Cell death in IBD is attributable to dysfunctional epithelial barrier function. Hypoxia is a key feature of IBD mucosal inflammation (Brown et al). However, research has not clarified the roles of hypoxia and hypoxia-induced cell machinery, such as transcriptional regulation by the hypoxia-inducible factor (HIF), in IBD.
Our study aims to investigate these roles with IBD patient biopsies and intestinal epithelial cell cultures. We hypothesise that a combination of hypoxia-induced pathways is responsible for modulating the hypoxic state and causing cell death. Certain pathways may be pro-apoptotic (detrimental) or anti-apoptotic (protective).
We will place investigatory focus on HIF-1α-mediated glycolytic lactate production. We will be determining the extent to which lactate production (and therefore its associated signalling capabilities) plays a role in IBD cell death.
Research will be conducted over 8 weeks.
Staurosporine = pro-apoptotic factor.
Annexin V staining = to determine the pro-apoptotic and anti-apoptotic properties of lactate.
2-Deoxy-D-glucose = artificial inhibitor of glycolysis.
We will also use Western blotting analysis on HIF-1α.
We will observe the differences in apoptotic assays to determine our results, for example:
To determine the extent to which hypoxia-induced cell machinery promotes or inhibits cell death (net effect):
1Normoxic cells without a pro-apoptotic factorNormal conditions
2Normoxic cells with a pro-apoptotic factorIncreased cell death
3Hypoxic cells without a pro-apoptotic factorHypoxia
4Hypoxic cells with a pro-apoptotic factor
Hypoxia + increased cell death
To determine the pro-apoptotic or anti-apoptotic effects of endogenous and/or exogenous lactate:
1Hypoxic cells with inhibited glycolytic lactate production No lactate
2Hypoxic cellsLactate is exclusively from hypoxia-induced glycolysis
3Hypoxic cells introduced with exogenously produced lactateLactate is from hypoxia-induced glycolysis + externally
4Hypoxic cells with inhibited glycolytic lactate production, introduced with exogenously produced lactateLactate is exclusively external