Shui Guan

Alpinia protocatechuic acid protects against oxidative damage in vitro and reduces oxidative stress in vivo.

In this study, the neuroprotective effects of Alpinia protocatechuic acid (PCA), a phenolic compound isolated from the dried fruits of Alpinia Oxyphylla Miq. was found. The protective effect of Alpinia PCA against H2O2-induced oxidative damage on PC12 cells was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Rats were injected intraperitoneally with Alpinia PCA at a dose of 5mg/kg per day for 7 days, behavioral testing was performed in Y-maze. In order to make clear the neuroprotective mechanism of Alpinia PCA, the activities of endogenous antioxidants and the content of lipid peroxide in brain were assayed. The results proved that Alpinia PCA significantly prevented the H2O2-induced reduction in cell survival, improved the cognition of aged rats, reduced the content of lipid peroxide, increased the activity of glutathione peroxidase and superoxide dismutase. All these suggested that Alpinia PCA was a potential neuroprotective agent and its neuroprotective effects were achieved at least partly by promoting endogenous antioxidant enzymatic activities and inhibiting free radical generation.

Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Anti‐ageing effects of protocatechuic acid from Alpinia on spleen and liver antioxidative system of senescent mice

The effects of Alpinia protocatechuic acid (PCA) on spleen and liver antioxidant system in aged rats have been studied. Alpinia PCA, a phenolic compound, was first isolated from the dried fruits of Alpinia Oxyphylla Miq. in our laboratory. Young and aged rats were injected intraperitoneally with Alpinia PCA at single doses of 5 mg kg−1 (low dose) or 10 mg kg−1 (high dose) per day for 7 days. The activities of endogenous antioxidants and the content of lipid peroxide in spleen and liver were assayed. Compared with young group, aged rats had significantly lower splenic weights, lower activities of glutathione peroxidase (GSH‐PX) and catalase (CAT), higher level of malondialdehyde (MDA) in spleen and liver. The results proved that Alpinia PCA significantly elevated the splenic weights, increased the activities of GSH‐PX and CAT and decreased the MDA level of aged rats. All these suggested that Alpinia PCA was a potential anti‐ageing agent, and its effects on spleen and liver were achieved at least partly by promoting endogenous antioxidant enzymatic activities and normalizing age‐associated alterations. It may be therapeutically useful to minimize age‐associated disorders where oxidative damage is the major cause. Copyright

Chitosan/gelatin porous scaffolds containing hyaluronic acid and heparan sulfate for neural tissue engineering

The novel chitosan (Cs)/gelatin (Gel) porous scaffolds containing hyaluronic acid (HA) and heparan sulfate (HS) were fabricated via freeze-drying technique, and their physicochemical characteristics including pore size, porosity, water absorption, and in vitro degradation and biocompatibility were investigated. It was demonstrated that the Cs/Gel/HA/HS composite scaffolds had highly homogeneous and interconnected pores with porosity above 96% and average pore size ranging from 90 to 140 μm and a controllable degradation rate. The scanning electron microscopic images, cell viability assay, and fluorescence microscopy observation revealed that the presence of HA and HS in the scaffolds significantly promoted initial neural stem and progenitor cells (NS/PCs) adhesion and supported long-time growth in three-dimensional environment. Moreover, NS/PCs also maintained mutilineage differentiation potentials with enhanced neuronal differentiation upon induction in the Cs/Gel/HA/HS composite scaffolds in relation to Cs/Gel scaffolds. These results indicated that the Cs/Gel/HA/HS composite scaffolds were suitable for neural cells’ adhesion, survival, and growth and could offer new and important options for neural tissue engineering applications.

Protocatechuic acid promotes the neuronal differentiation and facilitates survival of phenotypes differentiated from cultured neural stem and progenitor cells

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, plays crucial roles in the proliferation and neuroprotection of cultured neural stem and progenitor cells (NS/PCs) in our previous study. However, whether PCA modulates the differentiation of NS/PCs has remained to be elucidated. In this study, we show that PCA can promote the neuronal differentiation combined with fetal bovine serum (FBS) in vitro, although it cannot initiate the differentiation of NS/PCs by itself. Moreover, PCA is able to induce neuronal maturation and efficiently promote neurite outgrowth. On the other hand, PCA facilitates survival of phenotypes differentiated from cultured NS/PCs, which was associated with an increased percentage of the cellular viability and a decreased percentage of cells undergoing apoptosis under differentiation conditions. In addition, PCA-induced survival is also mediated with the activating of endogenous antioxidant enzymes. These results suggest that PCA may serve as a useful reference for future studies in designing stem cell strategies to promote brain recovery and repair in neurodegenerative diseases.

Protocatechuic acid from Alpinia oxyphylla promotes migration of human adipose tissue-derived stromal cells in vitro

Human adipose tissue-derived stromal cells (hADSCs) demonstrate promising potential in various clinical applications, including the transplantation to regenerate injured or degenerative tissues. The migration of engrafted hADSCs to the correct site of injure is essential for the curative effect of stem cell therapy. We found that protocatechuic acid (PCA) from Alpinia (A.) oxyphylla could promote the migration capacity of hADSCs through transwell coated with gelatin in vitro. PCA enhanced the cell migration rate in a dose-dependent and time-dependent manner. Meanwhile, RT-PCR and quantitative RT-PCR analysis revealed the elevation of membrane-type matrix metalloproteinase-1 (MT1-MMP) mRNA expression in 1.5 mM PCA-treated hADSCs. In the supernatants of these cells, the active matrix metalloproteinase-2 (MMP-2) increased compared with control cells with zymography. Moreover, the promotion of cell migration by PCA could be effectively and obviously inhibited by anti-MT1-MMP or anti-MMP-2 antibodies. Furthermore, flow cytometric analysis of the cell surface antigens, osteogenic induction, adipogenic induction and cardiomyocyte-like cell induction demonstrated that hADSCs retained their functional characteristics of multipotential mesenchymal progenitors after PCA treatment. These results suggest that PCA from A. oxyphylla promote the migration of hADSCs in vitro, which is partially due to the increased expression of MT1-MMP and the promotion of MMP-2 zymogen activation.

