High Concentrate Diet Induced Mucosal Injuries by Enhancing Epithelial Apoptosis and Inflammatory Response in the Hindgut of Goats

Current intensive production systems for ruminants feed large amounts of grain to animals to maximize energy intake and support high milk yields or rapid growth. These feeding programs might be useful to lower the cost in the short term, however, the excessive amounts of non-structural carbohydrates and highly fermentable forages result in rapid fermentation and organic acids accumulation in rumen and hindgut [1], [2]. Moreover, feeding excessive grain to ruminants likely leads to the accumulation of bacterial endotoxin (lipopolysaccharide, LPS) derived from gram-negative bacteria in the gastrointestinal (GI) tract [3], [4]. The increased LPS in the GI tract, especially in the hindgut, can increase the permeability for LPS [5], leading to the translocation of LPS from GI tract into circulatory system. Therefore, the accumulation and translocation of LPS might cause damages to the histological structure of mucosa barrier in GI tract [6].

Under normal conditions, the rumen epithelium prevents the translocation of toxic compounds into blood depending on the multi-cellular structure [7]. Recent findings suggest that the combination of low pH and high LPS concentration in the rumen may not cause the translocation of LPS [8]. However, there was a high risk of LPS translocation from the large intestine due to the monolayer structure when ruminants suffered a grain-induced subacute ruminal acidosis (SARA) challenge [9]. Tight junctions (TJs) play an important role in maintaining the barrier function of epithelial cells, preventing the translocation of LPS and other toxic compounds from the intestinal tract into the circulatory system [10]. The impairment of the epithelial TJ barrier can result in intestinal inflammation, including necrotizing enterocolitis, celiac disease, and Crohn's disease [11]–[13]. It's reported that the disruption of intestinal TJs can increase the permeability of intestinal epithelia, the translocation of LPS and even cause systemic inflammation [14].

Intestinal epithelial cells contribute to the regulation of inflammatory conditions and create an intestinal barrier against invading pathogens [15]. Toll-like receptors (TLRs) in the gut epithelium play a key role in maintaining the homeostasis by recognizing ligands known as microbial-associated molecular patterns (MAMPs) derived from both pathogenic and non-pathogenic bacteria [16]–[18]. After combining with TLR-4 on the host cell surface, LPS activates myeloid differentiating factor 88 (MyD88), and then elicits a pro-inflammatory NF-κB-dependent signaling cascade [19], [20]. As a major transcription factor and a first responder to harmful cellular stimuli, NF-κB plays a central role in inflammation through its ability to induce transcription of proinflammatory genes [21]. Systemic LPS administration caused a rapid intestinal epithelial cells apoptosis and the activation of caspases, paralleled a significant disruption in the intestinal villi and the epithelial cells shedding [22].

Several studies conducted in goat and dairy cattle showed that a high grain diet impaired barrier functions of the rumen epithelium [23], [24], inflammation and caused other health problems in host animals [25]. Several chaperone proteins with important cellular protective functions in epithelial tissues were collectively down-regulated in response to high dietary energy supply, which indicates that energy-rich diets and the resulting acidotic insult may facilitate the impairment of rumen barrier function by attenuating their cell defense system [26]. However, no information exists about the underlying mechanism behind the disruption of protective functions of the hindgut epithelium in response to high grain diet to ruminants. Based on the finding that the rumen digesta and presence of LPS make the hindgut tissues “leaky” [27], we hypothesized that feeding goats a diet enriched with a high level of concentrate could cause structure damages in hindgut epithelium via triggering epithelial cells apoptosis as well as the local inflammatory response. Therefore, the aim of this study was to investigate the changes of epithelial structure, apoptosis and inflammatory response in the hindgut of goats after feeding a high-concentrate diet for 6 wks.

