Hemodynamic data
In the present study, hemodynamic changes of rats were monitored at various time points (Table 1). No significant difference was found in the MAP of baseline in the sham-operated group and CPR group. In the CPR, CORM-2, and iCORM-2 groups, the MAP at each time point after resuscitation was markedly lower than that of baseline and this phenomenon was observed until 4 h after resuscitation (P<0.05; Table 1). The MAP at each time point after resuscitation in all the resuscitated-based groups was significantly lower than that at the same time point in the sham-operated group (P<0.05; Table 1). The MAP at each time point after resuscitation in the CORM-2 group was noticeably higher than that in the CPR and iCORM-2 groups (P<0.05; Table 1).
Effects of CORM-2 on the alleviation of damage to ileum tissues in rats after CPR
Figure 1 shows the histological changes in the intestinal tissues of rats in each group. Rats in the sham-operated group exhibited an intact ileal mucosa, neat intestinal villi, deep crypts, and a clear and complete gland structure. In the ileum of rats in the CPR group, villi arranged in a disorderly fashion, mucosal swelling, villi edema, partial villi destruction, and extensive necrosis in the intestinal epithelial cells. CORM-2 group showed a mild mucosal edema, a small amount of necrosis in the intestinal epithelial cells, and partial villi destruction. CORM-2, rather than iCORM-2, reduced intestinal epithelial damage, which improved morphological changes in the intestinal mucosa during CA-CPR. Compared with the sham-operated group, Chiu's scores for intestinal mucosal injury in all resuscitation-based groups were elevated (4.59±0.34, 2.38±0.38, and 3.64±0.53 vs. 0.40±0.13; P<0.05), and the Chiu scores in the CORM-2 group were significantly lower than those in the CPR and iCORM-2 groups (2.38±0.38 vs. 4.59±0.34 and 3.64±0.53, P<0.05).
TEM revealed ultrastructural changes in the intestinal mucosal tissues in all the groups. As illustrated in Figure 3, in the sham-operated group, the epithelial cells were closely arranged, and microvilli on the surface of epithelial cells were arranged in neat rows. The TJs and desmosomes were clear and complete, and the paracellular spaces were narrow. In the CPR group, the microvilli were sparse with an irregular length and arrangement (Figure 2B). The TJs and desmosomes were obscured or disappeared, mitochondria were swollen and vacuolated, and the paracellular spaces were wider. Compared with the CPR and iCORM-2 groups, CORM-2 improved ultrastructural integrity of intestinal mucosa in the CA-CPR group. However, no significant improvement by iCORM-2 treatment was detected.
Serum I-FABP level
I-FABP serves as a biomarker of enterocyte damage. The results showed that compared to the sham-operated group, the I-FABP level was significantly elevated in the CPR and iCORM-2 groups (585.64±119.84 and 414.12±36.13 vs. 230.19±37.01, P<0.05, Table 2). However, the CORM-2 treatment noticeably decreased the serum I-FABP level in the CORM-2 group (306.10±19.22 vs. 585.64±119.84, P < 0.05). Taken together, the data suggested that the CORM-2 treatment could reduce intestinal IRI in the CA-CPR group.
Effects of CORM-2 on the expression levels of claudin-3, occludin, and ZO-1 in ileum tissue
To study the mechanisms underlying the CO-mediated alleviation of the increased intestinal permeability in the CA/CPR group, the expression levels of claudin-3, ZO-1, and occluding, as markers of intestinal barrier function and permeability, were detected by Western blotting. Figure 3 displays that the expression levels of claudin-3, occludin, and ZO-1 in the intestinal mucosa were reduced in the CPR, CORM-2, and iCORM-2 groups (P<0.05) compared with those in the sham-operated group. However, the expression levels of occludin, claudin-3, and ZO-1 (P<0.05) were markedly elevated in the CORM-2 group compared with those in rats that received CPR or those underwent CPR + iCORM-2. The above-mentioned findings showed that CORM-2, rather than iCORM-2, increased the levels of TJ proteins in the CPR group.
Effects of CORM-2 on the expression levels of TNF-α, IL-10, and NF-кB p65 in ileum tissue
To indicate whether CO can inhibit the intestinal inflammation in rats that received CA/CPR, the expression levels of TNF-α, IL-10, and NF-кB p65 were detected by Western blotting. The expression levels of TNF-α and IL-10 in the CPR and iCORM-2 groups were significantly elevated compared with those in the sham-operated group (P<0.05, Figure 4). The expression level of TNF-α decreased in the CORM-2 group compared with that in the CPR group (P<0.05), and the expression level of IL-10 increased compared with that in the sham-operated group, while no significant difference was detected (P>0.05). Compared with the sham-operated group, the expression level of NF-кB p65 was significantly elevated in the CPR and iCORM-2 groups (P<0.05), However, the expression level of NF-кB in the CORM-2 group did not significantly change (P>0.05). Collectively, CO plays an anti-inflammatory role in intestinal injury. The anti-inflammatory role of CO is associated with inhibiting the activation of NF-κB and reducing the release of the pro-inflammatory factors.