Inonotus obliquus (IO) is parasitic mushroom that grows on birch and other trees in Russia, Korea, Europe and United States. However, IO is not readily available for consumption due to its high cost and difficult growth. In this regard, IO was inoculated on germinated brown rice (GBR) in the present study and the antioxidant and anti-inflammatory activities of the IO grown on germinated brown rice (IOGBR) extracts were evaluated extensively and compared with those for IO and GBR. IOGBR showed highest antioxidant activities with scavenging total intracellular ROS and MDA levels as well as increasing the antioxidant enzymes activity in the H₂O₂-stimulated mice liver. It also exhibited best inflammatory activities by suppressing the proinflammatory mediators such as NO, PGE₂, iNOS, COX-2, TNF-α, IL-1β, and IL-6 in an LPS-stimulated RAW 264.7 cell line. This study provides a comparative approach to find out an excellent natural source of antioxidants and anti-inflammatory agent as a dietary supplement.

Five new aromatic compounds, designed as lucidumins A-D (1-4) and lucidimine E (9), along with seven known aromatic compounds (5-8, 10-12) were isolated from Ganoderma lucidum. Their structures were determined by spectroscopic method. Bioactive evaluation showed that compounds 2-4 and 6-10 displayed remarkable neuroprotective activities against corticosterone-induced PC12 cell damage, with the cell viability ranging from 69.99% to 126.00%; and compounds 1-4, 9 and 10 exhibited significant anti-inflammatory activities against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages, with ICvalues ranging from 4.68 to 15.49 μM. In particular, compound 10 showed remarkable neuroprotection with ECvalue of 2.49 ± 0.12 μM, and potent anti-inflammation with ICvalue of 4.68 ± 0.09 μM.

High mobility group box 1 (HMGB1) is now recognized as a late mediator of sepsis. We tested hypothesis that ascorbic acid (AscA) induces heme oxygenase (HO)-1 which inhibits HMGB1 release in lipopolysaccharide (LPS)-stimulated cells and increases survival of septic mice. AscA increased HO-1 protein expression in a concentration- and time-dependent manner via Nrf2 activation in RAW 264.7 cells. HO-1 induction by AscA was significantly reduced by Nrf2 siRNA-transfected cells. Mutation of cysteine to serine of keap-1 proteins (C151S, C273S, and C288S) lost the ability of HO-1 induction by AscA, due to failure of translocation of Nrf-2 to nucleus. The PI3 kinase inhibitor, LY294002, inhibited HO-1 induction by AscA. Oxyhemoglobin (HbO2), LY294002, and ZnPPIX (HO-1 enzyme inhibitor) reversed effect of AscA on HMGB1 release. Most importantly, administration of AscA (200mg/kg, i.p.) significantly increased survival in LPS-induced endotoxemic mice. In cecal ligation and puncture (CLP)-induced septic mice, AscA reduced hepatic injury and serum HMGB1 and plasminogen activator inhibitor (PAI)-1 in a ZnPPIX-sensitive manner. In addition, AscA failed to increase survival in Nrf2 knockout mice by LPS. Thus, we concluded that high dose of AscA may be useful in the treatment of sepsis, at least, by activation of Nrf2/HO-1 signals.

Poria cocos Wolf (Polyporaceae) has been used as a medicinal fungus to treat various diseases since ancient times. This study aimed to investigate the anti-inflammatory chemical constituents of the sclerotia of P. cocos. Based on bioassay-guided fractionation using lipopolysaccharide (LPS)-stimulated Raw264.7 cells, chemical investigation of the EtOH extract of the sclerotia of P. cocos resulted in the isolation and identification of eight compounds including six triterpenoids, namely poricoic acid A (1), 3-O-acetyl-16α-hydroxydehydrotrametenolic acid (2), polyporenic acid C (3), 3β-hydroxylanosta-7,9(11),24-trien-21-oic acid (4), trametenolic acid (5), and dehydroeburicoic acid (6), as well as (-)-pinoresinol (7) and protocatechualdehyde (8). The structures of the isolated compounds were determined by spectroscopic analysis, includingH andC NMR spectra, and LC/MS analysis. The anti-inflammatory activities of the isolates were evaluated by estimating their effect on the production of nitric oxide (NO) and prostaglandin E(PGE) in LPS-stimulated Raw264.7 as well as on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compounds 1-5 inhibited NO production and iNOS expression in LPS-stimulated Raw264.7 cells. Among them, compound 1 exerted the highest anti-inhibitory activity and reduced PGElevels via downregulation of COX-2 protein expression. The findings of this study provide experimental evidence that the sclerotia of P. cocos are a potential source of natural anti-inflammatory agents for use in pharmaceuticals and functional foods. Furthermore, the most active compound 1, seco-lanostane triterpenoid, could be a promising lead compound for the development of novel anti-inflammatory agents.

