Dynamic ROS Production and Gene Expression of Heifers Blood Neutrophil in a Oligofructose Overload Model

Abstract

Alimentary oligofructose (OF) overload can induce several diseases in cattle, such as ruminal acidosis, laminitis, and synovitis. The role of blood polymorphonuclear neutrophil (PMN) remains unclear during OF overload. The aim of this study was to investigate the dynamic changes in reactive oxygen species (ROS) production and the expression profile of genes in blood PMN in a model of OF overload. Twelve clinically healthy and non-pregnant Chinese Holstein heifers, aged between 18 and 26 mo, weighing 335-403 kg, BCS (5-point scale) ranges 2.7-3.3 were used for the experiments. OF heifers (n = 6) received 17 g/kg of BW oligofructose dissolved in 2 L/100 kg of BW tap water and the CON heifers (n = 6) received 2 L/100 kg of BW tap water. Blood PMN was isolated for each heifer 0,6,12,18,24,36, 48, 60, and 72 h after administration. PMN was analyzed either by endogenous and phorbol myristate acetate (PMA)-induced ROS production or by quantitative real-time PCR. After 12 h, PMA-induced ROS production decreased, which was sustained until 48 h. The expressions of inflammation markers (IL1α, IL1β, IL6, IL10, TNFα, STAT3, TLR4, MMP9, and HP) and eicosanoids (ALOX5, ALOX5AP, and PLA2G4A) were upregulated. The expression of adhesion and migration (CXCR2, CXCL8, CD62L, ITGA4, ITGAM, and ITGB2) in OF heifers was increased compared with CON heifers. The expression of oxidative stress (SOD2 and S100A8) was upregulated, while SOD1 and MPO were downregulated. In metabolism and receptor genes, the expressions of GRα and INSR decreased after 12 h, while Fas increased until 6 h and then decreased at 18 h. The expression of LDHA and PANX1 did not show any differences after OF overload. These findings indicate that OF overload induced systemic activation of PMN, which provides a step toward a better understanding of the role of innate immune responses in response to oral OF administration.

Change in Proteolytic Profile in Heifers After Oligofructose Overload

“…All heifers in the OF-induced group exhibited lameness as expressed by >2 locomotion scores after 72 h (19). In addition, distinct symptoms of acute ruminal and systemic acidosis were observed in all heifers in this group, including depression, loss of appetence, watery diarrhea, and transient fever, consistent with a previous study (17).…”

“…Three days before the primary overload, 5% of the primary dose was provided to all heifers. For animal welfare, supportive therapy was provided in the form of calcium borogluconate after 18 h, and Ringer's solution (Heping Animal Medicine Co., Ltd, Harbin, China) and sodium bicarbonate 18 and 24 h after OF overload, as described in previous studies (17). In addition, Locomotion assessment was performed at each time point according to Sprecher et al (19).…”

“…However, the role of these proteinases was unclear in OF induced bovine laminitis. Previously, we demonstrated the activation of blood neutrophil and the inflammation reaction of lamellar tissue after OF overload (17,18). This study aimed to investigate the changes in the proteolytic profile in plasma and lamellar tissue in heifers with OF-induced laminitis.…”

Data_Sheet_1.docx

“…The primer sets utilized in this study were designed to recognize and amplify conserved nucleotide sequences encoding related bovine MPs and TIMPs. The primers used for housekeeping genes (β-actin, GAPDH, and UXT) were referred from previous studies (27,28). Primers for MMP-2, MMP-9, ADAM-10, ADAM-17, ADAMTS-1, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 were designed using the Primers 5 software (White Institute, Cambridge, MA, USA).…”

“…A reverse-transcription negative blank of each sample and a no-template blank were utilized as negative controls. The Ct value of each targeted mRNA was normalized to the geometric mean of three housekeeping genes, and the data were calculated according to the 2 − Ct method and considering quantitative PCR efficiency (19,28,29).…”