Mitochondrial transplantation exhibits neuroprotective effects and improves behavioral deficits in an animal model of Parkinson's disease.

Mitochondria are essential organelles for cell survival that manage the cellular energy supply by producing ATP. Mitochondrial dysfunction is associated with various human diseases, including metabolic syndromes, aging, and neurodegenerative diseases. Among the diseases related to mitochondrial dysfunction, Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neuronal loss and neuroinflammation. Recently, it was reported that mitochondrial transfer between cells occurred naturally and that exogenous mitochondrial transplantation was beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transfer on PD in vitro and in vivo. The results showed that PN-101 mitochondria isolated from human mesenchymal stem cells exhibited a neuroprotective effect against 1-methyl-4-phenylpyridinium, 6-hydroxydopamine and rotenone in dopaminergic cells and ameliorated dopaminergic neuronal loss in the brains of C57BL/6J mice injected 30 â€‹mg/kg of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally. In addition, PN-101 exhibited anti-inflammatory effects by reducing the expression of pro-inflammatory cytokines in microglial cells and suppressing microglial activation in the striatum. Furthermore, intravenous mitochondrial treatment was associated with behavioral improvements during the pole test and rotarod test in the MPTP-induced PD mice. These dual effects of neuroprotection and anti-neuroinflammation support the potential for mitochondrial transplantation as a novel therapeutic strategy for PD.

Veronica persica Ethanol Extract Ameliorates Dinitrochlorobenzene-Induced Atopic Dermatitis-like Skin Inflammation in Mice, Likely by Inducing Nrf2/HO-1 Signaling.

Atopic dermatitis (AD) is chronic allergic contact dermatitis with immune dysregulation. Veronica persica has pharmacological activity that prevents asthmatic inflammation by ameliorating inflammatory cell activation. However, the potential effects of the ethanol extract of V. persica (EEVP) on AD remain elusive. This study evaluated the activity and underlying molecular pathway of EEVP in two AD models: dinitrochlorobenzene (DNCB)-induced mice and interferon (IFN)-γ/tumor necrosis factor (TNF)-α-stimulated human HaCaT keratinocytes. EEVP attenuated the DNCB-induced increase in serum immunoglobulin E and histamine levels, mast cell counts in toluidine-blue-stained dorsal skin, inflammatory cytokine (IFN-γ, interleukin [IL]-4, IL-5, and IL-13) levels in cultured splenocytes, and the mRNA expression of IL6, IL13, IL31 receptor, CCR-3, and TNFα in dorsal tissue. Additionally, EEVP inhibited the IFN-γ/TNF-α-induced mRNA expression of IL6, IL13, and CXCL10 in HaCaT cells. Furthermore, EEVP restored the IFN-γ/TNF-α-induced downregulation of heme oxygenase (HO)-1 in HaCaT cells by inducing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. A molecular docking analysis demonstrated that EEVP components have a strong affinity to the Kelch-like ECH-associated protein 1 Kelch domain. In summary, EEVP inhibits inflammatory AD by attenuating immune cell activation and inducing the Nrf2/HO-1 signaling pathway in skin keratinocytes.

PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury.

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1-0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

Genomic profile of metastatic breast cancer patient-derived xenografts established using percutaneous biopsy.