INTRODUCTION:Cannabidiol (CBD) containing products are available in a plethora of flavors including oral, sublingual, and inhalable forms. Immunotoxicological effects of CBD containing liquids were assessed by hypothesizing that CBD regulates oxidative stress and lipopolysaccharide (LPS) induced inflammatory responses in macrophages, epithelial cells, and fibroblasts.

METHODS:Epithelial cells (BEAS-2B and NHBE), macrophages (U937), and lung fibroblast cells (HFL-1) were treated with varying CBD concentrations or exposed to CBD aerosols and reactive oxygen species (ROS), and the inflammatory mediators, were measured. Furthermore, monocytes and epithelial cells were stimulated with LPS in combination with CBD or dexamethasone to understand the anti-inflammatory effects of CBD.

RESULTS:CBD showed differential effects on IL-8 and MCP-1, and acellular and cellular ROS levels. CBD significantly attenuated LPS-induced NF-κB activity and IL-8 and MCP-1 release from macrophages. Cytokine array data depicted a differential cytokine response due to CBD. Inflammatory mediators, IL-8, serpin E1, CXCL1, IL-6, MIF, IFN-γ, MCP-1, RANTES, and TNF-α were induced, whereas MCP-1/CCL2, CCL5, eotaxin, IL-1ra, and IL-2 were reduced. CBD and dexamethasone treatments reduced the IL-8 level induced by LPS when the cells were treated individually, but showed antagonistic effects when used in combination via MCPIP (monocytic chemotactic protein-induced protein).

CONCLUSION:CBD differentially regulated basal pro-inflammatory response and attenuated both LPS-induced cytokine release and NF-κB activity in monocytes, similar to dexamethasone. Thus, CBD has a differential inflammatory response and acts as an anti-inflammatory agent in pro-inflammatory conditions but acts as an antagonist with steroids, overriding the anti-inflammatory potential of steroids when used in combination.

Cannabinoid (CB) ligands have demonstrated neuroprotective properties. In this study we compared the effects of a diverse set of CB ligands againstβ amyloid-mediated neuronal toxicity and activated microglial-conditioned media-based neurotoxicity in vitro, and compared this with a capacity to directly alter β amyloid (Aβ) fibril or aggregate formation. Neuroblastoma (SH-SY5Y) cells were exposed to Aβ1-42 directly or microglial (BV-2 cells)conditioned media activated with lipopolysaccharide (LPS) in the presence of the CB1 receptor-selective agonist ACEA, CB2 receptor-selective agonist JWH-015, phytocannabinoids Δ(9)-THC and cannabidiol (CBD), the endocannabinoids 2-arachidonoyl glycerol (2-AG) and anandamide or putative GPR18/GPR55ligands O-1602 and abnormal-cannabidiol (Abn-CBD). TNF-α and nitrite production was measured in BV-2 cells to compare activation via LPS or albumin with Aβ1-42. Aβ1-42 evoked a concentration-dependent loss of cell viability in SH-SY5Y cells but negligible TNF-α and nitrite production in BV-2 cells compared to albumin or LPS. Both albumin and LPS-activated BV-2 conditioned media significantly reduced neuronal cell viability but were directly innocuous to SH-SY5Y cells. Of those CB ligands tested, only 2-AG and CBD were directly protective against Aβ-evoked SH-SY5Y cell viability, whereasJWH-015, THC, CBD, Abn-CBD and O-1602 all protected SH-SY5Y cells from BV-2 conditioned media activated via LPS. While CB ligands variably altered the morphology of Aβ fibrils and aggregates, there was no clear correlation between effects on Aβ morphology and neuroprotective actions. These findings indicate a neuroprotective action of CB ligands via actions at microglial and neuronal cells.