In ruminants, SARA is a metabolic disease induced by feeding excessive amounts of highly fermentable forages and insufficient dietary coarse fiber [28], [29]. Several rumen pH thresholds (e.g., 6.0, 5.8, 5.6, and 5.5) have been used to define SARA [30][31], [32]. In the present study, ruminal pH in HC goats fed a diet with 65% concentrate decreased to less than 5.8 at 2 h after feeding and continued for approximately four hours during wk 2. However, after feeding 6 wks, although ruminal pH of HC goats was still significantly lower than the control goats, the value was increased to more than 6.0, indicating the adaptation to high concentrate diets after a long-term feeding. This adaptation phenomenon was also observed in Steele's experiment [6]. Similarly, feeding 60% grain diet to growing goats for 6 wks induced a lower ruminal and colonic pH, and also altered genes expression associated with nutrient transport and electrochemical gradients in rumen and colon epithelium, indicating an increase systemic absorption of VFA to counteract the consequences of luminal acidification on intracellular homeostasis in epithelial cells [33].

Previous studies suggested that ruminants fed diets enrich with starch resulted in the increase of rumen LPS concentration [34], [35]. In a good agreement, our results showed that LPS content in rumen fluids of HC goats was markedly higher than the control goats. Li et al also found that a grain-based SARA in dairy cow led to a huge increase of LPS concentration in cecal digesta and wet feces [9]. In growing goats, colonic LPS concentration significantly increased in response to the 60% grain diet for 6 wks, whereas its ruminal concentration decreased in response to the same diet [36]. Unfortunately, the concentration of LPS in hindgut digesta was not recorded in this study due to the insufficient amount of samples. As in the previous report [9], our results showed that all concentrations of LPS in blood plasma were below 0.04 EU/mL, and there was no significant difference between HC and LC goats. The grain-based SARA challenge caused translocation of LPS from the digestive tract but that LPS was detoxified before entering the peripheral blood circulation [9]. It is well known that acidic conditions in the abomasums lead to degradation of LPS [6] and the Kupfer cells in liver have high clearance rate of the LPS [37]. However, feeding 65% grain diets to the male goats for 7 weeks resulted in translocation of LPS into the blood with the concentrations of 0.8±0.20 EU/mL, suggesting a leakage of LPS into the blood [23]. Klevenhusen et al found that in growing goats, greater amounts of grain in the diet were associated with a quadratic increase in serum LPS concentration compared with controls (0.04 vs 0.08 EU/mL), whereas no signs of inflammation became apparent, as serum amyloid A (SAA) concentrations remained unaffected by diet [1]. Taken together, these results suggest that increased LPS release in the rumen or hindgut during lyses of gram-negative bacteria exposed to acidic conditions might have contributed to epithelial damage [9], [38].

The cellular structure of the ruminal epithelium is complex, consisting of a multilayered structure with intercellular tight junctions (TJs) in the two middle layers. However, hindgut epithelium only has a monolayer structure [38]. Histological differences between the rumen and the hindgut suggest that the barrier function of the hindgut epithelium is more easily compromised by high level of LPS [9]. Effects of high grain diet on ruminal defense function and epithelial permeability have been well documented [24], [27]. In the present study, obvious desquamation and severe cellular damage were observed in colon and cecum epithelium of HC goats, while all LC goats maintained normal and healthy epithelial structure. Our results also showed that the intercellular space was obviously widened in colon and cecum epithelium of HC goats, indicating the disruption and expansion of TJs in the hindgut tissues. It is reported that the TJs opening could regulate the paracellular pathway, and it might be related to disrupted permeability of digestive tract [39], [40]. In this study, as previously reported [41], the mechanical widening of the TJs (Figure 4) indicates the disruption of TJ proteins and the loss of TJ integrity. Several factors such as pH value and toxic compounds have been suggested to involve in the process of structure disruption in epithelial tissues caused by feeding high grain diet [9], [42]. The level of organic acids in colonic digesta was significantly increased in dairy cattle fed high grain diet, therefore, resulting in acidotic insult in the hindgut [9]. In a human colon adenocarcinoma cell line, acetate treatment in the pH range of 6.0 to 7.0 induced cell apoptosis rather than necrosis, while acetate treatment at pH 5.5 caused cell necrosis [7]. Additionally, a recent study conducted in growing goats showed that after feeding high grain diet for 6 wks, several chaperone proteins with important cellular protective functions were collectively down-regulated in ruminal epithelium, indicating an attenuated cell defense system [26].

