Δευτέρα 14 Οκτωβρίου 2019

Gelam honey promotes ex vivo corneal fibroblasts wound healing

Abstract

This study evaluated the effects of Gelam honey (GH) on ex vivo corneal fibroblast ulcer model via wound healing assay, gene expression and immunocytochemistry. Corneal fibroblasts from New Zealand white rabbits were culture expanded. The corneal fibroblast wound healing capacity was observed by creating a circular wound onto confluent monolayer cells cultured in basal medium (BM), BM with GH, serum-enriched basal medium (BMS) and BMS with GH respectively. Wound healing assay and phenotypic characterization of the corneal fibroblast were performed at different stages of wound closure. Expression of aldehyde dehydrogenase (ALDH), vimentin, α-smooth muscle actin (α-SMA), lumican, collagen I and matrix metalloproteinase 12 (MMP 12) were measured at day 1, day 3 and complete wound closure day. Corneal fibroblast cultured in BMS with GH demonstrated the fastest wound closure, at day 5 post wounding. The gene expressions of ALDH and vimentin were higher than control groups while α-SMA expression was lower, in GH enriched media. The expressions of lumican, collagen I and MMP 12 were also higher in cells cultured in GH enriched media compared to the control groups. GH was shown to promote in vitro corneal fibroblast wound healing and may be a potential natural adjunct in the treatment of corneal wound.

Antitumor effects of seleno-short-chain chitosan (SSCC) against human gastric cancer BGC-823 cells

Abstract

Seleno-short-chain chitosan (SSCC) is a derivative of chitosan. In the present study, we sought to investigate the underlying antitumor mechanism of SSCC on human gastric cancer BGC-823 cells in vitro. MTT assay suggested that SSCC exhibited a dose-dependent inhibitory effect on the proliferation of BGC-823 cells. We found the SSCC-treated cells showed typical morphological characteristics of apoptosis in a dose dependent manner by observing on microscope. Annexin V-FITC/PI double staining and cell cycle assay identified that SSCC could induce BGC-823 cells apoptosis by triggering G2/M phase arrest. Our research provided the first evidence that SSCC could effectively induce the apoptosis of BGC-823 cells via an intrinsic mitochondrial pathway, as indicated by inducing the disruption of mitochondrial membrane potential (MMP), the excessive accumulation of reactive oxidative species (ROS), the increase of Bax/Bcl-2 ratio and the activation of caspase 3, caspase 9 and cytochrome C (Cyt-C) in BGC-823 cells. These combined results clearly indicated that SSCC could induce BGC-823 cells apoptosis by the involvement of mitochondrial signaling pathway, which provided precise experimental evidence for SSCC as a potential agent in the prevention and treatment of human gastric cancer.

Infant cardiosphere-derived cells exhibit non-durable heart protection in dilated cardiomyopathy rats

Abstract

Stem cells provide a new strategy for the treatment of cardiac diseases; however, their effectiveness in dilated cardiomyopathy (DCM) has not been investigated. In this study, cardiosphere-derived cells (CDCs) were isolated from infants (≤ 24 months) and identified by the cell surface markers CD105, CD90, CD117 and CD45, which is consistent with a previous report, although increased CD34 expression was observed. The molecular expression profile of CDCs from infants was determined by RNA sequencing and compared with adult CDCs, showing that infant CDCs have almost completely altered gene expression patterns compared with adult CDCs. The upregulated genes in infant CDCs are mainly related to the biological processes of cell morphogenesis and differentiation. The molecular profile of infant CDCs was characterized by lower expression of inflammatory cytokines and higher expression of stem cell markers and growth factors compared to adult CDCs. After intramyocardial administration of infant CDCs in the heart of DCM rats, we found that infant CDCs remained in the heart of DCM rats for at least 7 days, improved DCM-induced cardiac function impairment and protected the myocardium by elevating the left ventricular ejection fraction and fraction shortening. However, the effectiveness of transplanted CDCs was reversed later, as increased fibrosis formation instead of angiogenesis was observed. We concluded that infant CDCs, with higher expression of stem cell markers and growth factors, exhibit non-durable heart protection due to limited residence time in the heart of DCM animals, suggesting that multiple administrations of the CDCs or post-regulation after transplantation may be the key for cell therapy in the future.