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, Delta(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD), decrease the production and release of proinflammatory cytokines, including interleukin-1beta, interleukin-6, and interferon (IFN)beta, from LPS-activated microglial cells. The cannabinoid anti-inflammatory action does not seem to involve the CB1 and CB2 cannabinoid receptors or the abn-CBD-sensitive receptors. In addition, we found that THC and CBD act through different, although partially overlapping, mechanisms. CBD, but not THC, reduces the activity of the NF-kappaB pathway, a primary pathway regulating the expression of proinflammatory genes. Moreover, CBD, but not THC, up-regulates the activation of the STAT3 transcription factor, an element of homeostatic mechanism(s) inducing anti-inflammatory events. Following CBD treatment, but less so with THC, we observed a decreased level of mRNA for the Socs3 gene, a main negative regulator of STATs and particularly of STAT3. However, both CBD and THC decreased the activation of the LPS-induced STAT1 transcription factor, a key player in IFNbeta-dependent proinflammatory processes. In summary, our observations show that CBD and THC vary in their effects on the anti-inflammatory pathways, including the NF-kappaB and IFNbeta-dependent pathways.

Hemp seed (Fructus cannabis) is rich in lignanamides, and initial biological screening tests showed their potential anti-inflammatory and anti-oxidative capacity. This study investigated the possible effects and underlying mechanism of cannabisin F, a hempseed lignanamide, against inflammatory response and oxidative stress in lipopolysaccharide (LPS)-stimulated BV2 microglia cells. Cannabisin F suppressed the production and the mRNA levels of pro-inflammatory mediators such as interleukin 6 (IL-6) and tumor necrosis factorα (TNF-α) in a concentration-dependent manner in LPS-stimulated BV2 microglia cell. Furthermore, cannabisin F enhanced SIRT1 expression and blocked LPS-induced NF-κB (Nuclear factor kappa B) signaling pathway activation by inhibiting phosphorylation of IκBα (Inhibit proteins of nuclear factor kappaB) and NF-κB p65. And the SIRT1 inhibitor EX527 significantly inhibited the effect of cannabisin F on pro-inflammatory cytokines production, suggesting that the anti-inflammatory effects of cannabisin F are SIRT1-dependent. In addition, cannabisin F reduced the production of cellular reactive oxygen species (ROS) and promoted the expression of Nrf2 (Nuclear factor erythroid-2 related factor 2) and HO-1 (Heme Oxygenase-1), suggesting that the anti-oxidative effects of cannabisin F are related to Nrf2 signaling pathway. Collectively, these results suggest that the neuro-protection effect ofcannabisin F against LPS-induced inflammatory response and oxidative stress in BV2 microglia cells involves the SIRT1/NF-κB and Nrf2 pathway.

The healthy benefits of hemp (L.) seed have often been attributed to its oils and proteins. Recent studies reveal that hemp seed phenylpropionamides could also show various bioactivities. Continuation of our study on hemp seed provided a phenylpropionamide, coumaroylaminobutanol glucopyranoside (CLG). This work investigated the neuroprotective effect of CLG and its underlying mechanism using lipopolysaccharide-induced BV2 microglia. Our study demonstrated that CLG increased adenosine monophosphate-activated protein kinase (AMPK) expression, suppressed the nuclear factor-kappa B (NF-κB) signaling pathway by inhibiting the phosphorylation of IκBα and NF-κB p65 and decreased proinflammatory cytokine levels in a concentration-dependent manner. Furthermore, CLG reduced the production of cellular reactive oxygen species and stimulated the nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway. Collectively, these results suggested that CLG effectively and simultaneously inhibited inflammatory responses and oxidative stress through the NF-κB and Nrf-2 signaling pathways. AMPK was also involved in the anti-inflammatory effect of CLG. This study provides new insights into the diverse bioactive constituents of hemp seed.

