Zhaoliang Shen

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.

Metformin preconditioning provide neuroprotection through enhancement of autophagy and suppression of inflammation and apoptosis after spinal cord injury.

Spinal cord injury (SCI) is one of the most serious nervous system disorders characterised by high morbidity and disability. Inflammatory and autophagy responses play an important role in the development of SCI. Metformin, a first-line drug for type-2 diabetes, features autophagy promotion as well as anti-inflammatory and anti-apoptotic properties in the nervous system. In this study, we investigated the neuroprotection effects of metformin preconditioning on rats after SCI. Results of Basso, Beattie and Bresnahan scores, HE staining and Nissl staining showed that the function and quantity of motor neurons were protected by metformin after SCI. Western blot revealed that the expression of Beclin-1 and LC3B-II was enhanced, and the phosphorylation levels of the mammalian target of rapamycin (mTOR) protein and p70S6K were reduced by metformin after SCI. Metformin significantly reduced the expression of NF-κB. Moreover, Western blot and immunofluorescence results indicated that caspase 3 activation was reduced, whereas bcl-2 level was significantly increased by metformin. Hence, metformin attenuated SCI by inhibiting apoptosis and inflammation and enhancing the autophagy via the mTOR/p70S6K signalling pathway.

Probucol inhibits neural cell apoptosis via inhibition of mTOR signaling pathway after spinal cord injury

Autophagy plays an essential role in neurodevelopment, axonal guidance, neuropathic pain remission, and neuronal survival. Inhibiting the mammalian target of rapamycin (mTOR) signaling pathway can induce the occurrence of autophagy. In this study, we initially detected the effect of probucol on autophagy after spinal cord injury (SCI) by intraperitoneally injecting spinal cord-injured rats with probucol for 7days. The levels of Beclin1 and LC3B were evidently enhanced at 7days post-operation. However, the increase in the phosphorylated AMP-activated protein kinase (AMPK) protein and the decrease in ribosomal protein S6 kinase p70 subtype (p70S6K) phosphorylation level simultaneously occurred after SCI. Moreover, the expression levels of apoptosis-related proteins of Caspase-3, Caspase-9, and Bax were significantly reduced. Immunofluorescence results indicated that the expression of Caspase-3 protein was evidently decreased and that of Beclin-1 protein was increased by probucol. Nissl staining and Basso, Beattie, and Bresnahan scores showed that the quantity and function of motor neurons were visibly preserved by probucol after SCI. This study showed that probucol inhibited the mTOR signaling pathway to induce autophagy, reduce neural cell apoptosis and promote recovery of neurological function after SCI.

Atorvastatin activates autophagy and promotes neurological function recovery after spinal cord injury

Atorvastatin, a lipid-lowering medication, provides neuroprotective effects, although the precise mechanisms of action remain unclear. Our previous studies confirmed activated autophagy following spinal cord injury, which was conducive to recovery of neurological functions. We hypothesized that atorvastatin could also activate autophagy after spinal cord injury, and subsequently improve recovery of neurological functions. A rat model of spinal cord injury was established based on the Allen method. Atorvastatin (5 mg/kg) was intraperitoneally injected at 1 and 2 days after spinal cord injury. At 7 days post-injury, western blot assay, reverse transcription-polymerase chain reaction, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining results showed increased Beclin-1 and light chain 3B gene and protein expressions in the spinal cord injury + atorvastatin group. Additionally, caspase-9 and caspase-3 expression was decreased, and the number of TUNEL-positive cells was reduced. Compared with the spinal cord injury + saline group, Basso, Beattie, and Bresnahan locomotor rating scale scores significantly increased in the spinal cord injury + atorvastatin group at 14-42 days post-injury. These findings suggest that atorvastatin activated autophagy after spinal cord injury, inhibited apoptosis, and promoted recovery of neurological function.

Melatonin Inhibits Neural Cell Apoptosis and Promotes Locomotor Recovery via Activation of the Wnt/β-Catenin Signaling Pathway After Spinal Cord Injury

The spinal cord is highly sensitive to spinal cord injury (SCI) by external mechanical damage, resulting in irreversible neurological damage. Activation of the Wnt/β-catenin signaling pathway can effectively reduce apoptosis and protect against SCI. Melatonin, an indoleamine originally isolated from bovine pineal tissue, exerts neuroprotective effects after SCI through activation of the Wnt/β-catenin signaling pathway. In this study, we demonstrated that melatonin exhibited neuroprotective effects on neuronal apoptosis and supported functional recovery in a rat SCI model by activating the Wnt/β-catenin signaling pathway. We found that melatonin administration after SCI significantly upregulated the expression of low-density lipoprotein receptor related protein 6 phosphorylation (p-LRP-6), lymphoid enhancer factor-1 (LEF-1) and β-catenin protein in the spinal cord. Melatonin enhanced motor neuronal survival in the spinal cord ventral horn and improved the locomotor functions of rats after SCI. Melatonin administration after SCI also reduced the expression levels of Bax and cleaved caspase-3 in the spinal cord and the proportion of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) positive cells, but increased the expression level of Bcl-2. These results suggest that melatonin attenuated SCI by activating the Wnt/β-catenin signaling pathway.

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.

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.

Berberine attenuated pro-inflammatory factors and protect against neuronal damage via triggering oligodendrocyte autophagy in spinal cord injury

Berberine exerts neuroprotective effect in neuroinflammation and neurodegeneration disease. However, berberine effect in acute spinal cord injury is yet to be elucidated. Herein, we investigated the neuroprotective effect of berberine in spinal cord injury (SCI). Sprague-Dawley rats were subjected to SCI by an intraperitoneal injection of berberine post-injury. The neurobehavioral recovery, cytokines of pro-inflammatory factors (TNF-α and IL-1β), autophagy-related proteins (LC3B, ATG16L, ATG7), and apoptosis-related protein cleaved caspase-3 were determined. The expressions of 2′, 3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase), marker of oligodendrocyte, autophagy-related proteins ATG5 and neurons at the ventral horn were assessed. In vitro, the contents of the pro-inflammatory factors, TNF-α and IL-1β, were detected in the lipopolysaccharide (LPS)-treated primary spinal neuron. Berberine significantly improved the neurobehavior BBB score and attenuated the cytokines of pro-inflammatory factors in cerebrospinal fluid post-SCI. In addition, berberine upregulated CNPase positive oligodendrocyte expressing ATG5, promoted neuronal survival and reduced the cleaved caspase-3 expression after SCI. In primary spinal neuron, the LPS-induced inflammatory factors could be reduced by berberine, whereas the autophagy inhibitor, 3-Methyladenine reverses the effect. Berberine attenuated inflammation of the injured spinal cord and reduced the neuronal apoptosis via triggering oligodendrocyte autophagy in order to promote neuronal recovery.