Semester of Graduation
Spring 2024
Degree
Master of Biological Science (MBioSci)
Department
Department of Veterinary Clinical Sciences
Document Type
Thesis
Abstract
Activation of the mTOR and STAT3 signaling pathways has been recently proposed to be essential in the development of equine sepsis- and insulin-associated laminitis. Both mTOR and STAT3 signaling pathways are important for cell proliferation and maintaining cytoskeleton structure and cellular adhesion dynamics. Understanding specific factors involved in activating these pathways in vitro could allow for the development of potential therapies. It was hypothesized that lipopolysaccharide (LPS), interleukin-6 (IL-6), insulin, and LPS-stimulated neutrophils would activate both the mTOR, demonstrated by downstream phosphorylation of RPS6, and STAT3 pathways in both equine epidermal and dermal cells when cultured individually.
Equine keratinocytes were cultured and stimulated for 24-hours with the following: EpiCM media (negative control), LPS (5 μg/mL and 500 ng/mL), reIL-6 (10 ng/mL), insulin (5 μg/mL and 2.5 μg/mL), and LPS-stimulated neutrophil supernatant (5 μg/mL and 500 ng/mL). Equine dermal fibroblast cells were cultured and stimulated for 24-hours with MEM media only (negative control), LPS (500 ng/mL), reIL-6 (10 ng/mL), insulin (5 μg/mL and 2.5 μg/mL), and-LPS stimulated neutrophil supernatant (5 μg/mL and 500 ng/mL). Protein was isolated and western hybridization was performed. Briefly, protein samples were loaded and separated on an SDS page gel and transferred to a PVDF membrane. Membranes were probed with antibodies for phospho-RPS6 (serine235/236 and serine240/244 moieties) or phospho-STAT3 (Tyr 705) followed by stripping of the blot and reprobing with total RPS6 and b-Actin antibodies. Images were acquired and band intensities were determined for each treatment group. Data was analyzed using a Kruskal-Wallis test with a Dunn’s multiple comparisons. Significance was set at p<0.05.
Only keratinocytes exposed to LPS-stimulated (500 ng/ml) neutrophil supernatant had a significant (p
Date
4-2-2024
Recommended Citation
Annison, Saige Holland, "The Potential Role of mTOR and STAT3 Signaling Pathways in Equine Laminitis" (2024). LSU Master's Theses. 5967.
https://repository.lsu.edu/gradschool_theses/5967
Committee Chair
Leise, Britta