Expression of RPL9 predicts the recurrence of non-muscle invasive bladder cancer with BCG therapy.

Numerous biomarkers and risk tables can be used to predict recurrence or progression of patients with primary or recurrent non-muscle invasive bladder cancer (NMIBC) receiving Bacillus Calmette-Guerin (BCG). However, few are suitable for BCG-unresponsive disease (i.e., recurrence or progression after BCG treatment). Therefore, identification of a novel marker that allows accurate prediction of prognosis, particularly risk of recurrence, is critically important in clinical practice. In the current study, gene ontology and gene set enrichment analyses of microarray datasets (GSE13507, n = 47) revealed that differentially expressed genes in recurred NMIBC patients after BCG treatment were associated with virus and ribosomal pathways. Among the core-enrichment genes, the expression of RPL9, a putative tumor suppressor, was lower in recurred NMIBC patients after BCG therapy than in patients without recurrence (P = 0.033) from the E-MTAT-4321 European cohort (n = 84). Data from The Cancer Genome Atlas (n = 406) showed that bladder cancer patients with higher RPL9 expression had a longer overall survival probability than patients with lower RPL9 expression (P = 0.011). Moreover, we used the latest digital PCR platform to examine 59 NMIBC patients and identified downregulation of RPL9 in patients with recurrence after BCG therapy (P = 0.031). The Kaplan-Meier survival estimator showed that NMIBC patients with higher expression of RPL9 had longer recurrence-free survival (log-rank test, P = 0.015). Therefore, we conclude that RPL9 expression is a prospective predictor of recurrence after BCG therapy in NMIBC patients.

Urinary hsv2-miR-H9 to hsa-miR-3659 ratio is an effective marker for discriminating prostate cancer from benign prostate hyperplasia in patients within the prostate-specific antigen grey zone.

PURPOSE: Tumor microRNAs (miRNAs) are released to biofluids directly or indirectly. Although urinary miRNAs are promising non-invasive biomarkers for the diagnosis of prostate cancer (PCa), their clinical application is challenging for technical reasons. We examined the efficacy of urinary hsv2-miR-H9 to hsa-miR-3659 ratio as a non-invasive diagnostic biomarker of PCa. MATERIALS AND METHODS: The expression of urinary miRNAs was quantified by real-time PCR in 116 samples from 53 patients with benign prostatic hyperplasia (BPH) and 63 patients with PCa. The miRNA expression ratio was calculated using an upregulated miRNA (hsv2-miR-H9) as the numerator and a downregulated miRNA (hsa-miR-3659) as the denominator. RESULTS: The urinary miR-H9 to miR-3659 ratio was significantly higher in PCa than in BPH controls (p<0.001). The diagnostic accuracy of the urinary miRNA expression ratio was comparable with that of prostate-specific antigen (PSA) (receiver operating characteristic [ROC] curve comparison, p=0.287). The area under the curve for urinary miRNA expression ratio was 0.862 and that for PSA was 0.642 in the "PSA gray zone" (3-10 ng/mL) (ROC curve comparison, p=0.034). The use of the urinary miRNA expression ratio would have prevented 70.6% of unnecessary prostate biopsies; however, 28.6% of PCa cases could be missed in patients within the PSA gray zone. CONCLUSIONS: The expression ratio of urinary miR-H9 to miR-3659 could be a relevant non-invasive biomarker for PCa diagnosis, particularly for patients within the PSA gray zone.

Precisely tuned photonic properties of crystalline nanocellulose biocomposite coatings by gradually tailored nanoarchitectures.

Inspired by nature, we fabricated optically tuned bio-organic films of cellulose nanofibers (CNFs) and chitosan by precisely structuring layer-by-layer (LBL) assembly techniques. Directly extracted from tunicate, highly crystalline CNFs were deposited into gradual nanoporous structures, which dictates wave-like behaviors of optical transmittance and cyclic patterns of structural coloration. The optical transmittance of a glass substrate increased from 91% to 98.2% at a wavelength of 550 nm by reflectivity attenuation controls. The monochromic structural colors were discretely varied from orange to purple, seen only at a specific angle of the incident light, demonstrating visibly hidden security potential. Furthermore, we could modulate the coloration cycles by accelerating the LBL processes with polydopamine-coated CNFs. The 3D nanoporous structures of CNFs also provide synergies, including superhydophilic surface-driven antifogging properties. Our optically versatile biofilms made by all-natural CNFs and chitosan provide various potential applications, including solar cells, securities, and eco-friendly colors.

High crystallinity of tunicate cellulose nanofibers for high-performance engineering films.

Tunicate cellulose nanofibers (CNFs) have received widespread attention as renewable and eco-friendly engineering materials because of their high crystallinity and mechanical stiffness. Here, we report the effects of disintegration process conditions on structure-property relationships of tunicate CNFs. By varying the hydrolysis time, we could establish a correlation between crystallinity of the CNFs with linearity and stiffness, which produces different molecular ordering within their nanostructured films. Despite having identical raw materials, tensile strength and thermal conductivity of the resulting layered films varied widely, ranging from 95.6 to 205 MPa and from 1.08 to 2.37 W/mK respectively. Furthermore, nanolayered CNF films provided highly anisotropic thermal conductivities with an in- and through-plane ratio of 21.5. Our systematic investigations will provide general and practical strategies in tailoring material properties for emerging engineering applications, including flexible paper electronics, heat sink adhesives and biodegradable, implantable devices.

