Cytotechnology最新文献

Immature mandarin orange is thinned in order to maturation of orange. To use immature mandarin orange as a cosmetic functional material, we investigated the seasonal fluctuation changes in hesperidin and narirutin levels of immature mandarin oranges, and the effects on human skin cells. The contents of hesperidin from Aoshima, Otsu, and Shonan gold, is higher at about a month after flowering. Shonan gold has higher content of narirutin to compere that of Aoshima and Otsu. We found the addition of immature mandarin orange extracts to the human skin fibroblasts and keratinocytes, gene expression level of hyaluronic acid synthase and the hyaluronic acid contents in the medium are higher than that of the control. It was suggested that hesperidin in immature mandarin orange enhances the ability of skin cells to produce hyaluronic acid. Our findings indicate that the immature mandarin orange is a characteristic material on cosmetics and functional foods.

LncRNA HOTAIR has been reported to be associated with metabolic diseases of the liver. However, the effect of HOTAIR on non-alcoholic fatty liver disease (NAFLD) inflammation and its potential mechanism have not been reported. Genes and proteins expression were detected by qRT-PCR and Western blot respectively. The level of inflammatory cytokines was assessed by ELISA. HepG2 cell viability was detected by MTT assay. TG level and lipid accumulation were measured by Assay Kit and Oil red O staining, respectively. Direct binding relationship between HOTAIR and Serine/arginine splicing factor 1 (SRSF1), SRSF1 and MLX interacting protein like (MLXIPL) were confirmed by RNA-pull down and RIP assay. HOTAIR was highly expressed in free fatty acids (FFA)-treated HepG2 cells. HOTAIR knockdown alleviated FFA-induced inflammation of HepG2 cells. Then further analysis showed that HOTAIR and SRSF1 had a mutual binding relationship, and HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells. Moreover, the inhibitory effect of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression. HOTAIR accelerates inflammation of FFA-induced HepG2 cells by recruiting SRSF1 to stabilize MLXIPL mRNA, which will help to find new effective strategies for NAFLD therapy.

Corneocytes and intercellular lipids form the stratum corneum. The content and composition of intercellular lipids in the stratum corneum significantly affect skin barrier function. The purpose of this study was to demonstrate the effect of Shotokuseki extract (SE) on intercellular lipid production and metabolism in human three-dimensional cultured human epidermis. SE or ion mixtures containing five common ions were applied to three-dimensional cultured human epidermis for 2–8 days for each assay. The mRNA expression levels of epidermal differentiation markers and lipid metabolism genes were quantified by real-time PCR. After extraction of lipids from the epidermis, ceramide, sphingosine, free fatty acids, and cholesterol were quantified by LC-MS/MS, GC-MS, or HPLC. The results showed that the application of SE increased the gene expression levels of epidermal differentiation markers keratin10 and transglutaminase. Elongation of very long-chain fatty acids protein 3, serine palmitoyl transferase, ceramide synthase 3, and acid ceramidase mRNA expression levels increased and fatty acid synthase mRNA expression decreased. The content of each lipid, [EOS] ceramide decreased and total sphingosine content increased on day 4. On day 8 of application, ceramide [NDS], [NP], and [EODS] increased and total free fatty acid content decreased. These results show that SE alters the lipid composition of the epidermis, increasing ceramides and decreasing free fatty acids in the epidermis. The composition of the ions in the SE may be responsible for the changes in lipid composition. These behaviors were different from those observed when the ion mixture was applied.

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Abstract

Cirrhosis is a familiar end-stage of multiple chronic liver diseases. The gene-modified mesenchymal stem cells (MSCs) have become one of the most promising schemes for the treatment of cirrhosis. MSCs exhibit their therapeutic role mainly by secreting hepatocyte growth factor (HGF). The aim of this research was to probe the anti-fibrosis role of exosomes secreted by HGF modified-mouse adipose MSCs (ADMSCs) on activated hepatic stellate cells (HSCs) and to preliminarily explore the possible mechanism. Firstly, mouse ADMSCs were isolated and identified. Quantitative real-time polymerase chain reaction verified the transfection efficiency of ADMSC transfected with HGF lentivirus. Exosomes derived from ADMSC transfecting negative control/HGF (ADMSC^NC-Exo/ADMSC^HGF-Exo) were extracted by density gradient centrifugation. HSCs were allocated to the control, TGF-β, TGF-β + ADMSC-Exo, TGF-β + ADMSC^NC-Exo, and TGF-β + ADMSC^HGF-Exo groups. Moreover, all mice were distributed to the control, CCl₄ (40% CCl₄ in olive oil), CCl₄+ADMSC-Exo, CCl₄+ADMSC^NC-Exo, and CCl₄+ADMSC^HGF-Exo groups. Exosomes derived from ADMSCs with or without HGF transfection suppressed HSC activation, as evidenced by attenuating cell viability and cell cycle arrest at S phase but inducing apoptosis. Moreover, ADMSC-Exo, ADMSC^NC-Exo, and ADMSC^HGF-Exo effectively repressed the gene and protein levels of α-SMA, Col-I, Rho A, Cdc42, and Rac1 in TGF-β-treated HSCs, and ADMSC^HGF-Exo had the best effect. ADMSC^HGF-Exo had a stronger regulatory effect on serum liver index than ADMSC^NC-Exo in CCl₄-induced mice. In conclusion, ADMSC^HGF-Exo alleviated liver fibrosis by weakening the Rho pathway, thus reducing collagen production.