Bioactive molecule Icariin inhibited proliferation while enhanced apoptosis and autophagy of rat airway smooth muscle cells in vitro

Abstract

Icariin is the main active compound extracted from epimedium Flavonoids (EFs) and involved in regulation of cell behaviors (proliferation, apoptosis, and autophagy etc.) for many cell types, but the effect of Icariin on airway smooth muscle cells (ASMCs) is still unknown. The aim of the present study is to examine the role of Icariin on rat ASMCs proliferation, apoptosis and autophagy. CKK8 assay showed that Icariin inhibited rat ASMCs growth in dose-time-dependent manner, and the flow cytometry assay showed that the Icariin interfered with ASMCs cell cycle, when treated with Icariin, S phase shortened while G2 phase extended, cyclin E1 and cyclinA1 gene and protein expression decreased. Next apoptosis was detected, Flow cytometry and TdTmediated dUTP Nick-End Labeling (TUNEL) assay showed that Icariin promoted ASMCs apoptosis, and enhanced apoptosis protein cleaved-caspase-3 expression. Finally, it was found Icariin induced rat ASMCs autophagy, with enhancement expression of autophagy marker LC3 II. In conclusion, Icariin inhibited ASMCs proliferation while promoted apoptosis and autophagy, revealing its potential role in treatment of airway remodeling in asthma.

Biological characterization of the UW402, UW473, ONS-76 and DAOY pediatric medulloblastoma cell lines

Abstract

Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent advances in molecular technologies allowed to classify MB in 4 major molecular subgroups: WNT, SHH, Group 3 and Group 4. In cancer research, cancer cell lines are important for examining and manipulating molecular and cellular process. However, it is important to know the characteristics of each cancer cell line prior to use, because there are some differences among them, even if they originate from the same cancer type. This study aimed to evaluate the similarities and differences among four human medulloblastoma cell lines, UW402, UW473, DAOY and ONS-76. The medulloblastoma cell lines were analyzed for (1) cell morphology, (2) immunophenotyping by flow cytometry for some specifics surface proteins, (3) expression level of adhesion molecules by RT-qPCR, (4) proliferative potential, (5) cell migration, and (6) in vivo tumorigenic potential. It was observed a relationship between cell growth and CDH1 (E-chaderin) adhesion molecule expression and all MB cell lines showed higher levels of CDH2 (N-chaderin) when compared to other adhesion molecule. ONS-76 showed higher gene expression of CDH5 (VE-chaderin) and higher percentage of CD144/VE-chaderin positive cells when compared to other MB cell lines. All MB cell lines showed low percentage of CD34, CD45, CD31, CD133 positive cells and high percentage of CD44, CD105, CD106 and CD29 positive cells. The DAOY cell line showed the highest migration potential, the ONS-76 cell line showed the highest proliferative potential and only DAOY and ONS-76 cell lines showed tumorigenic potential in vivo. MB cell lines showed functional and molecular differences among them, which it should be considered by the researchers in choosing the most suitable cellular model according to the study proposal.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

Abstract

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Evaluation of a cell model expressing βKlotho for screening FGF21 analogues

Abstract

βKlotho as the major role is a necessary auxiliary protein when fibroblast growth factor 21 (FGF21) binds FGF21 receptors (FGFR) for activating intracellular signaling pathways that ultimately generate biological effects. To achieve the aim of high throughput screening of FGF21 analogues, we established 3T3-L1-βKlotho cells that could stably express βklotho protein. The glucose uptake, expression of GLUT1 mRNA and activation of FGF signaling molecules ERK1/2 phosphorylation were detected by GOD-POD assay, real-time PCR analysis and western blotting assay in 3T3-L1-βKlotho cells and 3T3-L1 adipocytes, respectively. The results showed that FGF21 increased glucose uptake significantly in a dose-dependent and time-dependent manner in 3T3-L1-βKlotho cells. 3T3-L1-βKlotho cells stimulated with FGF21 up-regulated the transcriptional levels of GLUT1 mRNA obviously. FGF21 activated the FGF signaling molecules ERK1/2 in 3T3-L1-βKlotho cells. In addition, the same results were obtained in 3T3-L1 adipocytes. Furthermore, FGF21-stimulated elevation of glucose uptake, GLUT1 mRNA transcription and the phosphorylation of ERK1/2 were dramatically attenuated by pretreatment of cells with FGFR specific inhibitor SU5402 in 3T3-L1-βKlotho cells. This study demonstrated that the cell model could be applied to high throughput screen FGF21 analogues.