BACKGROUND: Metastatic breast cancer (mBC) is a complex and life-threatening disease and although it is difficult to cure, patients can benefit from sequential anticancer treatment, including endocrine therapy, targeted therapy and cytotoxic chemotherapy. The patient-derived xenograft (PDX) model is suggested as a practical tool to predict the clinical outcome of this disease as well as to screen novel drugs. This study aimed to establish PDX models in Korean patients and analyze their genomic profiles and utility for translational research. METHODS: Percutaneous core needle biopsy or punch biopsy samples were used for xenotransplantation. Whole exome sequencing and transcriptome analysis were performed to assess the genomic and RNA expression profiles, respectively. Copy number variation and mutational burden were analyzed and compared with other metastatic breast cancer genomic results. Mutational signatures were also analyzed. The antitumor effect of an ATR inhibitor was tested in the relevant PDX model. RESULTS: Of the 151 cases studied, 40 (26%) PDX models were established. Notably, the take rate of all subtypes, including the hormone receptor-positive (HR +) subtype, exceeded 20%. The PDX model had genomic fidelity and copy number variation that represented the pattern of its donor sample. TP53, PIK3CA, ESR1, and GATA3 mutations were frequently found in our samples, with TP53 being the most frequently mutated, and the somatic mutations in these genes strengthened their frequency in the PDX model. The ESR1 mutation, CCND1 amplification, and the APOBEC signature were significant features in our HR + HER2- PDX model. Fulvestrant in combination with palbociclib showed a partial response to the relevant patient's tumor harboring the ESR1 mutation, and CCND1 amplification was found in the PDX model. AZD6738, an ATR inhibitor, delayed tumor growth in a relevant PDX model. CONCLUSIONS: Our PDX model was established using core needle biopsy samples from primary and metastatic tissues. Genomic profiles of the samples reflected their original tissue characteristics and could be used for the interpretation of clinical outcomes.

WKYMVm ameliorates acute lung injury via neutrophil antimicrobial peptide derived STAT1/IRF1 pathway.

Formyl peptide receptors (FPRs) are mainly expressed on leucocytes and sense microbe-associated molecular pattern (MAMP) molecules, thereby regulating leukocyte chemotaxis and activation. The formyl peptide receptor 2 (FPR2) selective agonist WKYMVm (Trp-Lys-Met-Val-D-Met) has shown potent pro-angiogenic, anti-inflammatory, and anti-apoptotic properties. In this study, we investigated whether WKYMVm exhibits bactericidal activity during neutrophil accumulation in acute lung injury (ALI) in mice and determined its cellular signaling pathways in HL-60 neutrophil-like cells. A daily intraperitoneal treatment of ALI mice with WKYMVm (2.5- and 5 mg/kg/d) daily over four days decreased the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1ß, while it increased the MPO and NO release by differentiated HL-60 neutrophil-like cells. The IRF1 level and STAT1 phosphorylation at S727 were increased in the lungs of mice with ALI treated with WKYMVm. Lung histology induced by ALI was unaffected by treatment with WKYMVm. In vitro, WKYMVm increased MPO, NO, and SOD activity, as well as IRF1 and STAT1 phosphorylation at Ser727. Taken together, our data suggest therapeutic potential of WKYMVm, via FPR2-dependent regulation of STAT1/IRF1, in ALI.

Mesenchymal Stem Cell Transplantation Promotes Functional Recovery through MMP2/STAT3 Related Astrogliosis after Spinal Cord Injury.

BACKGROUND AND OBJECTIVES: Treatment with mesenchymal stem cells (MSC) in spinal cord injury (SCI) has been highlighted as therapeutic candidate for SCI. Although astrogliosis is a major phenomenon after SCI, the role of astrogliosis is still controversial. In this study, we determined whether acute transplantation of MSC improves the outcome of SCI through modulating astrogliosis. METHODS: Bone marrow derived rat MSCs were induced neural differentiation and transplanted after acute SCI rats. Matrix metalloproteinase (MMP) and neuro-inflammatory pathway were analyzed for acute astrogliosis at 1, 3 and 7 d after SCI in RT-PCR- and western blot analysis. Functional outcome was assessed serially at postoperative 1 d and weekly for 4 weeks. Histopathologic analysis was undertaken at 7 and 28 d following injury in immunohistochemistry. RESULTS: Transplantation of MSCs decreased IL-1α, CXCL-2, CXCL-10, TNF-α and TGF-ß in a rat model of contusive SCI. Protein level of NF-κB p65 was slightly decreased while level of STAT-3 was increased. In immunohistochemistry, MSC transplantation increased acute astrogliosis whereas attenuated scar formation with increased sparing white matter of spinal cord lesions. In RT-PCR analysis, mRNA levels of MMP2 was significantly increased in MSC transplanted rats. In BBB locomotor scale, the rats of MSC treated group exhibited improvement of functional recovery. CONCLUSIONS: Transplantation of MSC reduces the inflammatory reaction and modulates astrogliosis via MMP2/STAT3 pathway leading to improve functional recovery after SCI in rats.

