Cytotoxicity In RAW264 Research Articles

Effects of Antidesma thwaitesianum Müll. Arg. Fruit Extract on Radical Scavenging and Apoptotic Properties

Antidesma thwaitesianum Müll. Arg. is one of a tropical fruit which is a good source of phenolic compounds. Here, we investigated the in vitro radical scavenging activity and effect of chlorogenic acid (CGA) and A. thwaitesianum fruit extract (AFE) against H2O2-induced cytotoxicity in RAW264.7 macrophage cells. Chemical components in the extract were analyzed by colorimetric method and high performance liquid chromatography. The results exhibited that the amount of total phenolic and total flavonoid contents in AFE were 25.486 ± 0.370 mg of gallic acid equivalents (GAE)/g extract and 7.264 ± 0.312 mg of catechin equivalents (CE)/g extract, respectively, while 114 μg/g crude extract CGA was detected in AFE. AFE showed scavenging activity against ABTS and DPPH radicals, with a 50% inhibition concentration of 11.435 ± 0.381 and 2.063 ± 0.022 mg/mL, respectively. Pretreatment with both CGA (10 µg/mL) and AFE (400 µg/mL) significantly restored cell viability in H2O2-induced cytotoxicity in RAW264.7 macrophage cells, and only CGA at the concentrations of 5 and 10 µg/mL could protect cells from apoptosis. In conclusion, CGA-containing AFE has potent in vitro antioxidant activity and can increase the cell survival in H2O2-induced RAW264.7 macrophage cells. These results support the nutritional and medical value of A. thwaitesianum fruit for health-promoting benefits. Keywords: Antidesma thwaitesianum Müll. Arg., Chlorogenic acid, Antioxidant activity, Anti-apoptotic activity, RAW264.7 macrophage cells

Design and synthesis of pseudo-rutaecarpines as potent anti-inflammatory agents via regulating MAPK/NF-κB pathways to relieve inflammation-induced acute liver injury in mice

Pseudo-natural products (PNPs) design strategy provides a great valuable entrance to effectively identify of novel bioactive scaffolds. In this report, novel pseudo-rutaecarpines were designed via the combination of several privileged structure units and 46 target compounds were synthesized. Most of them display moderate to potent inhibitory effect on LPS-induced NO production and low cytotoxicity in RAW264.7 macrophage. The results of the anti-inflammatory efficacy and action mechanism of compounds 7l and 8c indicated that they significantly reduced the release of IL-6, IL-1β and TNF-α. Further studies revealed that they can strongly inhibit the activation of NF-κB and MAPK signal pathways. The LPS-induced acute liver injury mice model studies not only confirmed their anti-inflammatory efficacy in vivo but also could effectively relieve the liver injury in mice. The results suggest that compounds 7l and 8c might serve as lead compounds to develop therapeutic drugs for treatment of inflammation.

Anti-inflammatory and antioxidative effects of gallic acid on experimental dry eye: in vitro and in vivo studies

BackgroundTo investigate the anti-inflammatory and antioxidative effects of gallic acid (GA) on human corneal epithelial cells (HCECs) and RAW264.7 macrophages as well as its therapeutic effects in an experimental dry eye (EDE) mouse model.MethodsA cell counting kit-8 (CCK-8) assay was used to test the cytotoxicity of GA. The effect of GA on cell migration was evaluated using a scratch wound healing assay. The anti-inflammatory and antioxidative effects of GA in vitro were tested using a hypertonic model (HCECs) and an inflammatory model (RAW264.7 cells). The in vivo biocompatibility of GA was detected by irritation tests in rabbits, whereas the preventive and therapeutic effect of GA in vivo was evaluated using a mouse model of EDE.ResultsIn the range of 0–100 μM, GA showed no cytotoxicity in RAW264.7 cells or HCECs and did not delay the HCECs monolayer wound healing within 24 h. Ocular tolerance to GA in the in vivo irritation test was good after seven days. In terms of antioxidative activity, GA significantly reduced the intracellular reactive oxygen species (ROS) in lipopolysaccharide (LPS) activated RAW264.7 macrophages and HCECs exposed to hyperosmotic stress. Furthermore, after pre-treatment with GA, the expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NADPH quinone oxidoreductase-1 (NQO-1) were significantly upregulated in RAW264.7 macrophages. GA also exhibits excellent anti-inflammatory properties. This is mainly demonstrated by the ability of GA to effectively downregulate the nuclear transcription factor-κB (NF-κB) pathway in LPS-activated RAW264.7 macrophages and to reduce inflammatory factors, such as nitric oxide (NO), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α). In vivo efficacy testing results in a mouse model of EDE showed that GA can effectively prevent and inhibit the apoptosis of corneal epithelial cells (CECs), reduce inflammatory factors in the cornea and conjunctiva as well as protect goblet cells.ConclusionIn vitro and in vivo results indicate that GA possesses potent anti-inflammatory and antioxidative properties with no apparent cytotoxicity within the range of 0–100 μM. It is a promising eye drop formulation for the effective prevention and treatment of dry eye disease (DED).

