Yajiang Yuan

Neuroprotective Effect of Simvastatin via Inducing the Autophagy on Spinal Cord Injury in the Rat Model

Simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is invariably used to treat cardiovascular diseases. Simvastatin has been recently demonstrated to have a neuroprotective effect in nervous system diseases. The present study aimed to further verify the neuroprotection and molecular mechanism of simvastatin on rats after spinal cord injury (SCI). The expression of Beclin-1 and LC3-B was evidently enhanced at postoperation days 3 and 5, respectively. However, the reduction of the mTOR protein and ribosomal protein S6 kinase p70 subtype (p70S6K) phosphorylation level occurred at the same time after SCI. Simvastatin significantly increased the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). Meanwhile, immunofluorescence results indicated that the expression of chondroitin sulfate proteoglycan (CSPG) and caspase-3 protein was obviously reduced by simvastatin. Furthermore, Nissl staining and Basso, Beattie, and Bresnahan (BBB) scores showed that the quantity and function of motor neurons were visibly preserved by simvastatin after SCI. The findings of this study showed that simvastatin induced autophagy by inhibiting the mTOR signaling pathway and contributed to neuroprotection after SCI.

Simvastatin inhibits neural cell apoptosis and promotes locomotor recovery via activation of Wnt/β‐catenin signaling pathway after spinal cord injury

Statins exhibit neuroprotective effects after spinal cord injury (SCI). However, the molecular mechanism underlying these effects remains unknown. This study demonstrates that the hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin (Simv) exhibits neuroprotective effects on neuronal apoptosis and supports functional recovery in a rat SCI model by activating the Wnt/β‐catenin signaling pathway. In specific, Simv administration after SCI significantly up‐regulated the expression of low density lipoprotein receptor‐related protein 6 phosphorylation and β‐catenin protein, increased the mRNA expression of lymphoid enhancer factor‐1 and T‐cell factor‐1, and suppressed the expression of β‐catenin phosphorylation in the spinal cord neurons. Simv enhanced motor neuronal survival in the spinal cord anterior horn and decreased the lesion of spinal cord tissues after SCI. Simv administration after SCI also evidently reduced the expression levels of Bax, active caspase‐3, and active caspase‐9 in the spinal cord neurons and the proportion of transferase UTP nick end labeling (TUNEL)‐positive neuron cells, but increased the expression level of Bcl‐2 in the spinal cord neurons. However, the anti‐apoptotic effects of Simv were reduced in cultured spinal cord nerve cells when the Wnt/β‐catenin signaling pathway was suppressed in the lipopolysaccharide‐induced model. Furthermore, the Basso, Beattie, and Bresnahan scores indicated that Simv treatment significantly improved the locomotor functions of rats after SCI. This study is the first to report that Simv exerts neuroprotective effects by reducing neuronal apoptosis, and promoting functional and pathological recovery after SCI by activating the Wnt/β‐catenin signaling pathway.

VEGF inhibits the inflammation in spinal cord injury through activation of autophagy

Vascular endothelial growth factor (VEGF) is a secreted mitogen associated with angiogenesis and re-vascularization of spinal cord injury (SCI). VEGF has long been thought to be a potent neurotrophic factor for the survival of spinal cord neuron. However, the neuroprotective mechanism of VEGF is still unclear. The aim of this study was to investigate the effect of VEGF on spinal cord injury and its mechanisms. Young male Wistar rats were subjected to SCI and then VEGF165 were injected directly into the lesion epicenter 24 h post injury. We detected Basso, Beattie and Bresnahan (BBB) scores and numbers of motor neuron via Nissl staining. The expressions of autophagy related protein Beclin1 and LC3B were determined by Western blot and RT-PCR. We also detected the contents of inflammation factors interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α) and interleukin-10(IL-10) in LPS (Lipopolysaccharide) treated spinal neuron-glia co-culture by ELISA. We found that VEGF165 administration increased the BBB score and reduced the loss of motor neuron of rats induced by SCI. VEGF decreased the protein expressions of IL-1β, TNF-α and IL-10 and up-regulated the expressions of Beclin1 and LC3B of rats. In the in vitro study, VEGF165 decreased the levels of IL-1β, IL-10 and TNF-a in the medium of LPS treated spinal neuron-glia co-culture, which was partially blocked by 3-MA, the inhibitor of autophagy. In addition, VEGF165 up-regulate the expressions of Beclin1 and LC3B in co-culture cells. The results suggested that VEGF165 attenuated the spinal cord injury by inhibiting the inflammation and increasing the autophagy function.