Flavonoids and coumaric acid derivatives were identified from dandelion flower (Taraxacum officinale). Characteristics of chain-breaking antioxidants, such as extended lag phase and reduced propagation rate, were observed in oxidation of linoleic acid emulsion with the addition of dandelion flower extract (DFE). DFE suppressed both superoxide and hydroxyl radical, while the latter was further distinguished by both site-specific and non-specific hydroxyl radical inhibition. DPPH-radical-scavenging activity and a synergistic effect with alpha-tocopherol were attributed to the reducing activity derived from phenolic content of DFE. A significant (p < 0.05) and concentration-dependent, reduced nitric oxide production from acterial-lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells was observed with the addition of DFE. Moreover, peroxyl-radical-induced intracellular oxidation of RAW264.7 cells was inhibited significantly (p < 0.05) by the addition of DFE over a range of concentrations. These results showed that the DFE possessed marked antioxidant activity in both biological and chemical models. Furthermore, the efficacy of DFE in inhibiting both reactive oxygen species and nitric oxide were attributed to its phenolic content.

Acute exercise induces changes in peripheral mononuclear cells' (PBMCs) capabilities to produce cytokines. The aim was to investigate the effect of docosahexaenoic acid (DHA) diet supplementation on cytokine production, by lipopolysaccharide (LPS)-stimulated PBMCs after exercise, and the in vitro influence of temperature. Fifteen male soccer players were randomly assigned to a placebo or an experimental group. The experimental group consumed an almond-based beverage enriched with DHA (1.16 g DHA/day) for 8 weeks, whereas the placebo group consumed a similar non-enriched beverage. Blood samples were taken before and after the nutritional intervention in basal conditions and 2 h after acute exercise. Nutritional intervention significantly increased the DHA content in erythrocytes only in experimental group (from 34 ± 3.6 to 43 ± 3.6 nmols DHA/10(9) erythrocytes). Exercise significantly increased Toll-like receptor 4 (TLR4) in PBMCs but only in the placebo group (203 %). Exercise also significantly increased IL6, IL8, VEGF, INFγ, TNFα, IL1α, IL1β, MCP1, and EGGproduction rates by LPS-stimulated PBMCs, and this response was attenuated by DHA supplementation. Temperature but not DHA also affected the pattern of cytokine production increasing IL6, IL8, IL1β, and MCP1 synthesis. The higher change was evidenced in IL1β increasing the production rate at 39.5 °C from 3.19 ± 0.77 to 22.4 ± 6.1 pg/h 10(6) PBMC in placebo and from 2.36 ± 0.11 to 10.6 ± 0.38 pg/h 10(6) PBMC in the supplemented group. The profile of affected cytokines differs between temperature and exercise, suggesting a different PBMC activation pathway. DHA diet supplementation only attenuated cytokine production after exercise and not that induced by temperature.

Atropa belladonna and Echinacea angustifolia have been used in homeopathy as modulators of inflammatory processes, in simple potency or 'accord of potencies', as recommended by homotoxicology. We evaluated their effects on leukocyte migration and macrophage activity induced by experimental peritonitis in vivo. Mice were injected (i.p.) with LPS (1.0mg/kg) and treated (0.3ml/10g/day, s.c.) with different commercial forms of these medicines. Echinacea angustifolia D4--a simple potency preparation--and Belladonna Homaccord, Belladonna Injeel, Belladonna Injeel Forte, Echinacea Injeel and Echinacea Injeel Forte--all in 'accord of potencies'--were tested. The association of A. belladonna and E. angustifolia in 'accord of potencies' produced an increase of polymorphonuclear cell migration (Kruskal-Wallis, P = 0.03) and a decrease of mononuclear cell percentages (Kruskal-Wallis, P

Morchella conica is a species of rare edible mushroom whose multiple medicinal functions have been proven. However, reports barely mention the mechanisms of these functions. In this study, the effects of two polysaccharides from M. conica (PMCs) on nitric oxide (NO) production in lipopolysaccharide (LPS)-treated macrophages were investigated. The results showed that 50-200μg/ml of the extracellular polysaccharide (EPMC) and 25-200 μg/ml of the intracellular polysaccharide (IPMC) significantly inhibited NO production. Accordingly, the signal mechanisms were also explored. It was found that 100 μg/ml of EPMC and 25 μg/ml of IPMC could efficiently down-regulate theinducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) DNA-binding activity and up-regulate heme oxygenase 1 (HO-1) expression. Moreover, by using a HO-1 inhibitor NaPP to treat the cells, the PMC-inhibited NO production and iNOS expression, rather than NF-κB activation,were released partially, indicating that HO-1 probably medicates the inhibition of PMCs on iNOS and NO. Besides, EPMC also significantly suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38), c-jun N-terminal kinase, mitogen-activated protein kinase kinase 4, and expression ofNF-κB inducing kinase, while IPMC seemed to show no regular effect on p38. In conclusion, PMCs inhibited NO production in LPS-induced macrophages through regulating a series of signal pathways, suggesting that PMCs play a potential role on immunomodulation and treating related diseases.