Cytotoxicity and Autophagy Induced by Ivermectin via AMPK/mTOR Signaling Pathway in RAW264.7 Cells.

The widespread and excessive use of ivermectin (IVM) will not only cause serious environmental pollution, but will also affect metabolism of humans and other mammals that are exposed. IVM has the characteristics of being widely distributed and slowly metabolized, which will cause potential toxicity to the body. We focused on the metabolic pathway and mechanism of toxicity of IVM on RAW264.7 cells. Colony formation and LDH detection assay showed that IVM significantly inhibited the proliferation of and induced cytotoxicity in RAW264.7 cells. Intracellular biochemical analysis using Western blotting assay showed that LC3-B and Beclin-1 were upregulated and p62 was down-regulated. The combination of confocal fluorescence, calcein-AM/CoCl2, and fluorescence probe results showed that IVM could induce the opening of the mitochondrial membrane permeability transition pore, reduce mitochondrial content, and increase lysosome content. In addition, we focused on induction of IVM in the autophagy signal pathway. The Western blotting results showed that IVM increased expression of p-AMPK and decreased p-mTOR and p-S6K expression in protein levels, indicating that IVM activated the AMPK/mTOR signaling pathway. Therefore, IVM may inhibit cell proliferation by inducing cell cycle arrest and autophagy.

Cytotoxicity and Apoptotic Mechanism of 2-Hydroxyethyl Methacrylate via Genotoxicity and the Mitochondrial-Dependent Intrinsic Caspase Pathway and Intracellular Reactive Oxygen Species Accumulation in Macrophages.

Macrophages are mainly active cells of the immune system and play a role in the defense of pathogens. However, the overactivation of macrophages by fatal pathogens can result in toxic responses. 2-hydroxyethyl methacrylate (HEMA), which is a hydrophilic monomer, is used in dental adhesive reagents and composite resins as well as biocompatible hydrogels. The mechanisms underlying the genotoxicity engendered by HEMA-induced apoptosis that leads to cytotoxicity remain unclear. Accordingly, this study was conducted to clarify such mechanisms. The results showed that HEMA induced cell toxicity in RAW264.7 macrophages depending on the concentration. A higher HEMA concentration was associated with a higher level of apoptosis and genotoxicity. Moreover, HEMA induced a concentration-dependent increase in mitochondrial dysfunction and the intrinsic caspase pathway, including the activation of caspase-3 and caspase-9. HEMA was also found to upregulate intracellular reactive oxygen species generation and to decrease the activity of antioxidant enzymes, including superoxide dismutase and catalase. Taken together, the mitochondrial-dependent intrinsic caspase pathway and intracellular reactive oxygen species accumulation were found to mediate HEMA-induced genotoxicity and apoptosis, leading to cytotoxicity in RAW264.7 macrophages.

Antimicrobial peptide CC34 attenuates intestinal inflammation via downregulation of the NF-κB signaling pathway.

The investigational drug CC34 is a cation peptide with multiple bioactivities. Here, we studied the anti-inflammatory effects of CC34 in lipopolysaccharide (LPS)-treated mouse monocyte-macrophage cells (RAW264.7) and in mice with LPS-induced intestinal inflammation. In vitro, CC34 treatment with less than 50μg/mL for 24h did not induce cytotoxicity in RAW264.7 cells. Furthermore, CC34 significantly lowered the levels of select inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Intracellular levels of reactive oxygen species (ROS) were lower in RAW264.7 cells treated with CC34 + LPS than in cells treated with LPS alone. Additionally, CC34 treatment suppressed iNOS and COX-2 mRNA levels in LPS-treated cells. We also observed that CC34 exerted anti-inflammatory activity by suppressing the phosphorylation of IKKβ, IκBα, and NF-κB p65 in vitro. Moreover, CC34 downregulated the release of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the jejunum tissue and serum of LPS-treated mice. We also found that the myeloperoxidase (MPO) levels were decreased, and the pathological damages were effectively abated in the jejunum tissue of CC34 + LPS-treated mice. In summary, we demonstrated that CC34 exerted anti-inflammatory activities, associated with the neutralization of LPS, inhibition of ROS, inhibition the NF-κB signaling pathway, and down-regulating the secretion of inflammatory cytokines. Thus, CC34 may represent an effective therapeutic strategy for intestinal inflammation.

