Antiproliferative effects of levan polysaccharide against colorectal cancer cells mediated through oxidative stress-stimulated HOTAIR/Akt signaling pathway: In vitro

The overexpression of lncRNA HOTAIR can result in cancer progression through upregulation of the Akt signaling pathway. We aimed to evaluate the anticancer effects of levan polysaccharide from Erwinia herbicola on colorectal cancer cells (HT-29) and explore the role of the ROS-mediated HOTAIR/Akt signaling pathway. The HT-29 cancer cells were treated with levan either alone or along with N-acetyl-L-cysteine (NAC), as a potential antioxidant for 24 hrs and different assays including MTT, LDH, ROS, apoptosis, SOD activity, CAT activity, caspase-3 activity, qRT-PCR, and western blot analysis were performed. It was shown that levan treatment induces apparent reduction in cell viability, SOD and CAT activity, HOTAIR expression, the ratio of pAkt protein level and a significant increase in the ROS level, apoptosis induction, and caspase-3 activity, which were effectively suppressed by NAC co-treatment. These data indicated the effective antiproliferative effect of levan on colorectal cancer cells, in which downregulation of ROS-mediated HOTAIR/Akt plays an important role.     

Protection by the flavonoids quercetin and luteolin against peroxide- or menadione-induced oxidative stress in MC3T3-E1 osteoblast cells

Potential protective effects of the flavonoids quercetin and luteolin have been examined against the oxidative stress of MC3T3-E1 osteoblast-like cells. Although hydrogen peroxide and menadione reduced cell viability, the toxicity was prevented by desferrioxamine or catalase but not superoxide dismutase, suggesting the involvement of hydrogen peroxide in both cases. Quercetin and luteolin reduced the oxidative damage, especially that caused by hydrogen peroxide. When cultures were pre-incubated with quercetin or luteolin, protection was reduced or lost. Protection was also reduced when a 24 h pre-incubation with the flavonoids was followed by exposure to menadione alone. Pretreating cultures with luteolin impaired protection by quercetin, whereas quercetin pretreatment did not affect protection by luteolin. It is concluded that quercetin and luteolin suppress oxidative damage to MC3T3-E1 cells, especially caused by peroxide. The reduction in protection by pretreatment implies a down-regulation of part of the toxic transduction pathway.     

In Vitro Osteoblast Differentiation is Negatively Regulated by Hoxc8

Hoxc8 has multiple roles in normal skeletal development. In this paper, a MC3T3-E1 subclone 4 osteogenic cell differentiation model was used to examine expression of Hoxc8 at multiple stages of osteogenesis. We found that Hoxc8 expression levels do not change in the early stage but increase in the middle stage and decrease in the late stage of osteogenesis. A knockdown of Hoxc8 by small-interfering RNA transfection in C2C12 cells indicated that Hoxc8 is a negative regulator of osteogenesis. Similarly, expression of Hoxc8 in C2C12 cells decreases alkaline phosphatase levels induced by bone morphogenetic protein-2 (BMP-2). The results of this study showed that Hoxc8 is involved in BMP-2-induced osteogenesis, and osteoblast differentiation in vitro is negatively regulated by Hoxc8, suggesting that Hoxc8 regulation is essential for osteoblast differentiation.     

Biodegradable Polycaprolactone (PCL) Nanosphere Encapsulating Superoxide Dismutase and Catalase Enzymes

Biodegradable polycaprolactone (PCL) nanosphere encapsulating superoxide dismutase (SOD) and catalase (CAT) were successfully synthesized using double emulsion (w/o/w) solvent evaporation technique. Characterization of the nanosphere using dynamic light scattering, field emission scanning electron microscope, and Fourier transform infrared spectroscopy revealed a spherical-shaped nanosphere in a size range of 812?±?64 nm with moderate protein encapsulation efficiency of 55.42?±?3.7 % and high in vitro protein release. Human skin HaCat cells were used for analyzing antioxidative properties of SOD- and CAT-encapsulated PCL nanospheres. Oxidative stress condition in HaCat cells was optimized with exposure to hydrogen peroxide (H₂O₂; 1 mM) as external stress factor and verified through reactive oxygen species (ROS) analysis using H₂DCFDA dye. PCL nanosphere encapsulating SOD and CAT together indicated better antioxidative defense against H₂O₂-induced oxidative stress in human skin HaCat cells in comparison to PCL encapsulating either SOD or CAT alone as well as against direct supplement of SOD and CAT protein solution. Increase in HaCat cells SOD and CAT activities after treatment hints toward uptake of PCL nanosphere into the human skin HaCat cells. The result signifies the role of PCL-encapsulating SOD and CAT nanosphere in alleviating oxidative stress.     

