Transcriptomic analysis of the effect of remote ischaemic conditioning in an animal model of necrotising enterocolitis

Abstract

Necrotising enterocolitis (NEC) has a complex pathophysiology but the common end-point is ischaemia reperfusion injury (IRI) and intestinal necrosis. We have previously reported that RIC significantly reduces the intestinal injury in a rat model of NEC. Here we describe the changes in intestinal mRNA occurring in the intestine of animals exposed to IRI, both with and without RIC. Related rat-pups were randomly assigned to four groups: SHAM, IRI only, RIC only and RIC + IRI. IRI animals, underwent 40 min of intestinal ischaemia, and 90 min of reperfusion. Animals that underwent RIC had three cycles of 5 min of alternating ischaemia/reperfusion by means of a ligature applied to the hind limb. Samples from the terminal ileum were immediately stored in RNA-preserving media for later next generation sequencing and transciptome analysis using R v 3.6.1. Differential expression testing showed that 868 genes differentially expressed in animals exposed to RIC alone compared to SHAM and 135 in the IRI and RIC group compared to IRI alone. Comparison between these two sets showed that 25 genes were differentially expressed in both groups. Pro-inflammatory molecules: NF-ĸβ2, Cxcl1, SOD2 and Map3k8 all show reduced expression in response to RIC. Targeted gene analysis revealed increased expression in PI3K which is part of the so-called RISK-pathway which is a key part of the protective mechanisms of RIC in the heart. Overall, this transcriptomic analysis shows that RIC provides a protective effect to the intestine via anti-inflammatory pathways. This could be particularly relevant to treating and preventing NEC.

Figure 1

The differential expression results as identified by edgeR for RNA sequencing experiments on rat pups of different experimental groups. The volcano plots are coloured based on associated adjusted p value output of the edgeR differential expression testing method and the log fold change of the expression change. Orange signifies a result for the gene that is < 0.05 Q value. Red signifies a result for < 0.05 q value and > 1 log fold change. The heatmap represent the genes identified to be differentially expressed between the two groups (Q value < 0.05 when output from edgeR). The heatmaps are groups by condition. (A) Shows the differential expression results for rat pups exposed to Ischemia reperfusion injury vs SHAM surgery. (B) Shows the differential expression results for rat pups exposed to Remote ischaemic conditioning vs SHAM surgery. (C) Shows the differential expression results for rat pups exposed to Remote ischaemic injury and Ischaemic reperfusion injury vs Ischemia reperfusion injury. RIC Remote ischaemic conditioning; SHAM SHAM surgery, IRI Ischaemia reperfusion injury.

Figure 2

Venn diagram visualising the overlap of statistically significant genes between the experimental group comparisons of remote ischaemic conditioning vs SHAM surgery and remote ischaemic conditioning plus ischaemia reperfusion injury vs ischemia reperfusion injury. Statistically significant genes are defined by an adjusted p value (Q value) < 0.05 utilising the standard methodology for differential expression testing using edgeR. IRI Ischaemic reperfusion injury; RIC remote ischaemic conditioning; SHAM Sham surgery.

Figure 3

The expression differences in specific cell type markers for T-cells genes across the four experimental conditions. T tests were used to generate p-values across the groups. SHAM Underwent Sham surgery; RIC Remote ischaemic conditioning; IRI Ischaemic reperfusion injury; IRI + RIC Ischaemic reperfusion injury plus remote ischaemic conditioning.

Figure 4

The expression differences in specific cell type markers for hypoxic pathway genes across the four experimental conditions. T tests were used to generate p-values across the groups. SHAM Underwent Sham surgery, RIC Remote ischaemic conditioning; IRI Ischaemic reperfusion injury; IRI + RIC Ischaemic reperfusion injury plus remote ischaemic conditioning.

Figure 5

The expression differences in specific cell type markers for molecules known to be involved in necrotising enterocolitis genes across the four experimental conditions. T tests were used to generate p-values across the groups. SHAM Underwent Sham surgery; RIC Remote ischaemic conditioning; IRI Ischaemic reperfusion injury; IRI + RIC Ischaemic reperfusion injury plus remote ischaemic conditioning; NEC Necrotising enterocolitis.

Figure 6

The expression differences in specific cell type markers for the RISK pathway genes across the four experimental conditions. T tests were used to generate p-values across the groups. SHAM Underwent Sham surgery; RIC Remote ischaemic conditioning; IRI Ischaemic reperfusion injury; IRI + RIC Ischaemic reperfusion injury plus remote ischaemic conditioning.