BACKGROUND:Poria cocos Wolf, a medicinal fungus, is widely used in traditional medicines in East Asian countries owing to its various therapeutic potentials. Although several studies have demonstrated the anti-inflammatory activity of this fungus, its underlying mechanisms have not yet been clearly defined.

METHODS:In the present study, we have demonstrated the anti-inflammatory effects of ethanol extract of P. cocos (EEPC) in lipopolysaccaride (LPS)-stimulated RAW 264.7 macrophages. As inflammatory parameters, the productions of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1β and tumor necrosis factor (TNF)-α were evaluated. We also examined the EEPC's effect on the nuclear factor-kappaB (NF-κB) signaling pathway.

RESULTS:Our results indicated that EEPC exhibits a potent inhibitory effect on NO production and inhibits PGE2 release in LPS-induced macrophages without affecting cell viability. EEPC also significantly attenuated LPS-induced secretion of inflammatory cytokines IL-1β and TNF-α. Additionally, LPS-induced expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-1β, and TNF-α was decreased by pre-treatment with EEPC at the transcriptional level. Moreover, EEPC clearly inhibited LPS-induced nuclear translocation of NF-κB p65 subunits, which correlated with EEPC's inhibitory effects on inhibitor kappaB (IκB) degradation. Moreover, EEPC clearly suppressed the LPS-induced DNA-binding activity of NF-κB, as well as the nuclear translocation of the NF-κB p65, which correlated with EEPC's inhibitory effects on inhibitor kappaB (IκB) degradation.

CONCLUSIONS:Taken together, our data indicates that EEPC targets the inflammatory response of macrophages via inhibition of iNOS, COX-2, IL-1β, and TNF-α through inactivation of the NF-κB signaling pathway, supporting the pharmacological basis of P. cocos as a traditional herbal medicine for treatment of inflammation and its associated disorders.

Hepatocellular carcinoma (HCC) has a poor outcome. Most HCCs develop in the context of liver fibrosis and cirrhosis caused by chronic inflammation. Short-term fasting approaches enhance the activity of chemotherapy in preclinical cancer models, other than HCC. Multi-tyrosine kinase inhibitor Sorafenib is the mainstay of treatment in HCC. However, its benefit is frequently short-lived. Whether fasting can alleviate liver fibrosis and whether combining fasting with Sorafenib is beneficial remains unknown. A 24 hr fasting (2% serum, 0.1% glucose)-induced changes on human hepatic stellate cells (HSC) LX-2 proliferation/viability/cell cycle were assessed by MTT and flow cytometry. Expression of lypolysaccharide (LPS)-induced activation markers (vimentin, αSMA) was evaluated by qPCR and immunoblotting. Liver fibrosis and inflammation were evaluated in a mouse model of steatohepatitis exposed to cycles of fasting, by histological and biochemical analyses. A 24 hr fasting-induced changes were also analyzed on the proliferation/viability/glucose uptake of human HCC cells exposed to Sorafenib. An expression panel of genes involved in survival, inflammation, and metabolism was examined by qPCR in HCC cells exposed to fasting and/or Sorafenib. Fasting decreased the proliferation and the activation of HSC. Repeated cycles of short term starvation were safe in mice but did not improve fibrosis. Fasting synergized with Sorafenib in hampering HCC cell growth and glucose uptake. Finally, fasting normalized the expression levels of genes which are commonly altered by Sorafenib in HCC cells. Fasting or fasting-mimicking diet diets should be evaluated in preclinical studies as a mean to potentiate the activity of Sorafenib in clinical use.

