Histamine activates inflammatory response and depresses casein synthesis in mammary gland of dairy cows during SARA

Subacute ruminal acidosis (SARA) that is a common and frequent metabolic disorder disease can bring tremendous economic losses to dairy industry. The most important reason for SARA occurrence is that to maintain a high yield of milk, the nutrient density of dairy cow diets has increased [1]. The adjustment of the nutrient density is mainly through the feeding of HC diets. This feeding mode can increase the production of organic acids and cause a depression of the pH in the digestive tract over time [2, 3]. When the rumen pH value of cows falls below 5.6 for more than 3 h per day, SARA occurs. This decrease in pH may result in alterations in the type of fermentation and the composition of the microbiota in the rumen [4]. When the rumen has a low pH, the different types of bacteria make histidine for decarboxylization to produce histamine [5]. Histamine that is absorbed from the gastrointestinal tract can be rapidly detoxified by amine oxidases in healthy subjects, and it also can be produced by epithelial cell to regulate many physiological processes, such as cell growth and differentiation [6]. The acid-induced impairment of epithelial barrier function facilitates the translocation of histamine from the digestive tract into the bloodstream [7]. The increase of histamine in blood can cause systemic histaminosis such as laminitis, which is a popular dieses in dairy farm [1], and it is also closely associated with inflammation and lactation of mammary gland [6, 8], those observations suggested that histamine originated from gastrointestinal tract entering mammary gland may elicits local inflammation, and then affects lactation of mammary gland and milk quality.

Histamine (2-[4-imodazole]-ethylamine, HIS) is synthesized by the decarboxylation of histidine by L-histidine decarboxylase (HDC), which is dependent on the cofactor pyridoxal-5′-phosphate [9]. After histamine interacts with its receptors (H_sR), of which there are four subtypes (H₁R, H₂R, H₃R and H₄R) that all belong to the family of G protein-coupled receptors, it will exert many (patho-) physiological effects [10]. H₁R-coupled Gα_q/11 can generate inositol-1, 4, 5-triphosphate and diacylglycerol and increase the intracellular calcium content through stimulating the inositol phospholipid signalling pathways, which has been demonstrated to activate NF-κB [11, 12]. H₂R-coupled Gα_s can, through separate guanosine triphosphate-dependent mechanisms, activate adenylate cyclase and the phosphoinositide second messenger-cAMP to produce gastric acid and mucus [13, 14]. H₃R is mainly present in nervous system signal transduction, and it can lead to the inhibition of cAMP, the accumulation of Ca²⁺ and the activation of the mitogen-activated protein kinase pathway to release neurotransmitters when it is coupled with Gα_i/o [15]. H₄R has been reported to couple with Gα_i/o and play an important role in binding sites for interferon regulatory factor and NF-κB in the modulation of the immune system [16, 17].

Milk is a mixture of several ingredients, including water, fat, protein, lactose and inorganic salt. Meanwhile, casein is a complex particle containing calcium and phosphateand accounts for 80%~ 82% of the total amount of protein in milk. Casein has the following four groups: αS1-casein, αS2-casein, β-casein and κ-casein [18]. In dairy cows, the most abundantly expressed milk protein is αS1- casein. Janus kinase (JAK), the signal transducer and activator of transcription (STAT) signalling pathway and the mTOR signalling pathway are the primary pathways in milk protein expression. However, it has been reported that the role of the JAK-STAT signalling pathway is weak in regulating bovine milk protein expression [19], because the synthesis of αS1-casein is translational but not transcriptionally regulated. Previous studies have demonstrated that histamine can induce inflammation; however, it is unknown whether histamine derived from the digestive tract during SARA is capable of influencing inflammatory responses and casein synthesis in the mammary gland of dairy cows. Therefore, we hypothesized that the present study could investigate the effects of histamine derived from the digestive tract during SARA on the inflammatory response and casein synthesis in the mammary gland of lactating dairy cows fed high concentration (HC) diets.

All experimental procedures were approved by the Animal Experiment Committee of Nanjing Agricultural University, in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (The State Science and Technology Commission of China, 1988). All the experimental protocols were performed in accordance with the approved guidelines and regulations.

SARA effects are caused by a combination of ruminal pH and diet type [21]. The pH results of the present study demonstrated that the dairy cows had experimentally SARA at the end of experiment through the feeding of an HC diet for 18 weeks, as the defined by the definition of previous study [22]. When SARA occurs, the histamine produced by rumen bacteria can easily pass through the damaged rumen epithelium into the bloodstream and cause inflammation [23, 24].

