The Therapeutic Effects of Blueberry-Treated Stem Cell-Derived Extracellular Vesicles in Ischemic Stroke.

An ischemic stroke, one of the leading causes of morbidity and mortality, is caused by ischemia and hemorrhage resulting in impeded blood supply to the brain. According to many studies, blueberries have been shown to have a therapeutic effect in a variety of diseases. Therefore, in this study, we investigated whether blueberry-treated mesenchymal stem cell (MSC)-derived extracellular vesicles (B-EVs) have therapeutic effects in in vitro and in vivo stroke models. We isolated the extracellular vesicles using cryo-TEM and characterized the particles and concentrations using NTA. MSC-derived extracellular vesicles (A-EVs) and B-EVs were round with a lipid bilayer structure and a diameter of ~150 nm. In addition, A-EVs and B-EVs were shown to affect angiogenesis, cell cycle, differentiation, DNA repair, inflammation, and neurogenesis following KEGG pathway and GO analyses. We investigated the protective effects of A-EVs and B-EVs against neuronal cell death in oxygen-glucose deprivation (OGD) cells and a middle cerebral artery occlusion (MCAo) animal model. The results showed that the cell viability was increased with EV treatment in HT22 cells. In the animal, the size of the cerebral infarction was decreased, and the behavioral assessment was improved with EV injections. The levels of NeuN and neurofilament heavy chain (NFH)-positive cells were also increased with EV treatment yet decreased in the MCAo group. In addition, the number of apoptotic cells was decreased with EV treatment compared with ischemic animals following TUNEL and Bax/Bcl-2 staining. These data suggested that EVs, especially B-EVs, had a therapeutic effect and could reduce apoptotic cell death after ischemic injury.

Neuroprotective Effects of the Neural-Induced Adipose-Derived Stem Cell Secretome against Rotenone-Induced Mitochondrial and Endoplasmic Reticulum Dysfunction.

Mesenchymal stem cells (MSCs) have therapeutic effects on neurodegenerative diseases (NDDs) known by their secreted molecules, referred to as the "secretome". The mitochondrial complex I inhibitor, rotenone (ROT), reproduces α-synuclein (α-syn) aggregation seen in Parkinson's disease (PD). In this present study, we examined the neuroprotective effects of the secretome from neural-induced human adipose tissue-derived stem cells (NI-ADSC-SM) during ROT toxicity in SH-SY5Y cells. Exposure to ROT significantly impaired the mitophagy by increased LRRK2, mitochondrial fission, and endoplasmic reticulum (ER) stress (ERS). ROT also increased the levels of calcium (Ca2+), VDAC, and GRP75, and decreased phosphorylated (p)-IP3R Ser1756/total (t)-IP3R1. However, NI-ADSC-SM treatment decreased Ca2+ levels along with LRRK2, insoluble ubiquitin, mitochondrial fission by halting p-DRP1 Ser616, ERS by reducing p-PERK Thr981, p-/t-IRE1α, p-SAPK, ATF4, and CHOP. In addition, NI-ADSC-SM restored the mitophagy, mitochondrial fusion, and tethering to the ER. These data suggest that NI-ADSC-SM decreases ROT-induced dysfunction in mitochondria and the ER, which subsequently stabilized tethering in mitochondria-associated membranes in SH-SY5Y cells.

Autophagy Signaling by Neural-Induced Human Adipose Tissue-Derived Stem Cell-Conditioned Medium during Rotenone-Induced Toxicity in SH-SY5Y Cells.

