Fenzan Wu

The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in vivo.

Hypertrophic scars (HTS) and keloids are challenging problems. Their pathogenesis results from an overproduction of fibroblasts and excessive deposition of collagen. Studies suggest a possible anti-scarring effect of basic fibroblast growth factor (bFGF) during wound healing, but the precise mechanisms of bFGF are still unclear. In view of this, we investigated the therapeutic effects of bFGF on HTS animal model as well as human scar fibroblasts (HSF) model. We show that bFGF promoted wound healing and reduced the area of flattened non-pathological scars in rat skin wounds and HTS in the rabbit ear. We provide evidence of a new therapeutic strategy: bFGF administration for the treatment of HTS. The scar elevation index (SEI) and epidermal thickness index (ETI) was also significantly reduced. Histological reveal that bFGF exhibited significant amelioration of the collagen tissue. bFGF regulated extracellular matrix (ECM) synthesis and degradation via interference in the collagen distribution, the α-smooth muscle actin (α-SMA) and transforming growth factor-1 (TGF-β1) expression. In addition, bFGF reduced scarring and promoted wound healing by inhibiting TGFβ1/SMAD-dependent pathway. The levels of fibronectin (FN), tissue inhibitor of metalloproteinase-1 (TIMP-1) collagen I, and collagen III were evidently decreased, and matrix metalloproteinase-1 (MMP-1) and apoptosis cells were markedly increased. These results suggest that bFGF possesses favorable therapeutic effects on hypertrophic scars in vitro and in vivo, which may be an effective cure for human hypertrophic scars.

Exogenous Basic Fibroblast Growth Factor Inhibits ER Stress–Induced Apoptosis and Improves Recovery from Spinal Cord Injury

To investigate the mechanism of endoplasmic reticulum (ER) stress–induced apoptosis as well as the protective action of basic fibroblast growth factor (bFGF) both in vivo and in vitro.

Regulation of Autophagy and Ubiquitinated Protein Accumulation by bFGF Promotes Functional Recovery and Neural Protection in a Rat Model of Spinal Cord Injury

The role of autophagy in the recovery of spinal cord injury remains controversial; in particular, the mechanism of autophagy regulated degradation of ubiquitinated proteins has not been discussed to date. In this study, we investigated the protective role of basic fibroblast growth factor (bFGF) both in vivo and in vitro and demonstrated that excessive autophagy and ubiquitinated protein accumulation is involved in the rat model of trauma. bFGF administration improved recovery and increased the survival of neurons in spinal cord lesions in the rat model. The protective effect of bFGF is related to the inhibition of autophagic protein LC3II levels; bFGF treatment also enhances clearance of ubiquitinated proteins by p62, which also increases the survival of neuronal PC-12 cells. The activation of the downstream signals of the PI3K/Akt/mTOR pathway by bFGF treatment was detected both in vivo and in vitro. Combination therapy including the autophagy activator rapamycin partially abolished the protective effect of bFGF. The present study illustrates that the role of bFGF in SCI recovery is related to the inhibition of excessive autophagy and enhancement of ubiquitinated protein clearance via the activation of PI3K/Akt/mTOR signaling. Overall, our study suggests a new trend for bFGF drug development for central nervous system injuries and sheds light on protein signaling involved in bFGF action.

Nerve growth factor improves functional recovery by inhibiting endoplasmic reticulum stress-induced neuronal apoptosis in rats with spinal cord injury.

BackgroundEndoplasmic reticulum (ER) stress-induced apoptosis plays a major role in various diseases, including spinal cord injury (SCI). Nerve growth factor (NGF) show neuroprotective effect and improve the recovery of SCI, but the relations of ER stress-induced apoptosis and the NGF therapeutic effect in SCI still unclear.MethodsYoung adult female Sprague-Dawley rats’s vertebral column was exposed and a laminectomy was done at T9 vertebrae and moderate contusion injuries were performed using a vascular clip. NGF stock solution was diluted with 0.9% NaCl and administered intravenously at a dose of 20 μg/kg/day after SCI and then once per day until they were executed. Subsequently, the rats were executed at 1d, 3 d, 7d and 14d. The locomotor activities of SCI model rats were tested by the 21-point Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test and footprint analysis. In addition, Western blot analysis was performed to identify the expression of ER-stress related proteins including CHOP, GRP78 and caspase-12 both in vivo and in vitro. The level of cell apoptosis was determined by TUNEL in vivo and Flow cytometry in vitro. Relative downstream signals Akt/GSK-3β and ERK1/2were also analyzed with or without inhibitors in vitro.ResultsOur results demonstrated that ER stress-induced apoptosis was involved in the injury of SCI model rats. NGF administration improved the motor function recovery and increased the neurons survival in the spinal cord lesions of the model rats. NGF decreases neuron apoptosis which measured by TUNEL and inhibits the activation of caspase-3 cascade. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 are inhibited by NGF treatment. Meanwhile, NGF administration also increased expression of growth-associated protein 43 (GAP43). The administration of NGF activated downstream signals Akt/GSK-3β and ERK1/2 in ER stress cell model in vitro.ConclusionThe neuroprotective role of NGF in the recovery of SCI is related to the inhibition of ER stress-induced cell death via the activation of downstream signals, also suggested a new trend of NGF translational drug development in the central neural system injuries which involved in the regulation of chronic ER stress.