Bleomycin Inhibits Proliferation via Schlafen-Mediated Cell Cycle Arrest in Mouse Alveolar Epithelial Cells.

BACKGROUND: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. METHODS: Mouse AE II cell line MLE-12 were exposed to 1-10 µg/mL BLM and 0.01-100 µM baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. RESULTS: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor α, and transforming growth factor ß1. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. CONCLUSION: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

Laurus nobilis leaf extract controls inflammation by suppressing NLRP3 inflammasome activation.

Laurus nobilis Linn. (Lauraceae), commonly known as Bay, has been used as a traditional medicine in the Mediterranean and Europe to treat diverse immunological disorders. Although the effects of L. nobilis on immunosuppression have been reported, the detailed underlying mechanism remains unclear. In this study, to elucidate the anti-inflammatory mechanism of L. nobilis, we examined the effect of L. nobilis leaf extract on inflammasome activation in mouse bone marrow-derived macrophages. L. nobilis leaf extract inhibited NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation, which was associated with caspase-1 activation, interleukin-1ß secretion, and apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome complex formation. We also observed that 1,8-cineole, the major component of L. nobilis extract, consistently suppressed NLRP3 inflammasome activation. Furthermore, L. nobilis leaf extract attenuated the in vivo expression of proinflammatory cytokines in an acute lung injury mouse model. Our results provide the first evidence that L. nobilis leaf extract modulates inflammatory signaling by suppressing inflammasome activation.

Inhibition of RAGE Attenuates Cigarette Smoke-Induced Lung Epithelial Cell Damage via RAGE-Mediated Nrf2/DAMP Signaling.

The oxidative stress and cellular apoptosis by environmental factor including cigarette smoke induces alveolar airway remodeling leading to chronic obstructive pulmonary disease (COPD). Recently, the receptor for advanced glycan end products (RAGE) which is highly expressed in alveolar epithelium is emerging as a biomarker for COPD susceptibility or progression. However, it still remains unknown how RAGE plays a role in cigarette smoke extract (CSE)-exposed human alveolar type II epithelial cell line. Therefore, we determined the efficacy of RAGE-specific antagonist FPS-ZM1 in response to CSE-induced lung epithelial cells. CSE induced the elevated generation of RONS and release of pro-inflammatory cytokines, and impaired the cellular antioxidant defense system. Further, CSE induced the alteration of RAGE distribution via the activation of redox-sensitive DAMP (Damage-associated molecular patterns) signaling through Nrf2 in cells. Although pre-treatment with SB202190 (p38 inhibitor) or SP600125 (JNK inhibitor) failed to recover the alteration of RAGE distribution, treatment of FPS-ZM1 significantly exhibited anti-inflammatory and anti-oxidative/nitrosative effects, also inhibited the activation of redox-sensitive DAMP signaling through Nrf2 (nuclear factor erythroid 2-related factor 2) migration in the presence of CSE. Taken together, our data demonstrate that RAGE and Nrf2 play a pivotal role in maintenance of alveolar epithelial integrity.

L-carnosine induces apoptosis/cell cycle arrest via suppression of NF-κB/STAT1 pathway in HCT116 colorectal cancer cells.

L-carnosine, a dipeptide of the amino acids ß-alanine and histidine, is found in various tissues, such as the central nervous system and skeletal muscles. Recently, L-carnosine has been reported to possess anti-tumor activity; however, the molecular mechanism underlying its activity in colorectal cancer is still unknown. Therefore, we investigated the effect of L-carnosine using a human colorectal cancer cell line, HCT116. Treatment with L-carnosine (0, 100, or 200 mM) for 24 h gradually reduced cellular proliferation according to immunochemistry and 7-aminoactinomycin D (7-AAD) analyses and induced G0/G1 phase arrest. In the RT-PCR analysis, L-carnosine decreased the mRNA levels of cell cycle-related genes in HCT116 cells. In the Western blot analysis, levels of the cyclin D1, BAX/Bcl-2, cleaved caspase-3, p21, and p53 proteins were significantly increased in cells treated with L-carnosine. We next determined whether STAT1/NF-κB pathway is involved in regulation of cell cycle arrest- and cell death-associated gene in HCT116. The L-carnosine treatment significantly inhibited the phosphorylation of STAT1 on Tyr701 and NF-κB p65 on Ser276 and Ser536, and then, we exogenously blocked the NF-κB phosphorylation using Bay 11-7082. Based on our findings, L-carnosine induces cell cycle arrest and apoptosis in human colorectal cancer cells by suppressing of NF-κB/STAT1 signaling.