In fact, the concentrations of histamine in the rumen and plasma both increase during SARA. However, the absorption of histamine in the rumen is the direct cause of the increase in the concentration of histamine in the blood. Under normal conditions, dairy cows contain trace amounts of histamine [23], and when histamine is synthesized, it usually combines with polysaccharide sulphate, heparin and chondroitin sulphate to form inactive complexes stored in mast cell granules. The trace amount of histamine generally undergoes methylation or oxidation in the liver, where it is rapidly converted to a non-active substance in the urine. The results of the present study found that the histamine content is higher in the tissue of the mammary gland than in the tissue of the liver, which may be related to liver clearance. Meanwhile, the results illustrated that the liver has a higher tolerance to histamine than the mammary gland, as the mammary gland is more susceptible to the level of histamine [25].

Histamine can cause inflammation through the NF-κB signalling pathway, and this function is realized through combination with its receptors [26]. H₁R-coupledGαq_/11, H₃R-coupled Gα_i/o and H₄R-coupled Gα_i/o can activate PKC and H₂R-coupled Gα_s can activate PKA, both of which can further activate NF-κB through the classical pathway or the alternative pathway [27]. Most inflammatory genes contain NF-κB binding sites within their promoters, and therefore once NF-κB has been activated, it will lead to the secretion of proinflammatory cytokines such as TNF-α and IL-1β [28, 29]. Our research found that three histamine receptors, H₁R, H₂R and H₄R, were significantly increased during SARA, which may be closely related to their function. Histamine activation of H₁R can change vascular permeability by acting on vascular endothelial cells and causing contraction of vascular smooth muscle cells; these features may exacerbate the inflammatory response [30]. Meanwhile, the expression of H₁R genes can be up-regulated by histamine [31]. Compared with H₁R, the absence of histamine can down-regulate the expression of H₂R genes in a tissue-specific manner [32]. However, unlike H₁R, histamine activation of H₂R has effects on the relaxation of smooth muscle cells in the blood vessels and inhibition of cytokine production [33]. Recent work has highlighted the major role of H₄R in eosinophils, mast cells, neutrophils, T-cells and dendritic cells as well as the production of cytokines influenced by these cells, which confirms the importance of this receptor in inflammatory responses [34–37]. We also found that H₃R expression was not different between the LC and HC groups. According to previous studies, H₃R is an auto-receptor regulating histamine release, and a knock-out of H₃R in mice resulted in an increased severity of neuro-inflammatory diseases with enhanced cytokine release [38, 39]. The mRNA expression of NF-κB, TNF-α and IL-1β genes was significantly increased in the HC group, which was consistent with the results of the protein expression levels of NF-κBp65, NF-κBpp65 and IL-1β in the HC group. This showed that the PKC- NF-κB or PKA- NF-κB signalling pathway in the HC group was activated.

It has been reported that the phosphatidyl inositol 3 kinase(PI3K)-serine and threonine protein kinase(Akt)-mTOR signalling pathway plays a major role in casein synthesis. PI3K is activated to convert phosphoinositide 4, 5-bisphosphate (PIP2) into phosphoinositide 3, 4, 5-triphosphate (PIP3) [40]. Then, the growth factor activated kinase Akt is phosphorylated by the products of PI3K in the activation loop (Thr308) and recruited to the plasma membrane [41]. Accordingly, activated Akt can phosphorylate the negative regulator tuberous sclerosis protein-2 (TSC2) and dissociate the TSC complex into TSC1 and TSC2 through the lysosome [42]. Ras homologue enriched in brain (Rheb) is a small guanosine triphosphatase (GTPase) that can activate mTORC1 in its GTP-loaded state, while the GTPase-activating protein (GAP) activity of TSC2 hydrolyses Rheb-GTP into Rheb-GDP to inactivate mTORC1 [43]. The activated mTORC1 can phosphorylate protein translational regulators P70S6K and eukaryotic cells and translation initiation 4 e binding protein 1(4EBP1) [44]. Phosphorylated P70S6K can stimulate cell growth, while the function of 4EBP1 to stimulate protein translation is inhibited when it is phosphorylated. We found that the mRNA expression of mTOR, P70S6K and α-S1 was significantly decreased in the HC group, in agreement with the protein expression of mTOR, P70S6K and α-S1. Meanwhile, the phosphorylation level of mTOR and P70S6K protein in the HC group was also decreased. Collectively, the present study indicated that histamine derived from rumen caused the inflammatory response and reduction of casein synthesis in the mammary gland via activating the NF-κB and mTOR signalling pathway during SARA. It is well known SARA can cause diverse and complex consequences [1], and the present result is only one of consequences and may provide a new way to control inflammatory response of mammary gland elicited by SARA.

Our research showed that the milk protein of lactating cows is depressed after long-term feeding of HC at the individual level, which was paralleled at the gene and protein levels. The inflammatory response caused by histamine derived from the rumen is related to reduce casein synthesis. Our findings suggest new relationships between the inflammatory response and casein synthesis, but the understanding of the molecular mechanisms involved in this process will require further research.