Rotenone (ROT) inhibits mitochondrial complex I, leading to reactive oxygen species formation, which causes neurodegeneration and alpha-synuclein (α-syn) aggregation and, consequently, Parkinson's disease. We previously found that a neurogenic differentiated human adipose tissue-derived stem cell-conditioned medium (NI-hADSC-CM) was protective against ROT-induced toxicity in SH-SY5Y cells. In the present study, ROT significantly decreased the phospho (p)-mTORC1/total (t)-mTOR, p-mTORC2/t-mTOR, and p-/t-ULK1 ratios and the ATG13 level by increasing the DEPTOR level and p-/t-AMPK ratio. Moreover, ROT increased the p-/t-Akt ratio and glycogen synthase kinase-3ß (GSK3ß) activity by decreasing the p-/t-ERK1/2 ratios and beclin-1 level. ROT also promoted the lipidation of LC3B-I to LC3B-II by inducing autophagosome formation in Triton X-100-soluble and -insoluble cell lysate fractions. Additionally, the levels of ATG3, 5, 7, and 12 were decreased, along with those of lysosomal LAMP1, LAMP2, and TFEB, leading to lysosomal dysfunction. However, NI-hADSC-CM treatment increased the p-mTORC1, p-mTORC2, p-ULK1, p-Akt, p-ERK1/2, ATG13, and beclin-1 levels and decreased the p-AMPK level and GSK3ß activity in response to ROT-induced toxicity. Additionally, NI-hADSC-CM restored the LC3B-I level, increased the p62 level, and normalized the ATG and lysosomal protein amounts to control levels. Autophagy array revealed that the secreted proteins in NI-hADSC-CM could be crucial in the neuroprotection. Taken together, our results showed that the neuroprotective effects of NI-hADSC-CM on the autophagy signaling pathways could alleviate the aggregation of α-syn in Parkinson's disease and other neurodegenerative disorders.

Costaria costata Extract Suppresses Development of Atopic Dermatitis in chloro-2,4-dinitrobenzene-treated NC/Nga Mice.

We investigated the potential effects of Costaria costata (CC) on atopic dermatitis (AD) development in chloro-2,4-dinitrobenzene (DNCB)-treated NC/Nga mice. CC is a brown alga distributed across the seas of Korea, China, and Japan. A total of 40 mice were randomly assigned to 5 groups with 8 mice per group: untreated Balb/c mice, AD control (0.1% w/v DNCB-treated NC/Nga mice), positive control (i.e., DNCB-treated NC/Nga mice fed a dietary supplement of 66.6 mg/kg of body weight [b.w.] of CJLP133), DNCB-treated NC/Nga mice fed a dietary supplement of 100 mg/kg b.w. of CCE10 (CCE10 100), and DNCB-treated mice fed a dietary supplement of 300 mg/kg b.w. of CCE10 (CCE10 300) groups. The CCE10 100 and CCE10 300 treatment groups suppressed AD development including clinical and histopathological changes and a reduction in skin hydration induced by DNCB. In addition, Th2 cytokine production in primary splenocytes, serum IgE and histamine production, and mast cell infiltration into the skin were suppressed in the CCE10 300 mice compared to the CCE10 100 mice. Our finding demonstrated an inhibitory effect of CCE10 in AD development by means of improving the Th1/Th2 cytokine balance and anti-inflammatory effect in an in vivo model.

Isolation of high-purity and high-stability exosomes from ginseng.

Exosomes are nano-sized extracellular vesicles that regulate cell growth and defense by delivering bioactive cellular constituents. They are a promising material for biomedical and cosmetic utilization, especially in medicinal crops such as ginseng. One main hurdle to their usage is the need for a method to isolate stable exosomes with high purity. In this study, we first tested two methods to isolate exosomes from ginseng: ultracentrifugation, the most widely used method; and the ExoQuick system, a polymer-based exosome precipitation approach. We also designed and tested a third method in which we combined ultracentrifugation and ExoQuick methods. Size distribution analysis revealed that the exosome isolation purity by the ultracentrifugation and ExoQuick methods alone were 34.1% and 59.7%, respectively, while the combination method greatly improved exosome isolation purity (83.3%). Furthermore, we found that the combination method also increases the colloidal stability of isolated ginseng exosomes, and the increase was almost double that of the ultracentrifugation method. Lastly, we showed that the combination method can also be used to isolate high-purity and high-stability exosomes from the model plant Arabidopsis. Overall, our findings indicate that the combination method is suitable to isolate high-purity and high-stability exosomes from plants including ginseng.

Extracts of Porphyra tenera (Nori Seaweed) Activate the Immune Response in Mouse RAW264.7 Macrophages via NF-κB Signaling.

