Hemorrhage enhances cytokine, complement component 3, and caspase-3, and regulates microRNAs associated with intestinal damage after whole-body gamma-irradiation in combined injury

Abstract

Hemorrhage following whole-body γ-irradiation in a combined injury (CI) model increases mortality compared to whole-body γ-irradiation alone (RI). The decreased survival in CI is accompanied by increased bone marrow injury, decreased hematocrit, and alterations of miRNA in the kidney. In this study, our aim was to examine cytokine homeostasis, susceptibility to systemic bacterial infection, and intestinal injury. More specifically, we evaluated the interleukin-6 (IL-6)-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), Flt-3 ligand, and corticosterone. CD2F1 male mice received 8.75 Gy 60Co gamma photons (0.6 Gy/min, bilateral) which was followed by a hemorrhage of 20% of the blood volume. In serum, RI caused an increase of IL-1, IL-2, IL-3, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17A, IL-18, G-CSF, CM-CSF, eotaxin, IFN-γ, MCP-1, MIP, RANTES, and TNF-α, which were all increased by hemorrhage alone, except IL-9, IL-17A, and MCP-1. Nevertheless, CI further elevated RI-induced increases of these cytokines except for G-CSF, IFN- γ and RANTES in serum. In the ileum, hemorrhage in the CI model significantly enhanced RI-induced IL-1β, IL-3, IL-6, IL-10, IL-12p70, IL-13, IL-18, and TNF-α concentrations. In addition, Proteus mirabilis Gram(-) was found in only 1 of 6 surviving RI mice on Day 15, whereas Streptococcus sanguinis Gram(+) and Sphingomonas paucimobilis Gram(-) were detected in 2 of 3 surviving CI mice (with 3 CI mice diseased due to inflammation and infection before day 15) at the same time point. Hemorrhage in the CI model enhanced the RI-induced increases in C3 and decreases in CRP concentrations. However, hemorrhage alone did not alter the basal levels, but hemorrhage in the CI model displayed similar increases in Flt-3 ligand levels as RI did. Hemorrhage alone altered the basal levels of corticosterone early after injury, which then returned to the baseline, but in RI mice and CI mice the increased corticosterone concentration remained elevated throughout the 15 day study. CI increased 8 miRNAs and decreased 10 miRNAs in serum, and increased 16 miRNA and decreased 6 miRNAs in ileum tissue. Among the altered miRNAs, CI increased miR-34 in the serum and ileum which targeted an increased phosphorylation of ERK, p38, and increased NF-κB, thereby leading to increased iNOS expression and activation of caspase-3 in the ileum. Further, let-7g/miR-98 targeted the increased phosphorylation of STAT3 in the ileum, which is known to bind to the iNOS gene. These changes may correlate with cell death in the ileum of CI mice. The histopathology displayed blunted villi and villus edema in RI and CI mice. Based on the in silico analysis, miR-15, miR-99, and miR-100 were predicted to regulate IL-6 and TNF. These results suggest that CI-induced alterations of cytokines/chemokines, CRP, and C3 cause a homeostatic imbalance and may contribute to the pathophysiology of the gastrointestinal injury. Inhibitory intervention in these responses may prove therapeutic for CI and improve recovery of the ileal morphologic damage.

Fig 1. Hemorrhage enhances the CRP and C3 responses and the early corticosterone response to radiation in the serum, but not the late corticosterone or Flt-3 ligand response.

Mice received 8.75 Gy γ-photon radiation. Blood was collected at several time points after RI and CI from the surviving mice (N = 6 per group at each time point). Corticosterone (A), CRP (B), C3 (C), and Flt-3 ligand (D) in serum were measured. All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. Sham: free of any injury; RI. Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 2. CI increases cytokines and chemokines in serum more than RI. Mice received 8.75 Gy γ-photon radiation.

Blood was collected at several time points after RI (N = 6 per group at each time point). Cytokines and chemokines in serum were measured. The values were % to sham day 1. For IL-15 and IL-18, the sera on day 1 were used to measure these 2 cytokines because data with other cytokines and chemokines suggest a enhancement was present on day 1. All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. RI; #p<0.05 vs. sham and Hemo. Sham: free of any injury; Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 3. Ileal cytokines and chemokines are increase more in CI than RI.

Mice received 8.75 Gy γ-photon radiation. Ileum samples were collected on day 1 after RI (N = 6 per group). Cytokines and chemokines in ileum lysates on day 1 after RI and CI were measured. All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. Sham: free of any injury; RI. Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 4. CI induces ileum damage more than RI.

Mice received 8.75 Gy γ-photon radiation. Ileum samples were collected on day 15 after RI (N = 4 per group) for histology stained with H&E. All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. RI. Sham: free of any injury; Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 5. CI increases activated caspase-3 in ileal mucosa more than RI.

Mice received 8.75 Gy γ-photon radiation. Ileum samples were collected at several time points after RI (N = 6 per group at each time point). Activated caspase-3 concentrations in ileum lysates were measured. All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. RI. Sham: free of any injury; Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 6. CI increases iNOS, NF-κB, and STAT3 in ileum more than RI.

Mice received 8.75 Gy γ-photon radiation. Ileum lysates 1 day after RI were immunoblotted to detect iNOS, NF-κB p-65, p-50, and phosphorylated STAT3 (N = 4 per group). All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. RI. Sham: free of any injury; Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 7. CI increases MAPK activation in ileum more than RI.

Mice received 8.75 Gy γ-photon radiation. Ileum lysates 1 day after RI were immunoblotted to detect ERK, JNK, and p38 (N = 4 per group). All values are means±SEM. *p<0.05 vs. sham; ^p<0.05 vs. RI. Sham: free of any injury; Hemo: hemorrhage; RI: radiation injury; CI: RI+Hemo.

Fig 8. CI alters the miRNAs in ileum.

Ingenuity Pathway Analysis filtered experimentally validated and direct gene targets of (A) NF-κB, MAPK, and STAT3, and (B) cytokines/chemokine in ileum of CI mice.

Fig 9. CI alters the miRNAs in serum.

Ingenuity Pathway Analysis filtered experimentally validated and direct gene targets of cytokines/chemokine in serum after CI.

Fig 10. Potential scheme with possible intervention points.

RI and CI increase miR-34a, which regulates MAPK and NF-κB, and let-7g and miR-98, which regulate STAT3, which, in turn, transcribes the iNOS gene. Increases in caspase-3 activation result in subsequent apoptosis [8]. RI and CI also increase miR-15, miR-99, and miR-100, which target IL-6 and TNF.