An MC3T3-E1 Cell Line Biomembrane Extraction and HPLC–ESI-MS n Method for Simultaneous Analysis of Potential Anti-Osteoporosis Components of Epimedium koreanum

Aerial parts of Epimedium koreanum Nakai have been used as Herba Epimedii in China. Its anti-osteoporosis effect has attracted much attention in recent years. In this study, a method involving osteoblastic cell (MC3T3-E1 cell line) extraction and high-performance liquid chromatography–electrospray ionisation–mass spectrometry (HPLC–ESI-MS ⁿ ) was developed for screening of potential anti-osteoporosis agents in E.?koreanum. Four compounds identified as epimedin?A, epimedin?B, epimedin?C and icariin were found to interact with MC3T3-E1 cells and possessed potent osteoblast-stimulating activity as evaluated by cell proliferation [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay] and differentiation [alkaline phosphatase (ALP) activity and Ca content] in?vitro. The results suggest that these four flavonoids are the anti-osteoporosis constituents of Herba Epimedii and that the method combining MC3T3-E1 cell extraction with HPLC–ESI-MS ⁿ is rapid and effective in screening anti-osteoporosis agents from traditional Chinese medicines.     

In vitro Degradation and Biocompatibility of Ca-P Coated Magnesium Alloy

Calcium-phosphate compounds(Ca-P) coating was prepared on an Mg-Al alloy(AZ60). Biodegradation of Ca-P coated magnesium alloy was evaluated in simulated body fluid(SBF) by examining the changes in magnesium ion concentration and pH value, which indicated that the Ca-P coating on magnesium alloy strongly affected the corrosion of magnesium alloy. Osteoblast MC3T3-E1 cells were utilized to investigate the cellular cytocompatibility. The cytocompatibility was measured by carrying out a series of tests, such as cholecystokinin-octapeptide(CCK-8) test, alkaline phosphatase activity(ALP) test, cellular morphology of hematoxylin-eosin(HE) staining and the induction of apoptosis. It was found that the cell function showed better in the Ca-P coated Mg-alloy extract than in the uncoated magnesium alloy extract. In summary, the results indicate that the Ca-P coating can improve the corrosion resistance of magnesium alloy and elevate cellular proliferation and differentiation of osteoblast MC3T3-E1 cells.     

Micropatterning of cells using modulated magnetic fields

A new technique of cell micropatterning was presented. Mouse osteoblast cells (MC3T3-E1) were seeded on a substrate whose surface was exposed to a periodically modulated magnetic field (a line pattern with a 200- or 600-microm pitch) produced by a field modulator inserted into a homogeneous magnetic field of 1 T generated by an electromagnet. The cells were trapped consistent with the line profile of the modulated field. The trapping efficiency was enhanced by adding Mn(II)EDTA (paramagnetic) to the cultivation medium. The cells were subsequently incubated in the magnetic field. The same technique was applied to whole blood to pattern red blood cells.     

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7β-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.     

Regulation of cellular responses to macroporous inorganic films prepared by the inverse-opal method

Regenerative medicine for repairing damaged body tissues has recently become critically important. Cell culture scaffolds are required for the control of cell attachment, proliferation, and differentiation in in vitro cell cultures. A new strategy to control cell adhesion, morphology, and proliferation was developed by culturing mouse osteoblast-like MC3T3-E1 cells on novel cell culture scaffolds fabricated using ordered nanometer-sized pores (100, 300, 500, and 1000 nm). Results of this study indicate that after 72 h of incubation, the number of cells cultured on a silica film with a pore size of 1000 nm was similar to or slightly lower than that cultured on a non-porous control silica film. Films with 100-500 nm pore sizes, however, resulted in the cell growth inhibition. Morphology of the cultured cells revealed increased elongation and the formation of actin stress fibers was virtually absent on macroporous silica films with 100-500 nm pore size. Vinculin molecules expressed in cells cultured on the non-porous silica films showed many clear focal adhesions, whereas focal contacts were insufficiently formed in cells cultured on macroporous films. The influence of hydroxyapatite (HAp) and alumina scaffolds on the behavior of MC3T3-E1 cells was also evaluated. The proliferation rate of MC3T3-E1 cells cultured on HAp films with 1000 nm pore size was increased to approximately 20% above than that obtained of cells cultured on non-porous HAp films. These results demonstrate that the pore size and constituents of films play a role in controlling the morphology and proliferation rate of MC3T3-E1 cells.     