The tri-peptide GHK-Cu complex ameliorates lipopolysaccharide-induced acute lung injury in mice.

The tripeptide-copper complex glycyl-l-histidyl-l-lysine-Cu (II) (GHK-Cu) is involved in wound healing and tissue remodeling. Although GHK-Cu exhibits anti-aging and tissue renewing properties, its roles in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are still unknown. Therefore, we examined the effects of GHK-Cu in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages in vitro and ALI in mice in vivo. GHK-Cu treatment reduced reactive oxygen species (ROS) production, increased superoxide dismutase (SOD) activity while decreased TNF-α and IL-6 production through the suppression of NF-κB p65 and p38 MAPK signaling in vitro and in vivo model of ALI. Moreover, GHK-Cu attenuated LPS-induced lung histological alterations, suppressed the infiltration of inflammatory cells into the lung parenchyma in LPS-induced ALI in mice. Taken together, these findings demonstrate that GHK-Cu possesses a protective effect in LPS-induced ALI by inhibiting excessive inflammatory responses; accordingly it may represent a novel therapeutic approach for ALI/ARDS.

Functional characteristics of mesenchymal stem cells derived from the adipose tissue of a patient with achondroplasia.

Mesenchymal stem cells (MSCs) can be isolated from various tissues including bone marrow, adipose tissue, skin dermis, and umbilical Wharton's jelly as well as injured tissues. MSCs possess the capacity for self-renewal and the potential for differentiation into adipogenic, osteogenic, and chondrogenic lineages. However, the characteristics of MSCs in injured tissues, such as achondroplasia (ACH), are not well known. In this study, we isolated MSCs from human subcutaneous adipose (ACH-SAMSCs) tissue and circumjacent human adipose tissue of the cartilage (ACH-CAMSCs) from a patient with ACH. We then analyzed the characterization of ACH-SAMSCs and ACH-CAMSCs, compared with normal human dermis-derived MSCs (hDMSCs). In flow cytometry analysis, the isolated ACH-MSCs expressed low levels of CD73, CD90, and CD105, compared with hDMSCs. Moreover, both ACH- SAMSCs and ACH-CAMSCs had constitutionally overactive fibroblast growth factor receptor 3 (FGFR3) and exhibited significantly reduced osteogenic differentiation, compared to enhanced adipogenic differentiation. The activity of extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinases (p38 MAPK) was increased in ACH-MSCs. In addition, the efficacy of osteogenic differentiation was slightly restored in osteogenic differentiation medium with MAPKs inhibitors. These results suggest that they play essential roles in MSC differentiation toward adipogenesis in ACH pathology. In conclusion, the identification of the characteristics of ACH-MSCs and the favoring of adipogenic differentiation via the FGFR3/MAPK axis might help to elucidate the pathogenic mechanisms relevant to other skeletal diseases and could provide targets for therapeutic interventions.

Cigarette smoke-mediated oxidative stress induces apoptosis via the MAPKs/STAT1 pathway in mouse lung fibroblasts.