두릅 열수추출물의 항염 및 항헬리코박터 효과

Aralia elata contains several bioactive compounds, such as saponin, oleanolic acid, and beta-carotene, which are known widely to control blood pressure and diabetes. In this study, we investigated the anti-Helicobacter pylori, antioxidant, anti-inflammatory, and cytotoxic effects of the extract of A. elata which was procured from Sunchang-gun, South Korea. The extract was prepared using water bath at 80℃ for 5 h. The total polyphenolics content in A. elata hot-water extract was 186.8±2.7 mgGAE/g, and the total flavonoid content was 81.9±1.5 mgQE/g. In addition, the extract exhibited anti-Helicobacter pylori activity and the growth of the bacteria was decreased with increasing concentration of the extract. SC50 value of DPPH radical scavenging activity was 3,274.7±47.7 μg/mL, and ABTS radical scavenging activity was 2,660.1±50.3 μg/mL. Furthermore, A. elata hot-water extract reduced the production of nitric oxide (NO), TNF-α, IL-1β, and IL-6 in LPS-stimulated RAW264.7 cells. Cell viability assay revealed no cytotoxicity in RAW264.7 cells even at a concentration of 100 μg/mL. The results confirmed that A. elata hot-water extract could be used as an antioxidant, antibacterial, and anti-inflammatory agent.

Cellular evaluation of the metal-organic framework PCN-224 associated with inflammation and autophagy

Metal-organic frameworks (MOFs) are innovative porous structures consisting of metal ions and organic ligands, which have been verified for extraordinary applications in nanomedicine and pharmaceuticals. PCN-224 is a type of Zr-based MOFs, which has recently emerged as one of the most attractive nanomaterials for various applications, such as drug delivery, bioimaging and cancer therapy due to its favorable and fascinating physical-chemical properties. However, the safety evaluation and the potential toxicological properties remain unclear. In this study, the general cytotoxicity of PCN-224 were examined in both human hepatocytes L-02 cells and mouse macrophages RAW264.7. Furthermore, the effect of inflammation and autophagy were measured in L-02 cells. The results indicated that PCN-224 was engulfed in L-02 cells and subsequently resulted in morphological changes, cell membrane destruction, and oxidative stress in L-02 cells. PCN-224 might trigger inflammation by promoting the secretion of inflammatory factors such as Tumor necrosis factors (TNF-α) and Interleukin (IL-6). PCN-224 might induce autophagosome accumulation and subsequently autophagic dysfunction. Additionally, PCN-224 induced cytotoxicity in RAW264.7 cells and increased the protein levels of the inflammasome component NLR Family Pyrin Domain Containing 3 (NLRP3) molecular, which indicated its cellular effects in different cell types. All of these results will support the reasonable use of PCN-224.

Cytotoxic effects of dental prosthesis grinding dust on RAW264.7 cells

Respiratory diseases, including pulmonary fibrosis, silicosis, and allergic pneumonia, can be caused by long-term exposure to dental prosthesis grinding dust. The extent of the toxicity and pathogenicity of exposure to PMMA dust, Vitallium dust, and dentin porcelain dust differs. The dust from grinding dental prosthesis made of these three materials was characterized in terms of morphology, particle size, and elemental composition. The adverse effects of different concentrations of grinding dust (50, 150, 300, 450, and 600 μg ml−l) on RAW264.7 macrophages were evaluated, including changes in cell morphology and the production of lactate dehydrogenase (LDH) and reactive oxygen species (ROS). The dust particles released by grinding dental prosthesis made of these materials had different morphologies, particle sizes, and elemental compositions. They also induced varying degrees of cytotoxicity in RAW264.7 macrophages. A possible cytotoxicity mechanism is the induction of lipid peroxidation and plasma membrane damage as the dust particles penetrate cells. Therefore, clinicians who regularly work with these materials should wear the appropriate personal protection equipment to minimize exposure and reduce the health risks caused by these particulates.