Endoplasmic reticulum stress: relevance and therapeutics in central nervous system diseases.

Endoplasmic reticulum (ER) stress plays an important role in a range of neurological disorders, such as neurodegenation diseases, cerebral ischemia, spinal cord injury, sclerosis, and diabetic neuropathy. Protein misfolding and accumulation in the ER lumen initiate unfolded protein response in energy-starved neurons which are relevant to toxic effects. In neurological disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, ER dysfunction is well recognized, but the mechanisms remain unclear. In stroke and ischemia, spinal cord injury, and amyotrophic lateral sclerosis, chronic activation of ER stress is considered as main pathogeny which causes neuronal disorders. By targeting components of these ER signaling responses, to explore clinical treatment strategies or new drugs in CNS neurological diseases might become possible and valuable in the future.

Gelatin Nanostructured Lipid Carriers Incorporating Nerve Growth Factor Inhibit Endoplasmic Reticulum Stress-Induced Apoptosis and Improve Recovery in Spinal Cord Injury

Clinical translation of growth factor therapies faces multiple challenges; the most significant one is the short half-life of the naked protein. Gelatin nanostructured lipid carriers (GNLs) had previously been used to encapsulate the basic fibroblast growth factor to enhance the functional recovery in hemiparkinsonian rats. In this research, we comparatively study the enhanced therapy between nerve growth factor (NGF) loaded GNLs (NGF-GNLs) and NGF only in spinal cord injury (SCI). The effects of NGF-GNLs and NGF only were tested by the Basso-Beattie-Bresnahan (BBB) locomotion scale, inclined plane test, and footprint analysis. Western blot analysis and immunofluorescent staining were further performed to identify the expression of ER stress-related proteins, neuron-specific marker neuronal nuclei (NeuN), and growth-associated protein 43 (GAP43). Correlated downstream signals Akt/GSK-3β and ERK1/2 were also analyzed with or without inhibitors. Results showed that NGF-GNLs, compared to NGF only, enhanced the neuroprotection effect in SCI rats. The ER stress-induced apoptosis response proteins CHOP, GRP78 and caspase-12 inhibited by NGF-GNL treatment were more obvious. Meanwhile, NGF-GNLs in the recovery of SCI are related to the inhibition of ER stress-induced cell death via the activation of downstream signals PI3K/Akt/GSK-3β and ERK1/2.

Endoplasmic reticulum stress-induced neuronal inflammatory response and apoptosis likely plays a key role in the development of diabetic encephalopathy

We assumed that diabetic encephalopathy (DEP) may be induced by endoplasmic reticulum (ER)-mediated inflammation and apoptosis in central nervous system. To test this notion, here we investigated the neuronal ER stress and associated inflammation and apoptosis in a type 2 diabetes model induced with high-fat diet/streptozotocin in Sprague-Dawley rats. Elevated expressions of ER stress markers, including glucose-regulated protein 78 (GRP78), activating transcription factor-6 (ATF-6), X-box binding protein-1 (XBP-1), and C/EBP homologous protein, and phosphor-Jun N-terminal kinase (p-JNK) were evident in the hippocampus CA1 of diabetic rats. These changes were also accompanied with the activation of NF-κB and the increased levels of inflammatory cytokines, tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6). Mechanistic study with in vitro cultured hippocampus neurons exposed to high glucose (HG), which induced a diabetes-like effects, shown by increased ER stress, JNK and NF-κB activation, and inflammatory response. Inhibition of ER stress by 4-phenylbutyrate (4-PBA) or blockade of JNK activity by specific inhibitor or transfection of DN-JNK attenuated HG-induced inflammation and associated apoptosis. To validate the in vitro finding, in vivo application of 4-PBA resulted in a significant reduction of diabetes-induced neuronal ER stress, inflammation and cell death, leading to the prevention of DEP. These results suggest that diabetes-induced neuronal ER stress plays the critical role for diabetes-induced neuronal inflammation and cell death, leading to the development of DEP.

Regulation of Caveolin-1 and Junction Proteins by bFGF Contributes to the Integrity of Blood-Spinal Cord Barrier and Functional Recovery.

