointestinal dysfunction and intestinal mucosal lesion, which in turn may trigger systemic inflammation response [24]. From the body weight data, we can see clearly that all three doses of ST can cause body weight loss start with 48 h or 72 h in this experiment, which suggested that mice were in the sub-health conditions or getting sick after ST infection. Furthermore, we monitored the development of ST infection in Kunming mice and investigated the infection associated metabolic changes in serum and tissues including three segments of small intestine, and the jejunum proteome was analyzed. We then verified the results in vitro using IEC-6 cells. These results showed the intruding and damaging effects of ST on the host through the Cav-1/Akt/IKKα/NF-κB signaling pathway.
Since cytokines, chemokines and adhesion molecules, as effectors of the immune system, could contribute to amplifying and perpetuating the inflammatory reaction [13], we hypothesized that the ST pathogenic ability might be achieved through regulating the production of these factors. CRP, an acute inflammatory marker, increases dramatically in response to inflammation and infection [25]. To this end, the concentration of CRP, TNF-α, E-selectin, sICAM-1, IL-8 and MCP-1 were measured to analyze the inflammation conditions of mice. Our results showed that the concentrations of all inflammatory markers started to rise at 24 h in a dose-dependent manner. Among them, the concentrations of CRP, TNF-α, MCP-1 and IL-8 reached the peak at 48 h, while the concentrations of E-selectin and sICAM-1 reached the peak at 24 h. Similarly, CRP levels are known to increase during infections and inflammatory diseases, and return to normal when inflammation gradually improved [25–26]. In another study, the mRNA expression levels of cytokines (MCP-1 and IL-8) were increased significantly when porcine intestinal epithelial cells were stimulated with heat-stable Enterotoxigenic Escherichia coli [27]. In the rat models, LPS treatment upregulated the expression of ICAM-1 and vascular cell adhesion molecule‐1 (VCAM‐1) in liver cells, which contributed to immune cell infiltration and aggravated liver damage [28]. Moreover, increasing evidences have demonstrated that upregulation of adhesion molecules occurs in response to proinflammatory cytokines, specifically TNF [13, 29–30], which could explain our results of an increased concentration of adhesion molecules within a short time after ST infection.
To confirm whether the parenchymal organs were injured, we further studied the histopathology changes of small intestine, which is the site of chemical digestion and absorption of nutrients and minerals from food. 2.5×108 cfu/mL ST-infected mice exhibited severe disease in the duodenum and jejunum. The duodenum was characterized by infiltration of massive inflammatory cells and severe ulceration of lamina propria, while jejunum appeared necrosis and detachment of villus apical cells.
Persistent ST infection increases the susceptibility of mice to develop inflammation in the intestine [31], which was also supported exactly in our current study. However, it is noted that there is no significant damage to the ileum of infected mice, which is probably because there is more indigenous gut microbiota in the ileum than the duodenum and jejunum, and the microbiota are able to defend against overt foreign pathogens including salmonella [17, 32]. These results suggested that inflammation occurred of the mice after ST infection, which may be caused some disease accordingly. Along with time extension, mice inflammation attenuated.
Next, we utilized TMT-based quantitative proteomics to further clarify the pathogenic mechanism of ST infection. Based on the informatic analysis and associated signal pathway, we focused on three up-regulated and one down-regulated differential expressed protein in ST-infected mice. Cav-1, a major coat protein of caveolae found in cell membranes, regulates numerous signal pathways that plays critical role in modulation of inflammatory signaling pathways [33]. In mice challenged LPS/D-galactosamine (GalN), deletion of Cav‐1 suppressed the production of proinflammatory cytokine and chemokine, thereby alleviating liver damage, which was dependent on the modulation of NF-κB signal by Cav-1 [34]. NF-κB, an important nuclear transport factor, involves in the regulation of inflammation through multiple mechanisms to influence the magnitude and duration of the inflammatory response [35–36]. For instance, NF-κB regulates the expression of cytokines (eg. TNF-α, IL-1β, IL-6), chemokine (eg. MCP-1), and the adhesion molecule (eg. ICAM-1), affecting the initiation and progression of atherosclerosis [36]. The activation of NF-κB is dependent on IKK complex (containing three kinase subunits, IKKα, IKKβ and IKKγ) [37–38]. In unstimulated cells, inhibitor of κB (IκB) molecules sequester NF-κB in the cytosol, preventing its nuclear localization and transcriptional function [37, 39]. When inflammatory cascade signaling is initiated, IKK complex are activated to trigger NF-κB activation. Briefly, the phosphorylation and degradation of IKK contribute to release NF-κB, and released NF-κB translocates to the nucleus to promote the expression of target genes [37]. In addition, Akt, a serine threonine kinase, plays a key role in regulating various biological functions [40]. The activation of Akt facilitated IKK-mediated NF-κB transcription in hepatocellular carcinoma cells [41]. In the present study, we found that ST-treated mice exhibited the increased concentrations of inflammatory markers, the decreased expression level of IKKα and the increased expression levels of Cav-1, Akt and NF-κB p65 in the jejunum, which was verified in IEC-6 cell model.
To further investigate whether Cav-1, Akt, IKK and NF-κB was required for ST-mediated production of cytokines and chemokines, Cav-1, Akt, IKK and NF-κB expression was suppressed using inhibitions and the concentrations of LDH, CRP, TNF-α, IL-8 and MCP-1 was examined in vitro. We found that the ability of ST to increase their concentrations was significantly decreased in Cav-1 inhibition, Akt inhibition, IKK inhibition or NF-κB inhibition cells. Meanwhile, Cav-1 inhibition blocked the effects of ST on Akt, IKKα and NF-κB p65 activities, Akt inhibition blocked the effects of ST on IKKα and NF-κB p65 signaling activation, while inhibition of IKK activity by BMS-345541 resulted in the reduction of NF-κB p65 activity. This supports the previous findings that IKK inhibition (BMS-345541) binds at the variable site of the enzyme and blocks the transcription of NF-κB in mice [42] or human melanoma cells [43]. Besides, it has been reported that PI3K/Akt exists in the caveola and is regulated by Cav-1. When Cav-1 is inhibited, Akt activation is weakened and expression is also reduced [44]. These findings indicated that Cav-1/Akt/IKKα/NF-κB signaling pathway plays a critical role in ST-induced intestinal inflammatory response.