Procyanidin A2, a polyphenolic compound, exerts anti-inflammatory and anti-oxidative activity in lipopolysaccharide-stimulated RAW264.7 cells.

Procyandin A2 (PCA2) is a polyphenolic compound which is isolated from grape seeds. It has been reported that PCA2 exhibits antioxidative and anti-inflammatory effects, but its molecular mechanism is still poorly understood. This study tests the hypothesis that PCA2 suppresses lipopolysaccharide (LPS)-induced inflammation and oxidative stress through targeting the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and NF-E2-related factor 2 (Nrf2) pathways in RAW264.7 cells. PCA2 (20, 40, 80 μM) exhibited no significant cytotoxicity in RAW264.7 cells and showed an inhibitory effect on an LPS-induced nitrite level. Pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and reactive oxygen species (ROS) were suppressed by PCA2 with a concentration range of 0–80 μM. The mRNA levels of TNF-α and IL-6 were inhibited by PCA2 (80 μM). The hallmark-protein expression of the NF-κB (p-IKKα/β, p-IκBα, and p-p65) and MAPK (p-p38, p-JNK, and p-ERK) pathways were decreased by PCA2 in LPS-stimulated RAW264.7 cells. In addition, immunofluorescence results indicated that PCA2 (80 μM) promoted the translocation of NF-κB/p65 from the cytoplasm into the nucleus. PCA2 upregulated the expressions of Nrf2 and HO-1 and downregulated the expression of Keap-1. Simultaneously, PCA2 (80 μM) reversed LPS-induced Nrf2 translocation from the nucleus into the cytoplasm. Collectively, PCA2 protect cells against the damage from inflammation and oxidative injury, which suggest a potential therapeutic strategy for inflammatory and oxidative stress through targeting NF-κB, MAPK, and Nrf2 pathways in RAW264.7 cells.

Paeoniflorin suppresses IL-33 production by macrophages

Objective: Interleukin (IL)-33 has been attracting more and more attention as a new member of theIL-1 cytokine family in recent years. However, the underlying mechanisms referred to the regulation of endogenous IL-33 production are not fully illustrated. Paeoniflorin (PF) has been reported to possess multiple pharmacological activities, including anti-inflammation and anti-allergy. In this study, we aimed to investigate the effect of PF on IL-33 production by macrophages and explore the underlying mechanisms.Methods: In vivo, IL-33 production in mice after lipopolysaccharide (LPS) injection together with PF application was detected by enzyme-linked immunosorbent assay (ELISA). In vitro, MTT, Real-time PCR, ELISA, Calcium (Ca2+) imaging and Western blot were used to assess the cytotoxicity of PF, IL-33 expression at mRNA and protein levels, Ca2+ influx, protein kinase C (PKC) activity, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) activation in LPS-stimulated RAW264.7 macrophages with PF administration.Results: Our results indicated that PF (5 and 25 mg/kg) significantly reduced the production of TNF-a, IL-1β, and IL-33 in the peritoneal exudate of LPS-treated mice. In vitro assay, upregulation of PF concentration (≥ 20 μM) showed an increased cytotoxicity in RAW264.7 cells during the 24-h cell culture. PF (10 μM) inhibited IL-33 production, Ca2+ influx, PKC activity, NF-κB (p65) activation, and P38MAPK phosphorylation in LPS-treated macrophages. Notably, NF-κB inhibitor (BAY 11-7085), P38MAPK inhibitor (SB203580), and Ca2+ blocker (NiCl2) also curbed LPS-induced IL-33 production, respectively.Conclusions: PF suppresses IL-33 production by macrophages via inhibiting NF-κB and P38MAPK activation associated with the regulation of Ca2+ mobilization.

