In Vitro Cellular & Developmental Biology. Animal最新文献

Local anesthetics, such as ropivacaine (Ropi), are toxic to nerve cells. We aimed to explore the role of forkhead box O3 (FOXO3) in Ropi-induced nerve injury to provide a theoretical basis for reducing the anesthetic neurotoxicity. SK-N-SH cells were cultured and treated with different concentrations of Ropi. Cell viability, apoptosis, cytotoxicity (LDH/ROS/SOD), and levels of FOXO3, miR-126-5p, and tumor necrosis factor receptor-associated factor 6 (TRAF6) were detected. The enrichment of FOXO3 on the miR-126-5p promoter was analyzed. The binding relationships among FOXO3, miR-126-5p promoter sequence, and TRAF6 3'UTR sequence were verified. Combined experiments detected the regulatory role of FOXO3/miR-126-5p/TRAF6 in Ropi-induced nerve injury. FOXO3 was upregulated in Ropi-induced nerve cell damage. Inhibition of FOXO3 ameliorated Ropi-induced decreased cell viability, and increased apoptosis and cytotoxicity. FOXO3 bound to the miR-126-5p promoter and inhibited its expression, thereby counteracting miR-126-5p-induced repression. miR-126-5p inhibition and TRAF6 overexpression partially reversed the alleviative effect of FOXO3 inhibition on Ropi-induced nerve cell damage. In conclusion, FOXO3 aggravated the neurotoxicity of Ropi through miR-126-5p downregulation and TRAF6 upregulation, suggesting that FOXO3 inhibitor could be an adjuvant agent for local anesthetics, to alleviate local anesthetics-induced neurotoxicity.

The complement system plays an important role in biological defense as an effector to eliminate microorganisms that invade an organism and it is composed of more than 50 proteins, most of which are produced in the liver. Of these proteins, the mRNA expression of C3 and Cfb is known to be positively regulated by the nuclear receptor HNF4α. To investigate whether HNF4α regulates the complement system, we analyzed the hepatic expression of genes involved in the complement activation pathway and membrane attack complex (MAC) formation within the complement system using liver-specific Hnf4a-null mice (Hnf4a^ΔHep mice) and tamoxifen-induced liver-specific Hnf4a-null mice (Hnf4a^{f/f;AlbERT2cre} mice). We found that hepatic expression of many complement genes including C8a, C8b, C8g, and C9 that are involved in formation of the MAC was markedly decreased in Hnf4a^ΔHep mice and Hnf4a^{f/f;AlbERT2cre} mice. Furthermore, expression of C8A, C8B, and C8G was also decreased in human hepatoma cell lines in which the expression of HNF4α was suppressed, and expression of C8G and C9 was induced in a human immortalized hepatocyte cell line with forced expression of HNF4α. Transactivation of C8g and C9 was dependent on HNF4α expression of HNF4α binding sites, indicating that C8g and C9 are novel target genes of HNF4α. The results suggest that hepatic HNF4α plays an important role in regulation of the complement system, mainly MAC formation.

Ginsenoside Re (GS-Re) is a major saponin monomer found in Panax ginseng Meyer. It has been shown to exhibit a wide range of biological and pharmacological activities. This study aimed to investigate the effect of GS-Re on the proliferation of murine bone marrow–derived MSCs in vitro and to assess whether its effect is dependent on the estrogen receptor–mediated signal transduction. CFU colony formation assay, cell counting, and colorimetric MTT test were employed to examine effects of GS-Re on the in vitro proliferation of MSCs and the mechanisms of the underlying effect were detected by flow cytometric analysis, immunofluorescence staining for BrdU, and Western blotting. GS-Re dose-dependently promoted the in vitro proliferation of murine bone marrow–derived MSCs over a range of concentrations of 0.5 ~ 20 µmol/L, and this effect approached the maximal level at 10 µmol/L. Increases in the expression level of phosphorylated extracellular signal–regulated kinases 1/2 (p-ERK1/2) were observed in the passaged MSCs treated with 10 µmol/L of GS-Re. These effects of GS-Re on the MSCs were significantly counteracted by the addition of ICI 182, 780 (an estrogen receptor antagonist) to the culture media. We concluded that GS-Re is able to exert a proliferation-promoting effect on murine bone marrow–derived mesenchymal stem cells in vitro, and its action is involved in the estrogen receptor–mediated signaling.

