In modern ruminant production, an increased proportion content in the diet is widely used to improve production performance and economic benefits. Nevertheless, HC diets tend to induce adverse effects on feed intake (Nagaraja and Titgemeyer 2007) and lead to an accumulation of VFA, reducing buffering capacity in the rumen (Li et al. 2019). This promotes lower rumen pH, reduces ruminal microbial diversity and ultimately results in SARA (Mccann et al. 2016; Oba and Allen 2000). In this study, the results showed that the HC diet reduced the rumen pH in sheep. Although the rumen pH value did not reach a rumen acidosis standard of below 5.8 (Gozho et al. 2005), digestive disturbances still occurred, as evidenced by the reduced feed intake and high content of lactic acid in the rumen of the sheep. The HC diet supplemented with niacin increased the rumen pH and decreased the lactic acid compared to the HC diet. As previously reported, niacin supplementation in HC diets of beef cattle can improve ruminal pH and propionate concentration, decrease the content of acetate, and promote the growth of rumen microorganisms (Doreau and Ottou 1996; Luo et al. 2019a; Niehoff et al. 2008). The beneficial effect of niacin may be partially ascribed to the regulation of niacin of the growth and community diversity of rumen microorganisms by inhibiting starch utilization and stimulating fibre degradation (Luo et al. 2017). It is unclear if a higher concentration of niacin would fully reverse the effects of a HC diet. However, the previous in vitro study in our research group showed that the addition of 20–80 mM niacin attenuated cellular damage induced by butyrate and promoted cell proliferation. Higher concentrations (100 mM) of niacin lead to low cell viability (Luo et al. 2019b).
Feeding an HC diet to ruminants results in a massive release of bacterial endotoxins (such as LPS), which provoke rumen epithelial cells to generate a amount of reactive oxygen species (ROS) (Arroyo et al. 2017). The ROS causes oxidative damage in mitochondrial proteins, DNA, and lipids, and decrease the activities of GSH-Px and SOD (Martínez-Alfaro et al. 2011; Terpilowska and Siwicki 2019; Wang et al. 2015). In the present study, the HC diet decreased the activities of CAT, GSH-Px, SOD, and T-AOC and increased the MDA content in the rumen epithelium compared to the LC diet, which was consistent with previous work. Niacin is an important antioxidant. It can inhibit nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and reduce intracellular ROS production (Ganji et al. 2015). One previous review has summarized that niacin can effectively reduce the cellular ROS concentration, improve the activities of GSH-Px and CAT, and restore the activities of pyruvate dehydrogenase and succinate dehydrogenase (Ilkhani et al. 2016). Similarly, the HC diet supplemented with niacin increased the activities of CAT, GSH-Px, and T-AOC and decreased the MDA content.
The rumen mucosa, an important physiological barrier between rumen internal environment and blood circulation system, can effectively prevent various pathogens and harmful substances from entering blood (Plaizier et al. 2012). When rumen mucosal integrity is damaged, the functions of rumen digestion, absorption, and epithelial barriers are impaired (John et al. 2011). In this study, we observed that the rumen papillae length, width, and cuticular layer thickness in sheep fed with a HC diet were lower than in sheep fed a LC diet. This result was consistent with earlier reports suggesting that during SARA, exposure to an acidic environment coupled with high rumen LPS concentrations work together to cause rumen epithelial damage, resulting in a decrease in rumen papillae length, width, and cuticular layer (Hu 2008). In the present study, adding niacin to the HC diet increased the rumen papillae length, width, and cuticular layer thickness compared to the HC diet. The development of SARA is accompanied by inflammatory reaction, which due to that the destruction of ruminal epithelium, leading to abnormal harmful substances from the rumen entering the blood and resulting in an up-regulation of serum inflammatory cytokines by epithelial leakage (Dong et al. 2013; Plaizier et al. 2012). The results in the present study showed that the HC diet increased the serum inflammatory cytokines compared to the LC diet, and adding niacin to the HC diet decreased these factors. This effect may be partially due to niacin playing an anti-inflammatory role by reducing multiple inflammatory factors (including IL-6, TNF-α, and iNOS) via inhibition of the NF-κB signaling pathway (Chen et al. 2009).