Gadolinium-conjugated CB86: a novel TSPO-targeting MRI contrast agent for imaging of rheumatoid arthritis

TSPO is up-regulated in activated macrophages, and serves as an attractive target for macrophages molecular imaging and therapy. MRI may be an ideal technique in the clinical management of RA due to its excellent spatial resolution. In the present study, a novel TSPO-targeting MRI contrast agent was developed by conjugating a novel TSPO ligand CB86 with gadolinium chelate to visualise inflamed regions in RA mice model. A novel TSPO ligand CB86 was linked to DTPAA, followed by chelation with gadolinium to obtain MRI targeted contrast agent CB86-DTPA-Gd. CB86-DTPA-Gd was characterised by MRI relaxivity, cell cytotoxicity, cell specificity and in vitro stability analysis. The distribution and MRI intensity was evaluated in RA rat model. Synthesis of CB86-DTPA-Gd was completed successfully with MRI relaxivity of 11.05/mM/sec (9.4 T, 25 °C). CB86-DTPA-Gd exhibited a good stability in human serum, high RAW264.7 cells specificity and no cytotoxicity in RAW264.7 cells. The biodistribution and MRI studies showed that the accumulation and signal intensity of CB86-DTPA-Gd in the right RA ankles was higher and stronger than those of Gd‑DTPA. This study demonstrates that CB86-DTPA-Gd can identify phagocytic active macrophages in the synovial joints, and has potential as a promising targeting MRI contrast agent for imaging of peripheral inflammation.

Atmospheric pressure non-thermal plasma exposure reduces Pseudomonas aeruginosa lipopolysaccharide toxicity in vitro and in vivo

Lipopolysaccharide (LPS) is an endotoxin composed of a polysaccharide and lipid component. It is intrinsically responsible for the pathogenicity of Gram-negative bacteria and is involved in the development of bacterial sepsis. Atmospheric pressure non-thermal plasma is proposed as a potential new approach for the treatment of infected tissue such as chronic wounds, with both antibacterial and wound-healing activities extensively described. Using both the RAW264.7 murine macrophage cell line in vitro assays and the Galleria mellonella insect in vivo toxicity model, the effect non-thermal plasma exposure on LPS-mediated toxicity has been characterised. Short (60 s) non-thermal plasma exposures of Pseudomonas aeruginosa conditioned growth media, membrane lysates and purified P. aeruginosa LPS, resulted in a substantial detoxification and reduction of LPS-induced cytotoxicity in RAW264.7 murine macrophages. Non-thermal plasma exposure (60 s) of purified P. aeruginosa LPS led to a significant (p < 0.05) improvement in the G. mellonella health index (GHI) score, a measure of in vivo toxicity. These findings demonstrate the ability of short plasma exposures to significantly reduce LPS-induced cytotoxicity both in vitro and in vivo; attenuating the toxicity of this important virulence factor intrinsic to the pathogenicity of Gram-negative bacteria.

Cervus and cucumis peptides ameliorates bone erosion in experimental arthritis by inhibiting osteoclastogenesis

ObjectiveRheumatoid arthritis is an autoimmune disease characterised by inflammation and bone loss, leading to joint destruction and deformity. The cervus and cucumis polypeptide (CCP) injection, one of the traditional Chinese...

Analysis of NO-suppressing activity of Strawberry Wine supplemented with ball-milled achenes.

Inflammation is generally thought to be involved in the development of several chronical diseases, therefore, phytochemicals to modulate immune responses has attracted great interests. The objective of the present study was to evaluate the potential anti-inflammatory effects of wine supplemented using ball-milled achene on modulating NO production and inducible nitric oxide synthase (iNOS) expression. Ball-milled achenes were added in strawberry must prior to fermentation, and the wine samples were then concentrated and extracted with water and/or ethanol prior to analysis. Bioactivities of wine extracts were evaluated using the cell viability assay, cell cycle measurements, NO production and iNOS expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatments of achenes supplemented strawberry wine extract up to 100μg/mL inhibited the proliferation of LPS-stimulated RAW264.7 cell via affecting the progression of cell cycle. Moreover, no detectable cytotoxicity in RAW264.7 cells was observed. The supplemented wine extract suppressed the action of LPS and led to a decreased NO production in stimulated cells. The inhibitory effect of the wine extract on NO production was determined to be a 25-40% decrease in the level of 25-100μg/mL, in contrast to a 10% decrease for conventional wine samples. Additionally, an alcoholic wine extract (100μg/mL) led to a 40.31% decrease in iNOS expression in LPS-stimulated cells, which was more effective than the same dose of tocopherol. The results show that strawberry wine supplemented with ball-milled achenes causes a substantial inhibition of NO production, and this biofunction is exerted via the down-regulation of iNOS expression.