Oleanolic acid attenuated diabetic mesangial cell injury by activation of autophagy via miRNA-142-5p/PTEN signaling

Abstract

Oleanolic acid (OA), a potential drug for diabetic nephropathy (DN) treatment was found to downregulate the expression of microRNA (miR). The research aimed to investigate the effect of OA on autophagy mediated through miR-142-5p targeted PTEN signal. NRK-52E cells were cultured under normal or high glucose condition. DN model were induced by intravenous injection with streptozotocin (55 mg/kg). Renal fibrosis mice were detected by hematoxylin and eosin (HE) staining, Masson staining and immunohistochemistry assay. TargetScan and dual-luciferase reporter assay system was used to detect the target of miR-142-5p. Expression levels of microRNA and proteins were analyzed by real-time PCR and western blotting. Autophagy was decreased in the progression of renal fibrosis in diabetic nephropathy mice (in vivo) and in high glucose-induced NRK-52E cells (rat kidney epithelial cells) (in vitro) as the expression ofLC-3I and LC-3II (indicators of autophagy) were decreased mice MiR-142-5p was unregulated and PTEN was down-regulated in kidney mice and high glucose-induced NRK-52E cells. Targetscan prediction revealed that PTEN was a target of miR-142-5p. OA restricted HG-induced NRK-52E cell fibrosis through inhibition of miR-142-5p to promote PTEN expression and autophagy levels. To sum up, the research indicated that OA promoted autophagy through inhibition of PI3K/AKT/mTOR pathway. OA alleviated diabetic renal fibrosis by increasing autophagy through regulation of miR-142-5p/PTEN via PI3K/AKT/mTOR pathway in NRK-52E cells.

Sambucus nigra L. ameliorates UVB-induced photoaging and inflammatory response in human skin keratinocytes

Abstract

Sambucus nigra L. (Elderberry) is widely used as a dietary supplement in functional food and possesses many pharmacological activities to prevent ailments, such as the colds and fever, diabetes and cancer. However, research on its skin anti-aging effect is still limited. Here, we evaluated the recovery effects of elderberry extract (EB) in UVB-irradiated human skin keratinocytes (HaCaTs) and investigated whether EB represents a potential therapeutic agent against skin photoaging and inflammation. In this study, EB showed good efficiency on scavenging free radicals and dose-dependently reduced reactive oxygen species (ROS) generation. EB notably decreased UVB-induced matrix metalloproteinase-1 (MMP-1) expression and inflammatory cytokine secretion through the inhibition of mitogen-activated protein kinases/activator protein 1 (MAPK/AP-1) and nuclear factor-κB (NF-κB) signaling pathways, blocking extracellular matrix (ECM) degradation and inflammation in UVB-irradiated HaCaTs. In addition, EB improved nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling to increase oxidative defense capacity, and enhanced transforming growth factor beta (TGF-β) signaling activation to promote procollagen type I synthesis, relieving UVB-induced skin cell damage. These results indicated that EB has the potential to ameliorate UVB-induced skin photoaging and inflammation.

Alpha-linolenic acid confers protection on mice renal cells against cisplatin-induced nephrotoxicity

Abstract

Cisplatin is an antineoplastic agent used in the treatment of various types of solid tumors. Despite the dose-dependency of its antineoplastic effect, the high risk for nephrotoxicity frequently precludes the use of higher doses. α-Linolenic acid (ALA), a carboxylic acid having three cis double bonds, is an essential fatty acid required for health and can be acquired via foods that contain ALA or supplementation of foods high in ALA. Previous studies have shown that ALA demonstrates anti-cancer, anti-inflammatory, and anti-oxidative effects. In this study, we show the protective effect of ALA on cisplatin-induced renal toxicity associated with oxidative stress in mice using biochemical parameters. The mice were randomly assigned into four experimental groups. Group 1 (control group) were administered physiological saline solution for 9 days; group 2 (ALA group) received 200 mg/kg alpha-linolenic acid via gavage for 9 days; group 3 (CIS group) received 100 mg/kg intraperitoneal (i.p.) CIS for 9 days; and group 4 (ALA + CIS group) received 100 mg/kg i.p. CIS and followed by ALA 200 mg/kg via gavage for 9 days. Alpha-linolenic acid significantly reduced the expression of myeloperoxidase (MPO), phospholipase A2 (PLA2), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the ALA + CIS group compared to the CIS group. Furthermore, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) quantities were significantly elevated in the ALA + CIS group when compared to the CIS group. ALA significantly decreased the levels of Bax and cleaved caspase-3, while significantly increasing the level of bcl-2, an anti-apoptotic protein, in the ALA + CIS group than in the CIS group. Finally, histopathological examination in renal tissue showed that the significant edematous damage induced by CIS administration alone was reduced in ALA + CIS group. In conclusion, our findings show that ALA is beneficial to CIS-induced nephrotoxicity in mice via its anti-inflammatory and anti-oxidative effects.

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