Neuroprotective effect of rapamycin on spinal cord injury via activation of the Wnt/β-catenin signaling pathway

The Wnt/β-catenin signaling pathway plays a crucial role in neural development, axonal guidance, neuropathic pain remission and neuronal survival. In this study, we initially examined the effect of rapamycin on the Wnt/β-catenin signaling pathway after spinal cord injury, by intraperitoneally injecting spinal cord injured rats with rapamycin over 2 days. Western blot analysis and immunofluorescence staining were used to detect the expression levels of β-catenin protein, ca-spase-3 protein and brain-derived neurotrophic factor protein, components of the Wnt/β-catenin signaling pathway. Rapamycin increased the levels of β-catenin and brain-derived neurotrophic factor in the injured spinal cord, improved the pathological morphology at the injury site, reduced the loss of motor neurons, and promoted motor functional recovery in rats after spinal cord injury. Our experimental findings suggest that the neuroprotective effect of rapamycin intervention is mediated through activation of the Wnt/β-catenin signaling pathway after spinal cord injury.

Effect of implantation of biodegradable magnesium alloy on BMP-2 expression in bone of ovariectomized osteoporosis rats

The study was focused on the implantation of a biodegradable AZ31 magnesium alloy into the femoral periosteal of the osteoporosis modeled rats. The experimental results showed that after 4weeks implantation of AZ31 alloy in the osteoporosis modeled rats, the expression of BMP-2 in bone tissues of the rats was much enhanced, even higher than the control group, which should promote the bone formation and be beneficial for reducing the harmful effect of osteoporosis. Results of HE stains showed that the implantation of AZ31 alloy did not have obvious pathological changes on both the liver and kidney of the animal.

HMGB1/Advanced Glycation End Products (RAGE) does not aggravate inflammation but promote endogenous neural stem cells differentiation in spinal cord injury

Receptor for advanced glycation end products (RAGE) signaling is involved in a series of cell functions after spinal cord injury (SCI). Our study aimed to elucidate the effects of RAGE signaling on the neuronal recovery after SCI. In vivo, rats were subjected to SCI with or without anti-RAGE antibodies micro-injected into the lesion epicenter. We detected Nestin/RAGE, SOX-2/RAGE and Nestin/MAP-2 after SCI by Western blot or immunofluorescence (IF). We found that neural stem cells (NSCs) co-expressed with RAGE were significantly activated after SCI, while stem cell markers Nestin and SOX-2 were reduced by RAGE blockade. We found that RAGE inhibition reduced nestin-positive NSCs expressing MAP-2, a mature neuron marker. RAGE blockade does not improve neurobehavior Basso, Beattie and Bresnahan (BBB) scores; however, it damaged survival of ventral neurons via Nissl staining. Through in vitro study, we found that recombinant HMGB1 administration does not lead to increased cytokines of TNF-α and IL-1β, while anti-RAGE treatment reduced cytokines of TNF-α and IL-1β induced by LPS via ELISA. Meanwhile, HMGB1 increased MAP-2 expression, which was blocked after anti-RAGE treatment. Hence, HMGB1/RAGE does not exacerbate neuronal inflammation but plays a role in promoting NSCs differentiating into mature neurons in the pathological process of SCI.

Netrin-1 Improves Functional Recovery through Autophagy Regulation by Activating the AMPK/mTOR Signaling Pathway in Rats with Spinal Cord Injury

Autophagy is an process for the degradation of cytoplasmic aggregated proteins and damaged organelles and plays an important role in the development of SCI. In this study, we investigated the therapeutic effect of Netrin-1 and its potential mechanism for autophagy regulation after SCI. A rat model of SCI was established and used for analysis. Results showed that administration of Netrin-1 not only significantly enhanced the phosphorylation of AMP-activated protein kinase (AMPK) but also reduced the phosphorylation of mammalian target of rapamycin (mTOR) and P70S6K. In addition, the expression of Beclin-1 and the ratio of the light-chain 3B-II (LC3B-II)/LC3B-I in the injured spinal cord significantly increased in Netrin-1 group than those in SCI group. Moreover, the ratio of apoptotic neurons in the anterior horn of the spinal cord and the cavity area of spinal cord significantly decreased in Netrin-1 group compared with those in SCI group. In addition, Netrin-1 not only preserved motor neurons but also significantly improved motor fuction of injured rats. These results suggest that Netrin-1 improved functional recovery through autophagy stimulation by activating the AMPK/mTOR signaling pathway in rats with SCI. Thus, Netrin-1 treatment could be a novel therapeutic strategy for SCI.