Pulmonary fibrosis (PF) is a chronic lung disease that has a poor prognosis and a serious impact on the quality of life of patients. Here, we investigated the potential role of miR-92a-3p in PF. The mRNA level of miR-92a-3p was significantly increased in both the lung tissues of bleomycin (BLM)--treated mice and pulmonary microvascular endothelial cells (PMVECs). Overexpressing miR-92a-3p increased the mRNA and protein levels of α‑SMA, vimentin, and Col-1 but downregulated E-cadherin. Additionally, the protein and mRNA expression levels of KLF2 were significantly decreased in the lung tissues of BLM-treated mice, suggesting that KLF2 participated in the progression of BLM-induced PF. Downregulating miR-92a-3p upregulated the expression of KLF2 and inhibited the endothelial-to-mesenchymal transition (EndoMT) process, thus alleviating PF in vivo. Altogether, a miR-92a-3p deficiency could significantly reduce the development of myofibroblasts and ameliorate PF progression.

Abstract

In several forms of malignant tumors, nuclear enriched abundant transcript 1 (NEAT1), a lncRNA, has been identified to play an important role. NEAT1’s regulation patterns in prostate cancer (PCa) are, however, mainly unknown. This study was aimed to evaluate and study the roles and regulatory mechanisms of NEAT1 in PCa. NEAT1, miR-582-5p, and enhancer of zeste homolog 2 (EZH2) expression were detected by qRT-PCR. The PCa cells’ invasive, migrative, and proliferative activities in vitro were assessed using transwell migration and invasion, wound-healing, cloning creation, and CCK-8 assays. In the present study, impaired proliferative, migrative, and invasive capacities were observed in the NEAT1-deficient PCa (PC3 and LNCaP) cells. Further mechanistic studies found that NEAT1 performs its function through sponging miR-582-5p. Furthermore, EZH2 was confirmed to be the downstream target gene of miRNA-582-5p. The impaired progression caused by NEAT1 deficiency in PCa cells was significantly restored by the inhibition of miR-582-5p, while these effects were largely abolished by the deletion of EZH2. Finally, the xenograft nude mouse model showed that knocking down the expression of NEAT1 suppressed the growth of PCa. In conclusion, NEAT1 promotes the progression of PCa by controlling the miR-582-5p and miR-582-5p-mediated EZH2.

Abstract

While induced pluripotent stem (iPS) cells are expected to be a cell source for regenerative medicine, they also have tumorigenic properties owing to their proliferative potential. During the manufacturing of regenerative medicine products, undifferentiated iPS cells and malignant transformed cells may be mixed in the cell culture population. Therefore, it is essential to eliminate tumorigenic cells selectively. In this study, a mixed culture of normal human fetal hepatocytes (Hc cells) and human hepatocellular carcinoma cells (HuH-7 cells) was used as a cell population model to be used as regenerative medicine products, and the selective elimination of HuH-7 cells by hybrid liposomes (HL) was analyzed. HL tended to fuse and accumulate more in HuH-7 cells due to larger fluidity of plasma membrane for HuH-7 cells than that for Hc cells. In a mixed culture of Hc and HuH-7 cells, HL selectively eliminated HuH-7 cells while allowing Hc cells to remain viable. In addition, HL treatment for the mixed culture of Hc and HuH-7 cells suppressed the tumorigenicity of HuH-7 cells. Therefore, HL selectively fused and accumulated in tumorigenic cells in a mixed cell culture of normal and tumorigenic cells, and eliminated tumorigenic cells while allowing normal cells to remain viable. The results of this study suggest the potential of HL in eliminating tumorigenic cells during the manufacturing of regenerative medicine products. Thus, HL could be expected to contribute to the development of safe regenerative medical products.