The blood–spinal cord barrier (BSCB) plays important roles in the recovery of spinal cord injury (SCI), and caveolin-1 is essential for the integrity and permeability of barriers. Basic fibroblast growth factor (bFGF) is an important neuroprotective protein and contributes to the survival of neuronal cells. This study was designed to investigate whether bFGF is beneficial for the maintenance of junction proteins and the integrity of the BSCB to identify the relations with caveolin-1 regulation. We examined the integrity of the BSCB with Evans blue dye and fluorescein isothiocyanate–dextran extravasation, measured the junction proteins and matrix metalloproteinases, and evaluated the locomotor function recovery. Our data indicated that bFGF treatment improved the recovery of BSCB and functional locomotion in contusive SCI model rats, reduced the expression and activation of matrix metalloproteinase-9, and increased the expressions of caveolin-1 and junction proteins, including occludin, claudin-5, p120-catenin, and β-catenin. In the brain, in microvascular endothelial cells, bFGF treatment increased the levels of junction proteins, caveolin-1 small interfering RNA abolished the protective effect of bFGF under oxygen–glucose deprivation conditions, and the expression of fibroblast growth factor receptor 1 and co-localization with caveolin-1 decreased significantly, which could not be reversed by bFGF treatment. These findings provide a novel mechanism underlying the beneficial effects of bFGF on the BSCB and recovery of SCI, especially the regulation of caveolin-1.

Valproate Attenuates Endoplasmic Reticulum Stress-Induced Apoptosis in SH-SY5Y Cells via the AKT/GSK3β Signaling Pathway

Endoplasmic reticulum (ER) stress-induced apoptosis plays an important role in a range of neurological disorders, such as neurodegenerative diseases, spinal cord injury, and diabetic neuropathy. Valproate (VPA), a typical antiepileptic drug, is commonly used in the treatment of bipolar disorder and epilepsy. Recently, VPA has been reported to exert neurotrophic effects and promote neurite outgrowth, but its molecular mechanism is still unclear. In the present study, we investigated whether VPA inhibited ER stress and promoted neuroprotection and neuronal restoration in SH-SY5Y cells and in primary rat cortical neurons, respectively, upon exposure to thapsigargin (TG). In SH-SY5Y cells, cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and the expression of ER stress-related apoptotic proteins such as glucose‑regulated protein (GRP78), C/EBP homologous protein (CHOP), and cleaved caspase-12/-3 were analyzed with Western blot analyses and immunofluorescence assays. To explore the pathway involved in VPA-induced cell proliferation, we also examined p-AKT, GSK3β, p-JNK and MMP-9. Moreover, to detect the effect of VPA in primary cortical neurons, immunofluorescence staining of β-III tubulin and Anti-NeuN was analyzed in primary cultured neurons exposed to TG. Our results demonstrated that VPA administration improved cell viability in cells exposed to TG. In addition, VPA increased the levels of GRP78 and p-AKT and decreased the levels of ATF6, XBP-1, GSK3β, p-JNK and MMP-9. Furthermore, the levels of the ER stress-induced apoptosis response proteins CHOP, cleaved caspase-12 and cleaved caspase-3 were inhibited by VPA treatment. Meanwhile, VPA administration also increased the ratio of Bcl-2/Bax. Moreover, VPA can maintain neurite outgrowth of primary cortical neurons. Collectively, the neurotrophic effect of VPA is related to the inhibition of ER stress-induced apoptosis in SH-SY5Y cells and the maintenance of neuronal growth. Collectively, our results suggested a new approach for the therapeutic function of VPA in neurological disorders and neuroprotection.

NaHS restores mitochondrial function and inhibits autophagy by activating the PI3K/Akt/mTOR signalling pathway to improve functional recovery after traumatic brain injury

Traumatic brain injury (TBI) is one of the most serious public health problems in the world. TBI causes neurological deficits by triggering secondary injuries. Hydrogen sulfide (H2S), a gaseous mediator, has been reported to exert neuroprotective effects in central nervous system diseases, such as TBI. However, the molecular mechanisms involved in this effect are still unclear. The present study was designed to explore the ability of NaHS, a H2S donor, to provide neuroprotection in a mouse model of TBI and to discover the associated molecular mechanisms of these protective effects. Here, we found that administration of NaHS not only maintained the integrity of the blood brain barrier (BBB), protected neurons from apoptosis, and promoted remyelination and axonal reparation but also protected mitochondrial function. In addition, we found that autophagy was inhibited after treatment with NaHS following TBI, an effect that was induced by activation of the PI3K/AKT/mTOR signalling pathway. Our study indicated that H2S treatment is beneficial for TBI, pointing to H2S as a potential therapeutic target for treating TBI.