Erythropoietin attenuates motor neuron programmed cell death in a burn animal model

Abstract

Burn-induced neuromuscular dysfunction may contribute to long-term morbidity; therefore, it is imperative to develop novel treatments. The present study investigated whether erythropoietin (EPO) administration attenuates burn-induced motor neuron apoptosis and neuroinflammatory response. To validate our hypothesis, a third-degree hind paw burn rat model was developed by bringing the paw into contact with a metal surface at 75°C for 10 s. A total of 24 male Sprague–Dawley rats were randomly assigned to four groups: Group A, sham-control; Group B, burn-induced; Group C, burn + single EPO dose (5000 IU/kg i.p. at D0); and Group D, burn + daily EPO dosage (3000 IU/kg/day i.p. at D0–D6). Two treatment regimens were used to evaluate single versus multiple doses treatment effects. Before sacrifice, blood samples were collected for hematological parameter examination. The histological analyses of microglia activation, iNOS, and COX-2 in the spinal cord ventral horn were performed at week 1 post-burn. In addition, we examined autophagy changes by biomarkers of LC3B and ATG5. The expression of BCL-2, BAX, cleaved caspase-3, phospho-AKT, and mTOR was assessed simultaneously through Western blotting. EPO administration after burn injury attenuated neuroinflammation through various mechanisms, including the reduction of microglia activity as well as iNOS and COX-2 expression in the spinal cord ventral horn. In addition, the expression of phospho-AKT, mTOR and apoptotic indicators, such as BAX, BCL-2, and cleaved caspase-3, was modulated. Furthermore, the activity of burn-induced autophagy in the spinal cord ventral horn characterized by the expression of autophagic biomarkers, LC3B and ATG5, was reduced after EPO administration. The present results indicate that EPO inhibits the AKT-mTOR pathway to attenuate burn-induced motor neuron programmed cell death and microglia activation. EPO can modulate neuroinflammation and programmed cell death and may be a therapeutic candidate for neuroprotection.

HIF‑1α attenuates neuronal apoptosis by upregulating EPO expression following cerebral ischemia‑reperfusion injury in a rat MCAO model

“…In neonatal rats, delayed EPO administration stimulated oligodendrogenesis and attenuated white matter damage in hypoxic/ischemic brain injury (19). Furthermore, EPO attenuated inflammatory response by decreasing cyclooxygenase 2 and inducible nitric oxide synthase, suppressing microglial activation and inhibiting autophagy activation in burn-induced motor neuron neuroinflammation (20).…”

Erythropoietin Alleviates Burn-induced Muscle Wasting

“…Overexpressed EPO improved rat heart fibrosis through the PI3K/Akt/TLR4 pathway to suppress the release of mediators such as TGF-β1, proinflammatory cytokines, and matrix metalloproteinase [58]. EPO exerts neuroprotective effect in neurodegenerative diseases [104], ischemic brain injury [105,106], spinal cord injury [107] and motor neuron death post-burn [69]. EPO has also been found to improve muscular dysfunction in various experimental models, possible through increasing autophagy as well as decreasing apoptosis in type 2 diabetic skeletal muscles [65], reducing apoptosis in a crushing injury model [108] and preventing cancer-induced muscle alteration [67].…”

“…Afterwards, two groups were treated with daily or weekly EPO as scheduled. Our preparation of EPO and the dosage we used were based on our previous study [69]. Four weeks (W4) after burn, blood samples were collected to measure red blood cell (RBC) mass by an autoanalyzer (Bayer ADVIA 2120).…”

“…Western blotting of the tissue samples was performed as described previously [69]. The antibodies used were atrogin-1 (1: 1000; affinity biosciences, Changzhou, China), cleaved caspase 3 (1: 1000; Cell Signaling, Beverly, MA), apoptosisinducing factor (AIF) (1:1000, Abcam, Cambridge, MA), fibronectin (1:1000, Novus Biologicals, Littleton, CO, USA), type I collagen (1:1000, Novus Biologicals, Littleton, CO, USA), Type III collagen (1:1000, Origene, Rockville, Maryland, USA), transforming growth factor beta-1 (TGF-β1) (1:1000, Abcam, Cambridge, MA), connective tissue growth factor (CTGF) (1: 1000, Novus Biologicals, Littleton, CO, USA) and GADPH (1:2000, Sigma-Aldrich, Poole, Dorset, UK) were performed.…”

Mechanisms of Neuroprotective Effects of Peptides Derived from Natural Materials and Their Production and Assessment

“…Neuronal cell death is closely associated with apoptosis, and many apoptosis factors can be observed in neurodegenerative disease. Previous studies have reported that inhibition of apoptosis can help protect damaged neuronal cells (Balez et al., ; Chuang et al., ; Wu et al., ). Neuroprotective effects related to apoptosis are commonly demonstrated using PI or Hoechst 33342 staining, Annexin V/PI staining, immunochemistry, calcium concentration, and western blot.…”