A Combination Treatment Strategy for Hemorrhagic Shock in a Rat Model Modulates Autophagy

Abstract

Hemorrhagic shock leads to whole body hypoxia and nutrient deprivation resulting in organ dysfunction and mortality. Previous studies demonstrated that resveratrol, dichloroacetate, and niacin improve organ function and survival in rats following hemorrhagic shock injury (HI). We hypothesized that a combinatorial formula that collectively promotes survival will decrease the dose of individual compounds toward effective therapy for HI. Sprague-Dawley rats were subjected to HI by withdrawing 60% blood volume. NiDaR (Niacin-Dichloroacetate-Resveratrol; 2 mg/kg dose of each) or vehicle was administered following the shock in the absence of fluid resuscitation, and survival monitored. In order to study alterations in molecular mediators, separate groups of rats were administered NiDaR or vehicle along with resuscitation fluid, following HI. We observed significant improvement (p < 0.05) in survival following HI in animals that received NiDaR, in the absence of fluid resuscitation. In NiDaR treated animals that received resuscitation fluid, MAP was significantly increased compared to Veh-treated rats. HI-induced increase in systemic IL-6 levels and tissue expression of IL-6, IL-10, IL-1β, and IL-18 genes in the heart were attenuated with NiDaR treatment. NiDaR promoted autophagy following HI as demonstrated by reduced p-mTOR, increased p-ULK1 and p-Beclin. The combinatorial formula, NiDaR, reduced inflammation, promoted autophagy, and reduced doses of individual compounds used, and may be more effective in genetically heterogeneous population. In conclusion our experiments demonstrated that the combinatorial drug treatment has salutary effect in rats following HI.

Keywords: autophagy; hemorrhage; mitochondria; shock; trauma.

Figure 1

Chemical structure of niacin, dichloroacetate, and resveratrol. NiDaR is a combinatorial formulation of these three compounds. Drawn using Chemdoodle 2D sketcher.

Figure 2

NiDaR prolongs life after hemorrhagic shock. Mean survival time (minutes) following HI and treatment in each of the experimental groups (mean ± SEM); n = 5–6 in each group, *indicates p < 0.05 compared to HI + Veh. NiDaR (2 mg/Kg of each constituent) was administered immediately following shock period.

Figure 3

Effect of NiDaR on MAP and plasma lactate following hemorrhagic shock. (A) MAP following HI and treatment with NiDaR at maximum bleed out (MBO), start of resuscitation, end of resuscitation, 1 and 2 h post resuscitation. Bars represent mean ± SEM. *p < 0.05 vs. HI + Veh. (B) Plasma lactate levels in sham, HI + Veh, HI + NiDaR treatment groups; n = 5–6 in each group; bars represent mean ± SEM; *indicates p < 0.05.

Figure 4

NiDaR attenuates inflammatory response following hemorrhagic shock. (A) Plasma IL-6 levels measured by ELISA. (B) Cytokine gene expression was measured by realtime PCR in the heart. n = 5–6 in each group; results include 2 technical replicates; bars represent mean ± SEM; *Indicates p < 0.05.

Figure 5

NiDaR reduces NF-κb and NLRP3 levels following hemorrhagic shock. (A) Representative immunoblotting of NF-κB P65, p-P65, NLRP3 in heart tissues of sham, HI and NiDaR treated rats. GAPDH was used as the loading control. (B) NF-κB p-P65, P65 intensities were quantified using Image J software(NIH). (C) NLRP3 and GAPDH intensities were quantified using Image J software (NIH). n = 6 animals each group, 2–3 technical replicates. *Indicates p < 0.05.

Figure 6

NiDaR treatment reduces p-mTOR in the heart after hemorrhagic shock. (A) Representative immunoblotting of p-mTOR (S2448) and mTOR in sham, HI and NiDaR treated rat heart tissues. GAPDH was used as the loading control. (B) p-mTOR(S2448) and mTOR intensities were quantified using Image J software(NIH). n = 6 animals in each group; 2-3 technical replicates. *Indicates p < 0.05.

Figure 7

NiDaR promotes autophagy: effect on ULK1 and beclin. (A) Representative Immunoblotting of p-ULK1, ULK1, p-Beclin1, and Beclin1 in rat heart tissues. (B) p-ULK1 and ULK1 intensities were quantified using Image J software (NIH), *Indicates p < 0.05. n = 6. (C) p-Beclin1 and Beclin1 intensities were quantified using Image J software (NIH). n = 6 animals in each group; 2–3 technical replicates. *Indicates p < 0.05.

Figure 8

NiDaR promotes autophagy: effect on LC3-II and P62. (A) Representative Immunoblotting of LC3a/b and SQSTM1/P62 in rat heart tissues. GAPDH was used as the loading control. (B) LC3II and GAPDH intensities were quantified using Image J software (NIH), *Indicates p < 0.05. (C) P62 and GAPDH intensities were quantified using Image J software (NIH), There were 6 animals in each group, 2–3 technical replicates. *Indicates p < 0.05.