Feeding high grain diet to growing goats resulted in enhanced concentration of LPS in colonic digesta [38]. Increased release of LPS in colonic lumen exposed to acidic conditions might have contributed to epithelial damage by its ability to trigger inflammatory response. As one of the most potent inflammatory mediators and a major structural component of Gram-negative bacteria, LPS has been hypothesized to form an important risk factor of inflammatory bowel disease (IBD) [43]. LPS activates the host Toll-like receptor 4 (TLR4) to induce the production of pro-inflammatory cytokines and apoptosis of intestinal epithelial cells in IBD, which contribute to cytokine-mediated mucosal tissue damage, leading to a breakdown in the mucosal barrier [44]. MyD88-dependent pathway is the downstream signals of TLR-4, and initiation of MyD88-dependent pathway could lead to activation of NF-κB and transcription of several pro-inflammatory genes [16]. Diverse stimuli (e.g., microbial products, microbes, pro-inflammatory cytokines, and oxidative stress) can activate NF-κB, and the downstream cytokines have been used to assess inflammation [3], [23]. In this study, the mRNA transcript level of TLR-4 in colon mucosa was up-regulated in HC goats, and MyD88 mRNA expression showed a significant up-regulation with a 3-fold increase compared to control goats. TNF-α mRNA expression in colon epithelium of HC goats was increased by 1.5 fold compared to control. Moreover, the level of NF-κB p65 protein was significantly enhanced in colonic mucosa of HC goats. NF-κB activation has been detected in the mucosa of patients with IBD and in murine colitis model, and inhibition of NF-κB with a specific p65 antisense oligonucleotide is effective in preventing experimental models of IBD and efficiently down-regulates cytokine production [45]. Therefore, the activation of TLR-4 signaling pathway in colonic epithelium indicates the presence of an inflammatory response in HC goats after 6 wks feeding high concentrate diets. However, in cecum epithelium, except for a significant decrease of IL-1β mRNA expression there was no difference of TLR4, MyD88 mRNA expression, or NF-κB p65 protein translation between HC and control goats, suggesting other factors or pathways (e.g., apoptosis pathway) contributed to the epithelial damage.

Apoptosis was associated with activated caspase-3, -8, -9 and -10, and inactivation of caspase 10 or 3 was sufficient to block apoptosis in this pathway [46]. In epithelial tissues, tight junction damage or disruption is usually thought of as a downstream consequence of caspase cleavage during the apoptotic process [47]. In another study, signaling through tumor necrosis factor (TNF) leads to the recruitment of signaling complex that activates caspase-8, which then mediates apoptosis by activating caspases including caspase-3, suggesting the important roles of caspase-3 and -8 in mediating cell apoptosis and tissues damage. Our results showed that both mRNA transcription and the activities of caspase-3 and -8 were markedly enhanced in colon epithelium of HC goats compared to control. In cecum epithelium, the transcript level of caspase 3 showed a tendency to increase, and its activity was significantly enhanced in HC goats. It is reported that a high concentration of LPS increased localization epithelial apoptosis and permeability and these changes were dependent on caspase-3 activation [5]. Taken together, in the present study, the activation of caspase activity may contribute to the structural damage in colon and cecum epithelium of goats after feeding 6 wks of high concentrate diets.

We report herein for the first time that ruminal pH dropped to the threshold for defining SARA after feeding HC diet to goats for 2 wks. After 6 wks, although ruminal pH of HC goats was lower than that of LC goats, which was increased to above 6.0 due to the adaptation to high grain diet. LPS concentrations in ruminal fluids but not in plasma were significantly increased in HC goats compared to LC. Obvious structural disruption was observed in colon and cecum mucosa barrier in HC goats but not in LC counterparts. Activations of both cell apoptosis and inflammatory response contributed to the process of epithelial damage in colon mucosa, while in cecum mucosa the activation of cell apoptosis was the dominant mechanism responsible for epithelial disruption in HC goats.

All authors thank Dr. Hongli Wang for the analysis work of the histological damage scoring.

This work was supported by National Basic Research Program of China (Project No. 2011CB100802) and National Nature Science Foundation of China (Project No. 31272470) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.