Green tea polyphenols protect spinal cord neurons against hydrogen peroxide-induced oxidative stress

Green tea polyphenols are strong antioxidants and can reduce free radical damage. To investigate their neuroprotective potential, we induced oxidative damage in spinal cord neurons using hydrogen peroxide, and applied different concentrations (50–200 μg/mL) of green tea polyphenol to the cell medium for 24 hours. Measurements of superoxide dismutase activity, malondialdehyde content, and expression of apoptosis-related genes and proteins revealed that green tea polyphenol effectively alleviated oxidative stress. Our results indicate that green tea polyphenols play a protective role in spinal cord neurons under oxidative stress.

Activation of the Nrf2/ARE signaling pathway by probucol contributes to inhibiting inflammation and neuronal apoptosis after spinal cord injury

The nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway plays an essential role in the cellular antioxidant and anti-inflammatory responses. Spinal cord injury (SCI) results in a massive release of inflammatory factors and free radicals, which seriously compromise nerve recovery and axon regeneration. In this study, we examined the efficacy of probucol on anti-inflammatory responses and functional recovery after SCI by activating the Nrf2/ARE signaling pathway. We also investigated the mechanism by which inflammation is inhibited in this process. We found that treatment of injured rats with probucol significantly increased levels of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1), while levels of inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were decreased. This was associated with a reduction in neural cell apoptosis and promotion of nerve function recovery. These results demonstrate that the neuroprotective effects of probucol after SCI are mediated by activation of the Nrf2/ARE signaling pathway. These findings indicate that the anti-inflammatory effects of probucol represent a viable treatment for improving functional recovery following SCI.The nuclear erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway plays an essential role in the cellular antioxidant and anti-inflammatory responses. Spinal cord injury (SCI) results in a massive release of inflammatory factors and free radicals, which seriously compromise nerve recovery and axon regeneration. In this study, we examined the efficacy of probucol on anti-inflammatory responses and functional recovery after SCI by activating the Nrf2/ARE signaling pathway. We also investigated the mechanism by which inflammation is inhibited in this process. We found that treatment of injured rats with probucol significantly increased levels of Nrf2, heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1), while levels of inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were decreased. This was associated with a reduction in neural cell apoptosis and promotion of nerve function recovery. These results demonstrate that the neuroprotective effects of probucol after SCI are mediated by activation of the Nrf2/ARE signaling pathway. These findings indicate that the anti-inflammatory effects of probucol represent a viable treatment for improving functional recovery following SCI.

The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats

Our previous findings indicated that treatment with Netrin-1 could improve functional recovery through the stimulation of autophagy, by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway in rats following spinal cord injury (SCI). However, the underlying mechanisms were not elucidated. The purpose of this study was to investigate the underlying mechanisms by which Netrin-1 promotes autophagy and improves functional recovery after SCI. Following controlled SCI in Sprague-Dawley rats, we observed that the autophagic flux in neurons was impaired, as reflected by the accumulation of light chain 3-II (LC3-II)-positive and LC3-positive autophagosomes (APs), accompanied by the accumulation of the autophagic substrate, Sequestosome 1 (SQSTM1; also known as p62). Our results showed that treatment with Netrin-1 increases the levels of the lysosomal protease cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1), through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the AMPK/mTOR signaling pathway. In addition, this enhancement of lysosomal biogenesis correlated strongly with the restoration of autophagic flux, inhibition of neural apoptosis and improved functional recovery. Suppression of lysosomal biogenesis via the inhibition of the nuclear translocation of TFEB by Compound C abolished this restoration of autophagic flux and the functional recovery effects of Netrin-1 following SCI. Taken together, these results indicate that Netrin-1 enhances lysosomal biogenesis by regulating the nuclear translocation of TFEB via the AMPK/mTOR signaling pathway. Furthermore, the enhancement of lysosomal biogenesis by Netrin-1 following SCI promotes autophagic flux and improves functional recovery in rats. Thus, the regulation of lysosomal biogenesis by modulating the nuclear localization of TFEB might be a novel approach for the treatment of SCI.