The aim of this study was to investigate the anti-inflammatory properties of each fraction of Hericium erinaceus (HE). The ethanol extract from HE was partitioned with different solvents in the order of increasing polarity. The treatment with 10-100μg/mL of each fraction did not reduce RAW 264.7 cell viability except ethyl acetate fraction. Among the various extracts, the chloroform fraction showed the most potent activity against nitric oxide (NO), prostaglandin E(2) (PGE(2)) and reactive oxygen species (ROS). The western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that chloroform fraction from HE (CHE) significantly reduced the protein level of iNOS and cyclooxygenase-2 (COX-2) or mRNA levels of iNOS in lipopolysaccharide-induced macrophages. Furthermore, CHE inhibited the translocation of nuclear factor (NF)-κB p65 subunit, phsophorylation of I-κB, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in a dose-dependent manner. Furthermore, the activation of both activator protein-1 (AP-1) and NF κB in the nucleus were abrogated by CHE with luciferase assay. In conclusion, these results indicate that CHE may provide an anti-inflammatory effect by attenuating the generation of excessive NO, PGE(2), and ROS and by suppressing the expression of pro-inflammatory genes through the inhibition of NF-κB and JNK activity.

Ganoderma lucidum extract (GLE) is a potent ancient Asian remedy for the treatment of various diseases. This study investigated GLE preventive effects on LPS-stimulated inflammation of BV-2 microglial cells. The results show that pre-treatment with GLE decreased expression of pro-inflammatory cytokines: G-CSF, IL1-α, MCP-5, MIP3α, and, with a higher effect in MIP3α. In RT-PCR assays, pre-treatment with GLE decreased mRNA expression of CHUK, NFκB1/p150, and IKBKE (NFƙB signaling), which may be associated with the neuropathology of Alzheimer's disease. The data show GLE inhibiting ability on pro-inflammatory mediators' release and suggest a potential role of GLE in neurodegenerative disease prevention.

BACKGROUND:Steroids are a leading cause of femoral head osteonecrosis. Currently there are no medications available to prevent and/or treat steroid-associated osteonecrosis. Low-intensity pulsed ultrasound (LIPUS) was approved by the FDA for treating delayed union of bone fractures. Some studies have reported that LIPUS can enhance bone formation and local blood flow in an animal model of fracture healing. However, whether the effect of osteogenesis and neovascularization by LIPUS can enhance the repair progress in steroid-associated osteonecrosis is unknown.

QUESTIONS/PURPOSES:We hypothesized that LIPUS may facilitate osteogenesis and neovascularization in the reparative processes of steroid-associated osteonecrosis. Using a rabbit animal model, we asked whether LIPUS affects (1) bone strength and trabecular architecture; (2) blood vessel number and diameter; and (3) BMP-2 and VEGF expression.

METHODS:Bilateral femoral head necrosis was induced by lipopolysaccharide and methylprednisolone in 24 rabbits. The left femoral heads of rabbits received LIPUS therapy (200 mW/cm(2)) for 20 minutes daily and were classified as the LIPUS group. The right femoral heads of the same rabbits did not receive therapy and were classified as the control group. All rabbits were euthanized 12 weeks after LIPUS therapy. Micro-CT, biomechanical testing, histologic evaluation, immunohistochemistry, quantitative real-time PCR, and Western blot were used for examination of the effects of LIPUS.

RESULTS:Twelve weeks after LIPUS treatment, the loading strength in the control group was 355± 38 N (95% CI, 315-394 N), which was lower (p = 0.028) than that in the LIPUS group (441 ± 78 N; 95% CI, 359-524 N). The bone tissue volume density (bone volume/total volume) in the LIPUS group (49.29% ± 12.37%; 95 % CI, 36.31%-62.27%) was higher (p = 0.022) than that in the control group (37.93% ± 8.37%; 95 % CI, 29.15%-46.72%). The percentage of empty osteocyte lacunae in the LIPUS group (17% ± 4%; 95% CI, 15%-20%) was lower (p = 0.002) than that in the control group (26% ± 9%; 95% CI, 21%-32%). The mineral apposition rate (μm/day) in the LIPUS group (2.3 ± 0.8 μm/day; 95% CI, 1.82.8 μm/day) was higher (p = 0.001) than that in the control group (1.6 ± 0.3 μm/day; 95% CL, 1.4-1.8 μm/day). The number of blood vessels in the LIPUS group (7.8 ± 3.6/mm(2); 95% CI, 5.5-10.1 mm(2)) was greater (p = 0.025) than the number in the control group (5.7 ± 2.6/mm(2); 95% CI, 4.0-7.3mm(2)). Messenger RNA (mRNA) and protein expression of BMP-2 in the LIPUS group (75 ± 7, 95% CI, 70-79; and 30 ± 3, 95% CI, 28-31) were higher (both p<0.001) than those in the control groups (46± 5, 95% CI, 43-49; and 15 ± 2, 95% CI, 14-16). However, there were no differences (p = 0.114 and 0.124) in mRNA and protein expression of vascular endothelial growth factor between the control (26 ± 3, 95% CI, 24-28; and 22 ± 6, 95% CI, 18-26) and LIPUS groups (28 ± 2, 95% CI, 26-29; and 23 ±6, 95% CI, 19-27).

