ROCK Inhibition attenuates pro-apoptotic factors associated with Necrotizing Enterocolitis

Author(s):

Tyler Leiva; Katherine Snyder; Alena Golubkova; Camille Schlegel; Catherine Hunter

Background:

Necrotizing Enterocolitis (NEC) is a devastating disease of neonates. Although the pathogenesis is incompletely understood, it consists of an altered intestinal microbiome and dysregulation of cellular pathways, such as proliferation/apoptosis. The Rho-associated protein kinase (ROCK) pathway is a key regulator of differentiation and proliferation. The mitogen activated protein kinase’s (MAPK) are a family of proteins that integrate both intracellular and extracellular factors resulting in various responses involved in cellular processes, such as apoptosis. Two downstream proteins activated by MAPK’s include NF-κB and Caspase 9.

Hypothesis:

We hypothesize that inhibition of the ROCK pathway may attenuate the pro-apoptotic effects of NEC.

Methods:

The intestinal epithelial-derived cell line, Caco-2 BBe cells were grown to maturity and used for experimentation. These cell lines were then divided into 4 groups: a control group, an experimental NEC group, a ROCK Inhibitor (RI) treated group, and an experimental NEC group treated with RI.  Experimental NEC was achieved by adding 100µg/mL of lipopolysaccharide (LPS) to the media for 24 hours. Treatment with 10µM of RI, Y-27632 was administered to both experimental NEC and control cell lines. Cell pellets were then harvested and western blot analysis for NF-κB and Caspase 9 was performed. ANOVA was performed and significance was defined as a p-value < 0.05.

Results:

Induction of experimental NEC resulted in a statistically significant increase in both NF-κB and Caspase 9 proteins (p-value 0.011 and 0.003 respectively) (Figure 1). The addition of RI alone, resulted in a decrease in both proteins when compared to untreated cells. RI administration in cells exposed to experimental NEC resulted in no significant increase in both NF-κB and Caspase 9.

Conclusions:

NEC has been shown to increase the apoptosis-related factors NF-κB and Caspase 9 in vivo. RI administration attenuates this increase even in the presence of experimental NEC and preserves similar expression of both proteins. This signifies that ROCK inhibition may be protective against one of the key pathogenic processes involved in NEC.