Purification and Characterisation of κ-Carrageenan Oligosaccharides Prepared by κ-Carrageenase from Thalassospira sp. Fjfst-332

κ-Carrageenan oligosaccharides (KCOs) are promising agents for treating inflammatory diseases. However, the lack of purification and structural elucidation of KCOs has limited structure-function evaluation. In this study, using a system coupling medium pressure liquid chromatography (MPLC) with an evaporative light scattering detector (ELSD), four types of KCOs were separated. The total yield of the four KCO powders was ∼5.02% after purification (KCOs/κ-carrageenan, w/w). Their structural identities were characterised by ESI-MS, CID-MS/MS and NMR, as κ-neocarrabiose (α-DA-1,3-G4Srα/β), κ-neocarratetraose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β), κ-neocarrahexaose (α-DA-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β) and heterozygous κ/ι-neocarrahexaose (α-DA/DA2S-1,3-β-G4S-1,4-α-DA-1,3-β-G4S-1,4-α-DA-1,3-G4Srα/β). KCOs showed no cytotoxicity in RAW264.7 macrophages, and the anti-inflammatory activity was closely correlated with the degree of polymerisation and the number of sulfated groups. κ/ι-Neocarrahexaose exhibited the highest inhibition of ROS (Reactive Oxygen Species) production in LPS-induced RAW264.7 macrophages. The MPLC-ELSD system provides a platform for large-scale fabrication of purified KCOs and affords a route to these compounds that may regulate immune defense.

Antibacterial and detoxifying activity of NZ17074 analogues with multi-layers of selective antimicrobial actions against Escherichia coli and Salmonella enteritidis

NZ17074 (N1), an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is cytotoxic. To reduce its cytotoxicity, seven N1 analogues with different structures were designed by changing their disulfide bonds, hydrophobicity, or charge. The “rocket” analogue-N2 and the “kite” analogue-N6 have potent activity and showed lower cytotoxicity in RAW264.7 cells than N1. The NMR spectra revealed that N1, N2, and N6 adopt β-sheet structures stabilized by one or two disulfide bonds. N2 and N6 permeabilized the outer/inner membranes of E. coli, but did not permeabilize the inner membranes of S. enteritidis. N2 and N6 induced E. coli and S. enteritidis cell cycle arrest in the I-phase and R-phase, respectively. In E. coli and in S. enteritidis, 18.7–43.8% of DNA/RNA/cell wall synthesis and 5.7–61.8% of DNA/RNA/protein synthesis were inhibited by the two peptides, respectively. Collapsed and filamentous E. coli cells and intact morphologies of S. enteritidis cells were observed after treatment with the two peptides. Body weight doses from 2.5–7.5 mg/kg of N2 and N6 enhanced the survival rate of peritonitis- and endotoxemia-induced mice; reduced the serum IL-6, IL-1β and TNF-α levels; and protected mice from lipopolysaccharide-induced lung injury. These data indicate that N2 and N6, through multiple selective actions, may be promising dual-function candidates as novel antimicrobial and anti-endotoxin peptides.

복분자 자죽염의 마우스 대식세포주 및 복강 대식세포에 대한 면역증진 효과

Purple bamboo salt (PuBS) is commonly used as a medicinal food in Korea and has beneficial potentials such as antioxidant and anti-inflammatory effects. Rubus coreanus is called Bokbunja, which is used as a traditional medicine for treating asthma, impotence, and allergic diseases in Korea. The aim of present study was to investigate the immunostimulatory effect of PuBS composed with Rubus coreanus (PuBS-R). We performed comparative analysis between PuBS and PuBS-R in Raw264.7 cells, which is a mouse macrophage cell line, and peritoneal macrophages isolated primarily from the mouse peritoneal cavity. We evaluated cytotoxicity and the immune cytokine response in PuBS- and PuBS-R-treated cells. Both PuBS and PuBS-R did not have any cytotoxicity in Raw264.7 cells up to 500 μg/mL. Gene and protein levels of immune cytokines such as tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-10, and IL-12 were significantly elevated by PuBS-R more than PuBS in Raw264.7 cells. Moreover, we evaluated the immunostimulatory effects of PuBS-R on mouse primary peritoneal macrophages. Protein levels of inducible nitric oxide synthase, TNF-α, IFN-γ, IL-10, and IL-12 were significantly higher in PuBS-R-treated peritoneal macrophages than PuBS-treated peritoneal macrophages. These results suggest the potential immunostimulatory effect of PuBS-R for immunity against harmful infection.