Oxidative Stress Response of Blakeslea trispora Induced by Iron Ions During Carotene Production in Shake Flask Culture

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by iron ions during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD). The addition of 1.0 mM of FeCl₃ to the medium was associated with a mild oxidative stress as evidenced by remarkable increase of the specific activities of SOD and CAT. On the other hand, the addition 5.0 mM of FeCl₃ caused a strong oxidative stress resulting in a drastic decrease in carotene concentration. The oxidative stress in B. trispora changed the composition of the carotenes and caused a significant increase of γ-carotene ratio. The highest concentration of carotenes (115.0?±?3.5 mg/g dry biomass) was obtained in the basal medium without the addition of FeCl₃ after 8 days of fermentation. In this case, the carotenes consisted of β-carotene (46.3 %), γ-carotene (40.1 %), and lycopene (13.6 %). The addition of 1.0 mM of FeCl₃ into the medium did not change the concentration of carotenes. But, the composition of carotenes was changed with a drastic increase of γ-carotene ratio (61.6 %) and a decrease in β-carotene and lycopene ratio (31.2 and 7.2 %, respectively).     

Millettia ferruginea extract attenuates cisplatin-induced alterations in kidney functioning,DNA damage,oxidative stress,and renal tissue morphology

This study aimed to investigate the beneficial role of Millettia ferruginea extract (MF) in preventing cisplatin (Cisp) induced nephrotoxicity in rats. A total of 55 metabolites were identified using LC-MS analysis. The in vivo results indicated that MF pretreatment for 4 weeks (20 mg/kg b.w.) remarkably attenuated the altered renal biomarkers by decreasing the levels of plasma creatinine, urea, and uric acid when compared to the Cisp-group. The nephroprotective capacity of MF was further strengthened by histopathological observations, where Cisp + MF treated rats showed lower number of inflammatory cells and tubular degenerative changes than the Cisp-group. The harmful effects of cisplatin on renal oxidative stress indicators (MDA, SOD, CAT, and GPx), were restored by the treatment of MF. In addition, the reduction of inflammatory markers (IL-6 and TNF-α), associated with alleviating DNA fragmentation, highlighted the preventive effect of MF in kidney tissue. Additionally, MF components presented lower binding energies when docked into the active site of TNF-α and IL-6. The present findings concluded that M. ferruginea extract exhibited nephroprotective potential, which may be attributed to its antioxidant and anti-inflammatory properties. Further work is recommended to confirm the current results, explore the involved mechanism of action, and determine the therapeutic doses and time.     

The pharmacological effects of morroniside and loganin isolated from Liuweidihuang Wan, on MC3T3-E1 cells

Liuweidihuang wan (LW), initially a well-known formula for curing "wu chi wu ruan", is commonly used nowadays for clinical treatment of postmenopausal osteoporosis (PO), but the identity of the effective substance(s) remains unclear. The present study was designed to evaluate the effects of morroniside and loganin isolated from LW on the proliferation, differentiation and apoptosis of MC3T3-E1 cells, as well as the possible mechanism of action. Morroniside and loganin had no effects on the proliferation of MC3T3-E1 cells, but both susbtances could improve the activity of alkaline phosphatase (ALP), and increase the contents of collagen type I and osteocalcin. Simultaneously, the mRNA expression of caspase-3, capase-9, RANKL was down-regulated and that of bcl-2 was up-regulated, which partially explains the anti-osteoporosis mechanism in MC3T3-E1 cells. In conclusion, morroniside and loganin may directly promote the differentiation and inhibit the apoptosis of MC3T3-E1 cells, and accordingly indirectly reduce bone resorption, which makes them promising natural drugs leads for treating PO in the near future.     

