Lipopolysaccharide derived from the digestive tract triggers an inflammatory response in the uterus of mid-lactating dairy cows during SARA

Bilal et al. BMC Veterinary Research (2016) 12:284 DOI 10.1186/s12917-016-0907-1 RESEARCH ARTICLE Open Access Lipopolysaccharide derived from the digestive tract triggers an inflammatory response in the uterus of mid-lactating dairy cows during SARA Muhammad Shahid Bilal†, Juma Ahamed Abaker†, Zain ul Aabdin, Tianle Xu, Hongyu Dai, Kai Zhang, Xinxin Liu and Xiangzhen Shen* Abstract Background: The aims of the current study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrate-induced subacute ruminal acidosis (SARA) in mid-lactating dairy cows. Twelve mid-lactating Holstein cows with an average weight of 455 kg were allocated into two groups subjected to two diets for 18-weeks either a low-concentrate (LC) group containing 4:6 (NDF: NFC) and a high-concentrate (HC) group containing 6:4 (non-forage carbohydrates, NFC): (neutral detergent fiber, NDF) ratio based on dry matter. Results: The HC group showed lower ruminal pH and higher lipopolysaccharide (LPS) concentrations in both the rumen and peripheral plasma compared to the LC group. The LPS concentrations in the rumen fluid and the peripheral plasma were found significantly increased in the HC group compared to the LC group. The concentrations of IL-1β, TNF-α and IL-6 were significantly higher in the HC group compared to the LC group. The uterus of SARA cows revealed elevated mRNA concentrations of nuclear transcription factors and pro-inflammatory cytokines, which confirmed the presence of inflammation. The occurrence of uterine inflammation was further validated by the increased protein expression of NF-κB-p65 and its active phosphorylated variant in the uterus of SARA cows. Similarly, the inflammatory genes TLR4, LBP, MyD88, TRAF-6, NF-κB, IL-6, IL-8, TNF-α and IL-1β were significantly upregulated in the uterus of the HC versus the LC group. Conclusion: Therefore, the results indicated that LPS derived from the rumen triggered the genes associated with inflammation in the uterus of mid-lactating dairy cows fed a high-concentrate diet, causing endometritis. Keywords: Lipopolysaccharide, Sub-acute ruminal acidosis, Cow, LBP, Immune gene expression Background Feeding a high-concentrate diet for a longer period of time can cause subacute ruminal acidosis (SARA). It is a digestive disorder with a pH less than 5.6 lasted for more than 3 h per day [1, 2]. General clinical signs of SARA comprise of reduced dry matter intake (DMI), decreased ruminal pH and diarrhea [3, 4]. Another crucial outcome of SARA is the decline of milk quantity and quality [2, 3], and earlier studies also revealed that * Correspondence: † Equal contributors College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China SARA decreased milk protein yield [4, 5]. The persistent consumption of a HC diet by dairy animals enhances the production of organic acids and significantly results in a decline in pH values of the rumen and intestine [5–7]. This decrease in pH due to excessive high concentrate diet [8] may affect the alterations of the type of fermentation [9] and the structure of the microbes in the rumen [10, 11]. Moreover, the decline in pH also perturbs the balance of microbial population in the rumen causing substantial release of free endotoxin (lipopolysaccharide, LPS) from gram-negative bacteria [12]. Due to possible alterations in permeability and disruption of the gastrointestinal barrier, LPS can be translocated into © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bilal et al. BMC Veterinary Research (2016) 12:284 the blood stream [2, 13, 14]. There are also other reports revealed that elevated circulating LPS cause a systemic inflammatory response [15, 16]. Possibly, being an element of an immune response to LPS, SARA has been reported to increase the concentration of acute phase proteins in the peripheral blood, such as serum amyloid A (SAA) and haptogloblin (Hp) [2, 12, 14, 17]. The SAA has many functions but mainly alters innate immune responses, particularly in the migration of neutrophils and monocytes, while Hp has anti-inflammatory effects; LPS-binding protein (LBP) is activated by microbial infections and facilitates in the neutralization of LPS and in the triggering the release of pro-inflammatory cytokines, Such as TNF-α, IL-6 and IL-1β [6, 18–20]. The innate immune system is the first line of defence against invading pathogens; it involves various types of transmembrane and secreted molecules, recognized as pattern recognition receptors (PRR). Toll like receptors, such as TLR4, that join the LPS-LBP complex, are located on the outer surfaces of a variety of cells and contribute to the sensing of microbial pathogens in the intracellular endosomes and lysosomes [21, 22]. TLR4 recognizes LPS with the help of the LBP and cluster of differentiation antigen14 (CD14) [23, 24]. LPS activates myeloid differentiating factor 88 (MyD88) after joining with TLR4 on the host cell surface [25]. MyD88 activates TNF receptor-associated factor 6 (TRAF6), which stimulates the IκB kinase (IκK) complex, and phosphorylated NF-κB is consequently translocated from the cytoplasm to the nucleus [26, 27]. The NF-κB-dependent cascade pathway regulates the production of pro-inflammatory cytokines, including interleukin (IL)-1β, TNF-α, IL-6, chemokines (IL-8) and other inflammatory mediators [28]. The principal objectives of this study were to evaluate the inflammatory response in cow uterus and to explore the molecular mechanism triggered by high concentrateinduced SARA in mid-lactating dairy cows. Methods Animals, diets and experimental design Twelve healthy multiparous Holstein cows, which were purchased from the Experimental Farm of Nanjing Agricultural University, with an average body weight of 455 kg were selected for the current study. The experimental cows were randomly divided into two groups, each group containing six cows: the high-concentrate (HC) group and the low-concentrate (LC) group. The HC group was provided a high-concentrate diet containing 6:4 (non-forage carbohydrates, NFC): (neutral detergent fiber, NDF) ratio, while LC group was fed low concentrate diet containing 4:6 (NDF: NFC) for the 18week experimental period. The details of the nutritional ingredients and their compositions are shown in Table 1. The cows were surgically fixed with a ruminal fistula (...truncated)