CONCLUSIONS:The results of this study indicate that LIPUS promotes osteogenesis and neovascularization, thus promoting bone repair in this steroid-associated osteonecrosis model.

CLINICAL RELEVANCE:LIPUS may be a promising modality for the treatment of early-stage steroid-associated osteonecrosis. Further research, including clinical trials to determine whether LIPUS has a therapeutic effect on patients with early-onset steroid-associated osteonecrosis may be warranted.

Mushrooms are valuable sources of biologically active compounds possessing anticancer, antiplatelet, and anti-inflammatory properties. Phellinus baumii is a mushroom used in folk medicine for a variety of human diseases. However, its potential anti-inflammatory effect has remained unclear. Therefore, we studied the effect of P. baumii ethyl acetate extract (PBEAE) on inflammatory mediator and proinflammatory cytokine protein and/or mRNA expression levels using the nitric oxide (NO) assay, enzyme immunoassay (EIA), western blot, and reverse transcription polymerase chain reaction (RT-PCR) in lipopolysaccharide (LPS)-stimulated macrophage like RAW264.7 cells. PBEAE markedly inhibited NO generation and prostaglandin E(2) (PGE(2)) synthesis in a concentration-dependent pattern without any cytotoxic effect at the concentration range used. PBEAE also suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. In addition, LPS-induced iNOS and COX-2 mRNA expression levels were dose-dependently inhibited by PBEAE pretreatment. Furthermore, PBEAE attenuated the mRNA expression levels of proinflammatory cytokines, specifically interleukin (IL)-1β, IL-6, and granulocyte macrophage colony-stimulating factor (GM-CSF), in a concentration-dependent fashion. Our study suggests that P. baumii might exhibit anti-inflammatory properties by downregulating proinflammatory mediators. Thus, further study on compounds isolated from PBEAE is warranted to investigate the associated molecular mechanisms and identify the potential therapeutic targets.

BACKGROUND:Ganoderma lucidum (GL) has been widely used in Asian countries for hundreds of years to promote health and longevity. The pharmacological functions of which had been classified, including the activation of innate immune responses, suppression of tumour and modulation of cell proliferations. Effective fractions of Ganoderma lucidum polysaccharides (GLP) had already been reported to regulate the immune system. Nevertheless, the role of GLP in the microglia-mediated neuroinflammation has not been sufficiently elucidated. Further, GLP effect on microglial behavioural modulations in correlation with the inflammatory responses remains to be unravelled. The aim of this work was to quantitatively analyse the contributions of GLP on microglia.

METHODS:The BV2 microglia and primary mouse microglia were stimulated by lipopolysaccharides (LPS) and amyloid beta42 (Aβ42) oligomer, respectively. Investigation on the effect of GLP was carried by quantitative determination of the microglial pro- and anti-inflammatory cytokine expressions and behavioural modulations including migration, morphology and phagocytosis. Analysis of microglial morphology and phagocytosis modulations was confirmed in the zebrafish brain.

RESULTS:Quantitative results revealed that GLP down-regulates LPS- or Aβ-induced pro-inflammatory cytokines and promotes anti-inflammatory cytokine expressions in BV-2 and primary microglia. In addition, GLP attenuates inflammation-related microglial migration, morphological alterations and phagocytosis probabilities. We also showed that modulations of microglial behavioural responses were associated with MCP-1 and C1q expressions.

CONCLUSIONS:Overall, our study provides an insight into the GLP regulation of LPS- and Aβ-induced neuroinflammation and serves an implication that the neuroprotective function of GLP might be achieved through modulation of microglial inflammatory and behavioural responses.