Chitosan/gelatin porous scaffolds assembled with conductive poly(3,4-ethylenedioxythiophene) nanoparticles for neural tissue engineering

Electroactive biomaterials are widely explored as scaffolds for nerve tissue regeneration. Poly(3,4-ethylenedioxythiophene) (PEDOT) is a conductive polymer that has been chosen to construct tissue engineered scaffolds because of its excellent conductivity and non-cytotoxicity. In the present study, an electrically conductive scaffold was prepared by assembling PEDOT on a chitosan/gelatin (Cs/Gel) porous scaffold surface via in situ interfacial polymerization. The hydrophilic Cs/Gel hydrogel was used as a template, and PEDOT nanoparticles were uniformly assembled on the scaffold surface. The static polymerization of the 3,4-ethylenedioxythiophene (EDOT) monomer at the interface between the aqueous phase and the organic phase was accompanied by the formation of the PEDOT-assembled Cs/Gel scaffolds. PEDOT/Cs/Gel scaffolds were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. The results confirmed the deposition of PEDOT nanoparticles with the mean diameter of 50 nm on the Cs/Gel scaffold channel surface. Compared to the Cs/Gel scaffold, the incorporation of PEDOT on the scaffold increased the electrical conductivity, hydrophilicity, mechanical properties and thermal stability, whereas decreased the water absorption and biodegradation. For biocompatibility, PEDOT/Cs/Gel scaffolds, especially the 2PEDOT/Cs/Gel scaffold group, significantly promoted neuron-like rat pheochromocytoma (PC12) cell adhesion and proliferation. The results of both the gene expression and protein level assessments suggested that the PEDOT-assembled Cs/Gel scaffold enhanced the PC12 cellular neurite growth with higher protein and gene expression levels. This is the first report on the construction of a conductive PEDOT/Cs/Gel porous scaffold via an in situ interfacial polymerization method, and the results demonstrate that it may be a promising conductive scaffold for neural tissue engineering.

Pyrroloquinoline quinone against glutamate-induced neurotoxicity in cultured neural stem and progenitor cells

Pyrroloquinoline quinone (PQQ), as a well‐known redox enzyme cofactor, has been proven to play important roles in the regulation of cellular growth and development in mammals. Numerous physiological and medicinal functions of PQQ have so far been reported although its effect on neural stem and progenitor cells (NS/PCs) and the potential mechanism were even rarely investigated. In this study, the neuroprotective effects of PQQ were observed by pretreatment of NS/PCs with PQQ before glutamate injury, and the possible mechanisms were examined. PQQ stimulated cell proliferation and markedly attenuated glutamate‐induced cell damage in a dose‐dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PQQ pretreatment showed its significant effect on protecting NS/PCs against glutamate‐induced apoptosis/necrosis. PQQ neuroprotection was associated with the decrease of intracellular reactive oxygen species (ROS) production, the increase of glutathione (GSH) levels, and the decrease of caspase‐3 activity. In addition, pretreatment with PQQ also significantly enhanced the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in the NS/PCs exposed to glutamate. These results suggest that PQQ can protect NS/PCs against glutamate toxicity associated with ROS‐mediated mitochondrial pathway, indicating a useful chemical for the clinical application of NS/PCs.

Effect of the neurosphere size on the viability and metabolism of neural stem/progenitor cells

The objective of this study was to investigate the metabolic activity and viability of the mouse neural stem/progenitor cells (NSPCs) affected by the size of neurospheres. NSPCs dissociated from the forebrain of embryonic 14 days (E14) mice were cultured in flask for 120 h. During cultivation, the diameter distribution of neurospheres, cell viability and metabolic activities were monitored, together with the concentrations of glucose, lactate, glutamine and ammonia in the media. The results show that cell activity decreased with the increment of neurospheres size. When the diameter reached about 100 μm and the concentration of glucose and glutamine were 36.38 and 1.33 mmol/L, the growth of central cells in neurospheres began to surface. Furthermore, when the diameter reached about 100 to 150 μm and the concentrations of glucose and glutamine were 31.11 and 1.15 mmol/L, simultaneously, the death rate of NSPCs was larger than that of the newly born cells within the neurospheres. The metabolic activity of the cells declined to a very low level. This observation can be explained by diffusion limitation of nutrients and metabolic waste inside neurosphere. In conclusion, the mass transfer will be limited when the neurospheres size reaches a critical value of 100 to 150 μm and beyond this critical value, serious impact of nutrient supply and metabolites on the cell viability and metabolism occurs. Key words: Neural stem/progenitor cells (NSPCs), neurospheres, critical size, metabolism, mass transfer.

Mass Transfer Analysis of Growth and Substance Metabolism of NSCs Cultured in Collagen-Based Scaffold In Vitro

The aim of this study is to analyze the growth and substance metabolism of neural stem cells (NSCs) cultured in biological collagen-based scaffolds. Mass transfer and metabolism model of glucose, lactic acid, and dissolved oxygen (DO) were established and solved on MATLAB platform to obtain the concentration distributions of DO, glucose, and lactic acid in culture system, respectively. Calculation results showed that the DO influenced their normal growth and metabolism of NSCs mostly in the in vitro culture within collagen-based scaffolds. This study also confirmed that 2-mm thickness of collagen scaffold was capable of in vitro cultivation and growth of NSCs with an inoculating density of 1 × 106 cells/mL.