Exendin-4 improves neuron protection and functional recovery in experimental spinal cord injury in rats through regulating PCBP2 expression

Aims In the present research, we assessed the therapeutic effects of Exendin-4 (Ex-4) on rat models with spinal cord injury (SCI). Materials and methods 36 male Sprague–Dawley rats were randomly allocated into three groups, including sham operation group, SCI group and SCI+Ex-4 group (Ex-4 treatment (10 µg/rat) after SCI, i.p.). In the SCI group, a laminectomy was performed at the T10 vertebrae, followed by weight-drop contusion of the spinal cord. In the sham group, a laminectomy was carried out without SCI contusion. Key findings Our results showed that Basso-Beattie-Bresnahan scale scores were significantly decreased after SCI, and were obviously improved in SCI rats with Ex-4 administration. Additionally, the water content of spinal cord in SCI group was dramatically increased than that in sham group, and after Ex-4 treatment, degree of edema of spinal cord was remarkably reduced. And also, concentration levels of inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α) in the spinal cord were significantly elevated after SCI, and were remarkably reduced in SCI rats with Ex-4 administration. Subsequently, cell apoptosis rate in the injured spinal cord was significantly increased, and after Ex-4 treatment, cell apoptosis rate was remarkably decreased. We also revealed that levels of PCBP2 mRNA and protein were significantly up-regulated after SCI, and were dramatically dropped in SCI rats with Ex-4 administration. Significance Take altogether, our findings disclosed that Ex-4 plays a role in promoting neurological function recovery and inhibiting neuronal apoptosis through effecting PCBP2 expression in SCI rat models.


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The pathophysiological changes of spinal cord injury (SCI), a highly devastating 54 pathology that seriously harms human health worldwide (1), is categorized into two 55 temporally-related mechanisms: the primary injury and the secondary injury. The 56 primary injury refers to a short-time impairment caused by the initial mechanical 57 trauma, which leads to irreversible damage of neurons. The secondary injury, induced 58 by multiple biological events including oxidative stress, immune dysfunction and 59 neuronal apoptosis (2-5), often causes a great number of neurological, behavioral, 60 emotional, and cognitive deficits. Despite great achievements in the therapeutic 61 approaches, the prognosis of SCI patients still remains poor due to severe 62 neurological deficits. Accordingly, it is necessary for us to explore the main 63 mechanisms of neuronal apoptosis in SCI and identify a novel anti-apoptotic drug for 64 SCI treatment. 65 Oxidative stress is one of the important factors that cause the damage of SCI neurons. 66 Oxidative stress response can cause the levels of reactive oxygen species and 4 4 67 inflammatory factors increase, thereby inducing neuronal damage and apoptosis (6) 68 (7). Nrf-2 / OH-1 is an antioxidant pathway that plays an important role in the body's 69 antioxidant response, inhibition of the Nrf-2 / OH-1 pathway will aggravate SCI, and 70 activate the Nrf-2 / OH-1 pathway play a protective effect on neurons (8,9). It is 71 showed that Nrf-2 / OH-1 pathway can be activated by Exendin-4 (Ex-4) (10). Ex-4, a 72 39-amino acid peptide originally extracted from helodermasuspectum venom (11), is 73 extensively considered to be an effective drug for treating diabetes in the past decades 74 (12-14). In addition, more and more investigations showed that Ex-4 might serve a showed that PCBP2 was down-expressed in oral cancer cells, and up-regulation of 87 PCBP2 could contribute to a significant increase in the number of apoptotic cells. 88 Moreover, previous evidence indicated that PCBP2 might play important roles in 5 5 89 neuronal apoptosis and astrocyte proliferation after SCI (24).

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In the present article, we aimed to explore whether Ex-4 could exert a therapeutic 91 effect in SCI through regulating PCBP2 expression and Nrf-2 / OH-1 pathway. Our 92 research might provide a potential therapeutic strategy for patients with SCI. 12-hour light/dark cycle and were given ad libitum access to standard diet and water.

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The rats were randomly divided into three groups (n=12 per group), including (1)  Establishing SCI model 112 The rat models of SCI in this study were established based on the method by Allen as 113 described previously (25). All rats in this study were anesthetized with intraperitoneal 114 injection of 10% chloral hydrate (0.3 ml/kg, i.p.) and placed in a stereotactic frame.

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The skin and muscle were incised and a laminectomy was carried out to explore the 116 thoracic vertebra level 10 (T10) of rats. Then a 10-g weight impactor was vertically 117 dropped from a 20-mm height onto the exposed cord. After the operation above, the 118 incision were closed with suture. In addition, the rats in sham operation group were 119 only received the laminectomy without SCI. All animals were then placed on a warm   Foster City, CA).The relative expression of PCBP2 mRNA was normalized to 156 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and calculated using 157 the 2 −ΔΔCt method (ΔCt = Ct target gene -Ct internal control ) (27).
158 Table 1 The sequences of primers.

Gene name
Primer sequences Western blot assay

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Relative optical density was calculated relative to GAPDH.

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TUNEL assay 170 Rats were anesthetized by intraperitoneal injection of about chloral hydrate (4%) and 171 the rats were sacrificed. Spinal cord tissue was collected and embedded in paraffin.

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The sample was cut into 4 μm-thick slices, dewaxed with xylene, and incubated with The data were presented as mean ± standard deviation (SD). All the data were

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To investigate the degree of edema of spinal cord in three groups, wet/dry weight 194 ratio test was performed to calculate the water content of spinal cord in this study. As  cell apoptosis. In the present study, RT-qPCR analysis was performed to investigate 218 whether mRNA expression of PCBP2, Nrf-2 and OH-1 had an association with SCI.

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As shown in Figure 5A and 6A, compared with that in sham group, the mRNA