Porphyra tenera, also known as nori, is a red algal species of seaweed. It is cultivated in Asia for culinary purposes. We report that P. tenera extract (PTE) enhances the immune response in mouse macrophages. We found that P. tenera extract regulates the NF-κB IκB kinase (IKK) signaling pathway, and we assessed the expression and translocation of p65, a subunit of NF-κB, in RAW264.7 mouse macrophage cells after treatment with PTE. We also investigated the effects of 10% ethanol PTE (PTE10) in RAW264.7 cells. The production of IL-10, IL-6, TNF-α, and IFN-γ was induced by PTE treatment of the macrophages, and PTE also enhanced p-IκB and p-AKT. PTE10 showed no cytotoxicity at 10-20 µg/mL in RAW264.7 cells. PTE10, in fact, increased cell viability at 24 h, stimulated macrophage cells, and induced the phosphorylation of Akt. Akt stimulates IKK activity through the phosphorylation of IKKα and enhances immune activity through the activation of NF-κB. In this study, NF-κB activation was induced by increasing p-NF-κB and p-IKK. A subunit of NF-κB, p65, was located in the nucleus and increased the expression of various cytokines. PTE thus enhanced the immune response through IκB-α immunostimulation signaling in RAW264.7 cells. PTE10 has potential therefore for development of future treatments requiring immune system stimulation.

Laminarin favorably modulates gut microbiota in mice fed a high-fat diet.

We investigated the anti-obesity effects of the potential prebiotic, laminarin, on mice fed a high-fat diet. A metagenomics approach was applied to characterize the ecological and functional differences of gut microbiota among mice fed a normal diet (CTL), a high-fat diet (HFD), and a laminarin-supplemented high-fat diet (HFL). The HFL mice showed a slower weight gain than the HFD mice during the laminarin-feeding period, but the rate of weight gain increased after the termination of laminarin supplementation. Gut microbial community analysis showed clear differences between the CTL and HFD mice, whereas the HFL mice were between the two. A higher abundance of carbohydrate active enzymes was observed in the HFL mice compared to the HFD mice, with especially notable increases in glycoside hydrolase and polysaccharide lyases. A significant decrease in Firmicutes and an increase in the Bacteroidetes phylum, especially the genus Bacteroides, were observed during laminarin ingestion. Laminarin ingestion altered the gut microbiota at the species level, which was re-shifted after termination of laminarin ingestion. Therefore, supplementing laminarin could reduce the adverse effects of a high-fat diet by shifting the gut microbiota towards a higher energy metabolism. Thus, laminarin could be used to develop anti-obesity functional foods. Our results also suggest that laminarin would need to be consumed regularly in order to prevent or manage obesity.

Dopaminergic differentiation of neural progenitors derived from placental mesenchymal stem cells in the brains of Parkinson's disease model rats and alleviation of asymmetric rotational behavior.

Parkinson's disease (PD) is caused by the progressive loss of dopaminergic neurons in the mesencephalic substantia nigra and is accompanied by behavioral abnormalities. Pharmacological administration of L-dihydroxyphenylalanine (l-dopa) improves the abnormalities in the early phase of the illness, but numerous adverse effects hinder long-term administration. Transplantation of fetal mesencephalic tissues has been suggested as an alternative to l-dopa treatment; however, the use of human fetal tissues is controversial. Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a promising substitute for fetal tissue for the replacement of diseased tissues or organs. Previously, this group isolated 17 independent MSCs from the first trimester human placenta (termed first trimester placental MSCs, or fPMSCs) and reported their successful in vitro differentiation into fPMSC-derived neural progenitors (fPMSC-NPs) (Park et al., Placenta 2011; 32:269-276). In the current study, the in vitro-generated fPMSC-NPs were transplanted into the striatum of a rat model of PD to evaluate whether they could undergo terminal differentiation and mediate behavioral recovery. As early as 2 weeks after transplantation, a minor but significant amelioration of rotational asymmetry was observed, and near-normal motor function was achieved at 24weeks. Immunohistochemical and positron emission tomography (PET) analyses provided experimental evidence for the dopaminergic differentiation of the transplanted progenitors. These results show that in vitro-generated fPMSC-NPs are capable of terminal differentiation in vivo and can attenuate motor defects associated with PD. Hence, the placenta is an auspicious source of stem cells for the therapeutic treatment of neurological disorders.