Cigarette smoking is the major aetiologic factor in chronic obstructive pulmonary disease (COPD). Lung fibroblasts are key participants in the maintenance of the extracellular matrix within the lung parenchyma. However, it still remains unknown how pulmonary fibroblasts are affected by cigarette smoking. Therefore, in this study, we isolated lung fibroblasts from mice and determined the apoptotic mechanism in response to cigarette smoke extract (CSE). When the lung fibroblasts were exposed to CSE, the generation of ROS was increased as shown by H2-DCFDA staining and Flow Cytometry. By immunocytochemistry, Ki67 expressing cells gradually decreased in a dose-dependent manner. The nitrite concentration in the supernatants increased, while the SOD activity and GSH recycling decreased in response to CSE. CSE increased the mRNA levels of TNF-α and COX-2, and the secretory proteins TNF-α and IL-6 increased as measured by ELISA. We next determined whether this inflammatory process is associated with the Bax/Bcl-2 apoptosis pathway. The Bax/Bcl-2 mRNA ratio increased, and cleaved caspase-3 protein was activated in the lung fibroblasts treated with CSE. Moreover, CSE induced the phosphorylation of STAT1 at Tyr701/Ser727 and increased the activation of ERK1/2, p38, and JNK in the MAPK pathway. Taken together, these data suggest that CSE-mediated inflammation alters the redox regulation via the MAPK-STAT1 pathway, leading to intrinsic apoptosis of lung fibroblasts.

Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization.

Recent studies have reported that stem cells can be isolated from a wide range of tissues including bone marrow, fatty tissue, adipose tissue and placenta. Moreover, several studies also suggest that skin dermis could serve as a source of stem cells, but are of unclear phenotype. Therefore, we isolated and investigated to determine the potential of stem cell within human skin dermis. We isolated cells from human dermis, termed here as human dermis-derived mesenchymal stem cells (hDMSCs) which is able to be isolated by using explants culture method. Our method has an advantage over the enzymatic method as it is easier, less expensive and less cell damage. hDMSCs were maintained in basal culture media and proliferation potential was measured. hDMSCs were highly proliferative and successfully expanded with no additional growth factor. In addition, hDMSCs revealed normal karyotype and expressed high levels of CD90, CD73 and CD105 while did not express the surface markers for CD34, CD45 and HLA-DR. Also, we confirmed that hDMSCs possess the capacity to differentiate into multiple lineage including adipocyte, osteocyte, chondrocyte and precursor of hepatocyte lineage. Considering these results, we suggest that hDMSCs might be a valuable source of stem cells and could potentially be a useful source of clinical application.

Nicotine inhibits the proliferation by upregulation of nitric oxide and increased HDAC1 in mouse neural stem cells.

Cigarette smoking (CS) is considered one of the major risk factors to cause neurodegenerative disorders. Nicotine is the main chemical in CS which is responsible for dysfunction of the brain as a neuroteratogen. Also, nicotine dependency is a real mental illness and disease. Recently, chronic nicotine exposure has been shown to cause oxidative/nitrosative stress leading to a deleterious condition to cellular death in different brain regions. However, little is known about the effects of nicotine on mouse neural stem cells (mNSCs). The aim of this study is to investigate the effects of nicotine on mNSCs and elucidate underlying mechanisms involved in expression of a diversity of genes regulated by nicotine. When mNSCs were isolated from the whole brain of embryonic day 16 mice treated with nicotine at vehicle, 100, 400, and 800 µM for 5 d, nicotine significantly decreased the number and size of neurospheres. In immunocytochemistry, nicotine-exposed mNSCs expressing nestin showed the shortened filaments and condensed nuclei. In RT-PCR, messenger RNA (mRNA) levels of proliferating cell nuclear antigen (PCNA) and sirtuin1 (SIRT1) were significantly decreased, while the production of nitric oxide and mRNA levels of cyclooxygenase2 (COX-2), tumor necrosis factor-alpha TNF-α, and histone deacetylase 1 (HDAC1) were increased in a dose-dependent manner. In addition, sodium butyrate and valproic acid, HDAC inhibitors, partially rescue proliferation of mNSCs via inhibition of HDAC1 expression and NO production. Taken together, these data demonstrate that prolonged exposure of nicotine decreased proliferation of mNSCs by increased NO and inflammatory cytokine through increased HDAC1. Furthermore, this study could help in the development of a therapy for nicotine-induced neurodegenerative disorder and drug abuse.