The molecular mechanism underlying niacin regulation of rumen epithelial papillae morphology is not clear. It is well known that the stability of rumen epithelial cells depends on a specific balance between cellular apoptosis and proliferation. In the current study, the HC diet increased the apoptotic index of rumen epithelial cells in sheep over the LC diet, which might suggest that the disruption of the rumen mucosal barrier is associated with rumen epithelial cell apoptosis induced by the HC diet. Dai et al. (2020) demonstrated that a high grain diet could promote rumen epithelial cell apoptosis in dairy cows. During SARA, the rumen is in a state of abnormal stress due to the accumulation of organic acids and the decrease in pH, which may result in an increase in the apoptosis of rumen epithelial cells. Similarly, acetic acid-induced mucosal injury in a rat model is often used to study the therapeutic effects of anti-apoptotic drugs on gastric ulcers (Li et al. 2017; Nakamura et al. 2003). When the content of butyrate exceeds the rumen papillae absorbing ability, it will inhibit the proliferation of epithelial cells (Luo et al. 2019b). In this study, niacin supplementation in the HC diet decreased the apoptotic index of rumen epithelial cells. It is suggested that lower VFA (especially of butyrate) and higher ruminal pH in sheep in the HCN treatment may be responsible for stress relief in the rumen epithelium.
As a cascade reaction, apoptosis is regulated by intracellular genes and extracellular factors (Bergmann 2007). Among the many apoptosis-related genes, Fas, Bcl-2, and Bax have attracted more attention in the literature. Fas (also known as Apo-1/CD95) an one cell surface protein, belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family. Fas activation by FasL and its receptor (Fas-associated death domain, FADD) activate caspases-3, -8, and − 9, leading to apoptosis (Freiberg et al. 1996; Hodge et al. 1998; Walker et al. 1998). Bcl-2 is highly homologous to Bax. Expression of Bcl-2 has been shown to suppress apoptosis, whereas Bax promotes apoptosis in response to different stimuli, and a high Bcl-2/Bax ratio indicates resistance to apoptosis (Zhang et al. 2011). A crucial tumor suppressor protein, p53 plays a vital role in inducing cell cycle arrest, DNA repair, and apoptosis (Thakur et al. 2012). Previous studies have revealed that a medium concentrate diet (35% concentrate) increased the mRNA expression of apoptotic genes (caspase-3, caspase-8, caspase-9, p53, and Bax), and reduced the ratio of Bcl-2 to Bax (Bcl-2/Bax) expression compared to a LC diet (10% concentrate) (Gui and Shen 2016). Similarly, in the present study, the HC diet increased the mRNA expression of rumen epithelium apoptosis-related genes (Fas, Bcl-2, Bax, Caspase-3, Caspase-8, Caspase-9, and p53) over the LC diet. However, Bcl-2/Bax expression in the HC group was elevated, probably because programmed cell death is not regulated by Bcl-2 family proteins but rather depends on caspase activity and Cyt-C release. Niacin supplementation in the HC diet decreased the mRNA expression of apoptosis genes (Fas, Bcl-2, Bax, Caspase-3, Caspase-8, Caspase-9, and p53). The down-regulation of apoptotic genes by niacin may be partially because niacin promotes ATP generation, DNA synthesis, and repair, and thereby improves the levels of cell viability (Kennedy 2016). Furthermore, oxidative stress-mediated by ROS is a well-known inducer of cell apoptosis (Raj et al. 2011). In this study, niacin increased the antioxidant capacity of rumen epithelium cells. It reduced the ROS production, which may contribute to the inhibition of apoptosis induced by the HC diet. More studies establishing the SARA model in vitro will be conducted soon to observe the effects of niacin on DNA repair and ROS generation in rumen epithelial cells, and to investigate the associated molecular signalling pathways to uncover the molecular mechanism of niacin in regulating rumen epithelial cell apoptosis.
In conclusion, the HC diet reduced the rumen pH, decreased the rumen epithelial papillae morphology and size and antioxidant capacity, and increased the apoptotic index of rumen epithelial cells and apoptosis-related gene expression, promoting the inflammatory response in sheep. Adding 800 mg/kg niacin tended to reverse the negative effects induced by the HC diet in terms of improving rumen epithelial papillae morphology and size, rumen epithelial antioxidant capacity, and inhibiting rumen epithelial cell apoptosis.