新型氯膦酸二钠脂质体灌肠剂诱导小鼠巨噬细胞凋亡

目的：探讨一种氯膦酸二钠脂质体灌肠剂的制备及其诱导巨噬细胞凋亡的作用。方法：以DPPC和胆固醇为主要组分并添加带负电荷的磷酸二鲸蜡酯，用薄膜水化法制备脂质体；用MTT法测定该氯膦酸二钠脂质体对离体培养的小鼠巨噬细胞的致死活性；以免疫印迹法和流式细胞术对该氯膦酸二钠脂质体诱导的细胞凋亡进行了研究。结果：薄膜水化法制备所得的脂质体粒径适于巨噬细胞吞噬。用离体培养的小鼠巨噬细胞对氯膦酸二钠脂质体灌肠剂的细胞致死活性进行评估，该氯膦酸二钠脂质体制剂可有效地杀伤巨噬细胞，其细胞毒性随浓度而增加；免疫印迹法可从氯膦酸二钠脂质体处理的细胞提取物中检测到显著增加的活性半胱天冬酶-3，表明氯膦酸二钠脂质体可诱导细胞凋亡；流式细胞术检测Annexin V的结果确证氯膦酸二钠脂质体可诱导巨噬细胞凋亡。结论：该氯膦酸二钠脂质体制剂可诱导巨噬细胞凋亡从而清除巨噬细胞，有望用于某些免疫性疾病和炎症性疾病的辅助治疗。 Aim: To investigate apoptotic effect of a novel clodronate liposomal enema. Methods: A novel clodronate liposomal enema was developed by thin-film hydration using DPPC, cholesterol, and negatively charged dicetylphosphate. Lethality of clodronate liposomes was determined by MTT assay using RAW264.7 murine macrophages. Clodronate liposome-induced apoptosis was investigated by Western blotting and fluorescence-activated cell sorting (FACS). Results: A novel clodronate liposomal enema was developed by thin-film hydration. This liposomal enema had a size range that is suitable for phagocytosis. MTT assay showed that this clodronate liposomal enema efficiently caused cytotoxicity of RAW264.7 murine macrophages in a dose-dependent manner. Western blots showed remarkably increased cleaved caspase-3 in clodronate liposome-treated RAW264.7 cells compared to controls, indicating apoptosis induced by clodronate liposomes. In addition, FACS analysis for Annexin V confirmed that clodronate liposomes induced apoptosis in RAW264.7 macrophages. Conclusion: This clodronate liposomal enema is capable of inducing macrophage apoptosis, and may be used for macrophage depletion in certain diseases as an adjuvant therapy.

The Protective Effect of Bafilomycin A1 Against Cobalt Nanoparticle-Induced Cytotoxicity and Aseptic Inflammation in Macrophages In Vitro.

Co ions released due to corrosion of Co nanoparticles (CoNPs) in the lysosomes of macrophages may be a factor in the particle-induced cytotoxicity and aseptic inflammation accompanying metal-on-metal (MOM) hip prosthesis failure. Here, we show that CoNPs are easily dissolved under a low pH, simulating the acidic lysosomal environment. We then used bafilomycin A1 to change the pH inside the lysosome to inhibit intracellular corrosion of CoNPs and then investigated its protective effects against CoNP-induced cytotoxicity and aseptic inflammation on murine macrophage RAW264.7 cells. XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} assays revealed that bafilomycin A1 can significantly decrease CoNP-induced cytotoxicity in RAW264.7 cells. Enzyme-linked immunosorbent assays showed that bafilomycin A1 can significantly decrease the subtoxic concentration of CoNP-induced levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), but has no effect on anti-inflammatory cytokines (transforming growth factor-β and interleukin-10) in RAW264.7 cells. We studied the protective mechanism of bafilomycin A1 against CoNP-induced effects in RAW264.7 cells by measuring glutathione/oxidized glutathione (GSH/GSSG), superoxide dismutase, catalase, and glutathione peroxidase levels and employed scanning electron microscopy, transmission electron microscopy, and energy dispersive spectrometer assays to observe the ultrastructural cellular changes. The changes associated with apoptosis were assessed by examining the pAKT and cleaved caspase-3 levels using Western blotting. These data strongly suggested that bafilomycin A1 can potentially suppress CoNP-induced cytotoxicity and aseptic inflammation by inhibiting intracellular corrosion of CoNPs and that the reduction in Co ions released from CoNPs may play an important role in downregulating oxidative stress in RAW264.7 cells.