In vitro Evaluation of Osteoblast Response to Sol-Gel Derived Gelatin-Siloxane Hybrids

Cytotoxicity and cytocompatibility of porous gelatin-siloxane hybrids were evaluated due to osteoblastic cell (MC3T3-E1) proliferation, ALP activity, or their responses to the hybrids and their extracts. The hybrids were found intoxic, and appropriate incorporation of calcium ions stimulated proliferation and differentiation in vitro. Cells were seeded into the porous hybrids and the cell morphology was studied. The hybrids involving calcium ions favored osteoblast growth and differentiation.     

Antioxidant, Anti-inflammatory, and Anti-senescence Activities of a Phlorotannin-Rich Natural Extract from Brown Seaweed Ascophyllum nodosum

Aging at the cellular level is characterized by oxidative stress, inflammation, and cell senescence. An extract of the brown seaweed Ascophyllum nodosum rich in phlorotannins has been studied for its inhibitory activity against oxidative stress, inflammation, and senescence. A. nodosum extract at 0.2?% prevented tBHP-induced reactive oxygen species production (evaluated using the H2DCF-DA test in cytofluorometry) in epithelial cells and LPS-induced TNF-?? and IL-6 release (evaluated using ELISA technique) in macrophages. A. nodosum extract also increased nuclear SIRT1 activity in epithelial cells. Altogether, these beneficial cellular effects of phlorotannin-rich A. nodosum extract could be used in topical therapeutic formulations against aging.     

Effect of Ginkgo leaf parenteral solution on blood and cochlea antioxidant and immunity indexes in OM rats

Oxidative stress is involved in the development and progression of otitis media (OM). In this study, we investigated the effect of Ginkgo leaf parenteral solution on blood and cochlea antioxidant and immunity indexs in OM rats. In OM model rats, blood and cochlea malondialdehyde (MDA), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and interleukin-10 (IL-10) levels were significantly increased, whereas antioxidant enzymes activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR)) were significantly decreased compared with normal rats. Treatment with Ginkgo leaf parenteral solution restored the altered parameters in a dose-dependent manner. We conclude that Ginkgo leaf parenteral solution confers protection against oxidative injuries in OM rats by increasing activities of antioxidants and immunity, suggesting a potential drug for the prevention and therapy of OM.     

Isolation and Characterization of Levan from Moderate Halophilic Bacteria Bacillus licheniformis BK AG21

Levan is fructose polymer as the result of biosynthesis by levansucrase. This study is aimed to explore the potential of moderate halophilic bacteria Bacillus licheniformis BK AG21 isolated from Bledug Kuwu, Purwodadi Central Java, in producing levan. This bacteria displayed positive potential in producing levan based on the appearance of slime mucoid on a sucrose medium. The optimum production of levan was attained when the culture medium containing sucrose and peptone as respective carbon and nitrogen sources was shaking incubated with speed of 150 rpm for 24 hours at 37 ^oC. Thermal gravimetric analysis revealed that levan produced by B. licheniformis BK AG21 decomposed at 214 ^oC. The structure of the isolated levan was elucidated with FTIR, ¹H and ¹³C NMR spectroscopies. Based on the obtained spectroscopic data, the isolated levan was composed of β-(2,6)-linkages of fructose residues.     

Ethanolic Extract from Pteris wallichiana Alleviates DSS-Induced Intestinal Inflammation and Intestinal Barrier Dysfunction by Inhibiting the TLR4/NF-κB Pathway and Regulating Tight Junction Proteins

The aim of the research was to determine the protective effect and mechanism of Pteris wallichiana J. Agardh extract (PWE) on DSS-induced ulcerative colitis (UC) in mice. In this research, PWE is rich in flavonoids and diterpenoids by UPLC-MS/MS analysis. In LPS-induced RAW264.7 cells, PWE reduced the productions of inflammatory factors (i.e., NO, TNF-α, IL-6, and IL-1β). In DSS-induced UC in mice, PWE improved disease activity index (DAI) score, attenuated oxidative stress by decreasing MPO and MDA activities and activating GSH and SOD levels, and inhibited TNF-α, IL-6, and IL-1β expressions in the colonic tissues. PWE also improved the intestinal barrier by upregulating the expressions of tight junction proteins, including occludin and ZO-1. Moreover, PWE extract alleviated intestinal inflammation by suppressing the TLR4/MyD88/NF-κB signaling pathway. Conclusion: PWE can alleviate DSS-induced UC in mice by increasing the expressions of intestinal tight junction proteins and inhibiting the TLR4/NF-κB inflammatory pathway.