Abstract

Fructobacillus is a lactic-acid bacterium recently identified in fructose-rich environments. Fructobacillus is also known to exhibit unusual growth characteristics due to an incomplete gene encoding alcohol/acetaldehyde hydrogenase, which results in an imbalance in the nicotinamide adenine mononucleotide (NAD⁺)/NADN levels. Recently, the addition of d-fructose to the culture medium of Fructobacillus strains increased the intracellular nicotinamide mononucleotide (NMN) content. In the present study, we evaluated the functionality of Fructobacillus that produces high levels of NMN, using one substrain (Fructobacillus fructosus OS-1010). Therefore, in this study, we examined its functionality in the interaction between intestinal cells and muscle cells. The results showed that supernatant derived from intestinal epithelial cells (Caco-2 cells) treated with F. fructosus OS-1010 activated muscle cells (C2C12 cells). Further analysis revealed that Caco-2 cells treated with F. fructosus OS-1010 secreted exosomes known as extracellular vesicles, which activated the muscle cells. Furthermore, pathway analysis of the target genes of miRNA in exosomes revealed that pathways involved in muscle cell activation, including insulin signaling and cardiac muscle regulation, neurotrophic factors, longevity, and anti-aging, can be activated by exosomes. In other words, F. fructosus OS-1010 could activate various cells such as the skin and muscle cells, by secreting functional exosomes from the intestinal tract.

Abstract

Degradation of extracellular matrix (ECM), reactive oxygen species (ROS) production, and inflammation are critical players in the pathogenesis of intervertebral disc degeneration (IDD). Evodiamine exerts functions in inhibiting inflammation and maintaining mitochondrial antioxidant functions. However, the biological functions of evodiamine and its related mechanisms in IDD progression remain unknown. The IDD-like conditions in vivo were stimulated via needle puncture. Hematoxylin and eosin staining, Safranin O/Fast Green staining and Alcian staining were performed to determine the degenerative status. The primary nucleus pulposus cells (NPCs) were isolated from Sprague–Dawley rats and then treated with tert-butyl peroxide (TBHP) to induce cellular senescence and oxidative stress. The cell viability was assessed by cell counting kit-8 assays. The mitochondria-derived ROS in NPCs was evaluated by MitoSOX staining. The mitochondrial membrane potential in NPCs was identified by JC-1 staining and flow cytometry. The expression of collagen II in NPCs was measured by immunofluorescence staining. The levels of mRNAs and proteins were measured by RT-qPCR and western blotting. The Nrf2 expression in rat nucleus pulposus tissues was measured by immunohistochemistry staining. Evodiamine alleviated TBHP-induced mitochondrial dysfunctions in NPCs. The enhancing effect of TBHP on the ECM degradation was reversed by evodiamine. The TBHP-stimulated inflammatory response was ameliorated by evodiamine. Evodiamine alleviated the IDD process in the puncture-induced rat model. Evodiamine promoted the activation of Nrf2 pathway and inactivated the MAPK pathway in NPCs. In conclusion, evodiamine ameliorates the progression of IDD by inhibiting mitochondrial dysfunctions, ECM degradation and inflammation via the Nrf2/HO-1 and MAPK pathways.

Atherosclerosis constitutes a proverbial pathogenic mechanism for cardio-cerebrovascular disease that accounts for the most common cause of disability and morbidity for human health worldwide. Endothelial dysfunction and inflammation are the key contributors to the progression of atherosclerosis. Glutaredoxin 2 (GLRX2) is abundantly existed in various tissues and possesses a range of pleiotropic efficacy including anti-oxidative and anti-inflammatory responses. However, its role in atherosclerosis is still undefined. Here, down-regulation of GLRX2 was validated in lipopolysaccha (LPS)-induced vascular endothelial cells (HUVECs). Moreover, elevation of GLRX2 reversed the inhibition of cell viability in LPS-treated HUVECs and decreased LPS-induced increases in cell apoptosis and caspase-3 activity. Additionally, enhancement of GLRX2 expression antagonized oxidative stress in HUVECs under LPS exposure by inhibiting ROS, lactate dehydrogenase and malondialdehyde production and increased activity of anti-oxidative stress superoxide dismutase. Notably, GLRX2 abrogated LPS-evoked transcripts and releases of pro-inflammatory cytokine (TNF-α, IL-6, and IL-1β), chemokine MCP-1 and adhesion molecule ICAM-1 expression. Furthermore, the activation of Nrf2/HO-1 signaling was demonstrated in LPS-stimulated HUVECs. Importantly, blockage of the Nrf2 pathway counteracted the protective roles of GLRX2 in LPS-triggered endothelial cell injury, oxidative stress and inflammatory response. Thus, these data reveal that GLRX2 may alleviate the progression of atherosclerosis by regulating vascular endothelial dysfunction and inflammation via the activation of the Nrf2 signaling, supporting a promising therapeutic approach for atherosclerosis and its complications.