In this study, we investigated the potential therapeutic mechanism of ginsenoside Rg1 (GRg1) in chronic heart failure (CHF), focusing on its regulation of ERK1/2 protein phosphorylation. H9c2 cardiomyocytes and SD rats were divided into the control group, CHF (ADR) group, and CHF+ginsenoside Rg1 group using an isolated cardiomyocyte model and an in vivo CHF rat model induced by adriamycin (ADR). Cell viability, proliferation, apoptosis, and the expression of relevant proteins were measured to assess the effects of GRg1. The results showed that treatment with GRg1 increased cell activity and proliferation, while significantly reducing levels of inflammatory and apoptotic factors compared to the CHF (ADR) group. Moreover, the CHF+ginsenoside Rg1 group exhibited higher levels of Bcl-2 mRNA and protein expression, as well as lower levels of Caspase3 and Bax mRNA and protein expression, compared to the CHF (ADR) group. Notably, the CHF+ginsenoside Rg1 group displayed decreased serum NT-proBNP levels and heart weight/body weight (HW/BW) index. Furthermore, the electrocardiogram of rats in the CHF+ginsenoside Rg1 group resembled that of rats in the control group. Overall, our findings suggested that GRg1 alleviated CHF by inhibiting ERK1/2 protein phosphorylation, thereby inhibiting apoptosis, enhancing cell activity and proliferation, and reducing cardiac inflammatory responses.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes β-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.

Decellularized tissues are an attractive scaffolds for 3D tissue engineering. Decellularized animal tissues have certain limitations such as the availability of tissue, high costs and ethical concerns related to the use of animal sources. Plant-based tissue decellularized scaffolds could be a better option to overcome the problem. The leaves of different plants offer a unique opportunity for the development of tissue-specific scaffolds, depending on the reticulate or parallel veination. Herein, we decellularized spinach leaves and employed these for the propagation and osteogenic differentiation of dental pulp stem cells (DPSCs). DPSCs were characterized by using mesenchymal stem cell surface markers CD90, CD105 and CD73 and CD34, CD45 and HLA-DR using flow cytometry. Spinach leaves were decellularized using ethanol, NaOH and HCL. Cytotoxicity of spinach leaf scaffolds were analysed by MTT assay. Decellularized spinach leaves supported dental pulp stem cell adhesion, proliferation and osteogenic differentiation. Our data demonstrate that the decellularized spinach cellulose scaffolds can stimulate the growth, proliferation and osteogenic differentiation of DPSCs. In this study, we showed the versatile nature of decellularized plant leaves as a biological scaffold and their potential for bone regeneration in vitro.

Human dental pulp stem cells (DPSCs) have become an important component for bone tissue engineering and regenerative medicine due to their ability to differentiate into osteoblast precursors. Two miRNA chip datasets (GSE138180 and E-MTAB-3077) of DPSCs osteogenic differentiation were analyzed respectively to find the expression of miR-483-3p significantly increased in the differentiated groups. We further confirmed that miR-483-3p continued to overexpress during osteogenic differentiation of DPSCs, especially reaching its peak on the 7th day. Moreover, miR-483-3p could significantly promote the expression of osteogenic markers including RUNX2 and OSX, and activate MAPK signaling pathway by inducing phosphorylation of ERK, p38, and JNK. In addition, as a significant gene within the MAPK signaling pathway, ARRB2 was identified as the target gene of miR-483-3p by bioinformatic prediction and experimental verification. In conclusion, we identified miR-483-3p could promote osteogenic differentiation of DPSCs via the MAPK signaling pathway by targeting ARRB2.