Cynaroside protects human periodontal ligament cells from lipopolysaccharide-induced damage and inflammation through suppression of NF-κB activation.

OBJECTIVE: To investigate whether cynaroside protects human periodontal ligament (hPDL) cells from lipopolysaccharide (LPS)-induced damage and inflammation and to analyze the underlying mechanism. METHODS: LPS was used to stimulate hPDL and RAW264.7 cells. MTT assay was used to detect cell viability, and protein expression levels were measured via western blot analysis. Nitrite oxide and prostaglandin E2 were used to quantify the inflammatory response. Alizarin Red S staining was used to detect mineralized nodules. RESULTS: Cynaroside inhibited the expression of iNOS, COX-2, TNF-α, and IL-6 in LPS-stimulated hPDL and RAW264.7 cells without cytotoxicity. Furthermore, cynaroside significantly suppressed LPS-induced protein expression of matrix metalloproteinase 3. Additionally, cynaroside prevented LPS-induced NF-κB p65 subunit translocation to the nucleus by inhibiting the phosphorylation and degradation of IκB-α. Moreover, cynaroside could restore the mineralization ability of hPDL cells reduced by LPS. CONCLUSION: Cynaroside protected hPDL cells from LPS-induced damage and inflammation via inhibition of NF-κB activation. These results suggest that cynaroside may be a potential therapeutic agent for the alleviation of periodontitis.

Adverse Initial CT Findings Associated with Poor Prognosis of Coronavirus Disease.

BACKGROUND: The predictors of poor prognosis in patients with coronavirus disease 2019 (COVID-19) using computed tomography (CT) have not been investigated in a large cohort. Therefore, the purpose of this study was to investigate the adverse initial CT features to predict poor prognosis in COVID-19. METHODS: From February to April 2020, 281 COVID-19 patients who underwent CT at the time of admission were included. We divided the patients into the severe and non-severe disease groups. The severe group included patients with severe pneumonia or critical events. Intensive care unit admission or death were the critical events in this study. We compared the clinical and CT findings between the severe and non-severe groups and investigated the prognostic factors and critical events of the severe group using the regression analysis. RESULTS: Among the 281 patients, 36 (12.8%) patients were in the severe group and 245 (87.2%) patients were in the non-severe group. Critical events occurred in 10 patients (3.6%). In the severe group, patients showed significantly more pneumonia with consolidation, crazy-paving appearance, pleural effusion, and higher CT scores than those in the non-severe group (all, P < 0.05). In the multivariate regression, pleural effusion (odds ratio [OR], 8.96; 95% confidence interval [CI], 1.81-44.42; P = 0.007), CT score > 5 (OR, 3.70; 95% CI, 1.44-9.53; P = 0.007), old age (> 77 years, OR, 9.96; 95% CI, 3.78-26.28; P < 0.001), and elevated C-reactive protein (OR, 4.15; 95% CI, 1.62-10.6; P = 0.003) were significant prognostic factors of severe disease. CT score > 5 (OR, 7.29; 95% CI, 1.37-38.68; P = 0.020), pleural effusion (OR, 5.67; 95% CI, 1.04-30.8; P = 0.045) and old age (OR, 8.6; 95% CI, 1.80-41.0; P = 0.007) were also significant predictors of critical events. CONCLUSION: Pleural effusion and the extent of pneumonia on initial CT scans are associated with poor prognosis in patients with COVID-19.

Characteristics of COVID-19 Patients Who Progress to Pneumonia on Follow-Up Chest Radiograph: 236 Patients from a Single Isolated Cohort in Daegu, South Korea.

OBJECTIVE: We investigated the prevalence of pneumonia in novel coronavirus disease 2019 (COVID-19) patients using chest radiographs to identify the characteristics of those with initially negative chest radiographs, who were positive for pneumonia on follow-up. MATERIALS AND METHODS: Retrospective cohort data of 236 COVID-19 patients were reviewed. Chest radiography was performed on admission, with serial radiographs obtained until discharge. The 'positive conversion group' was defined as patients whose initial chest radiographs were negative but were positive for pneumonia during follow-up. Patients with initially positive chest radiographs were defined as the 'initial pneumonia group.' Patients with negative initial and follow-up chest radiographs were defined as the 'non-pneumonia group.' Clinical and laboratory findings were compared between groups, and predictors of positive conversion were investigated. RESULTS: Among 236 patients, 108 (45.8%) were in the non-pneumonia group, 69 (29.2%) were in the initial pneumonia group, and 59 (25%) were in the positive conversion group. The patients in the 'initial pneumonia group' and 'positive conversion group' were older, had higher C-reactive protein (CRP) and lactate dehydrogenase levels, and lower absolute lymphocyte counts than those in the 'non-pneumonia group' (all p < 0.001). Among patients with negative initial chest radiographs, age ≥ 45 years (odds ratio [OR]: 3.93, 95% confidence interval [CI]: 1.76-8.75, p = 0.001), absolute lymphocyte count < 1500 cells/µL (OR: 2.25, 95% CI: 1.03-4.89, p = 0.041), and CRP > 0.5 mg/dL (OR: 3.91, 95% CI: 1.54-9.91, p = 0.004) were independent predictors for future development of pneumonia. CONCLUSION: More than a half of COVID-19 patients initially had normal chest radiographs; however, elderly patients (≥ 45 years of age) with abnormal laboratory findings (elevated CRP and low absolute lymphocyte counts) developed pneumonia on follow-up radiographs.

Chondroprotective Effect of Cynaroside in IL-1ß-Induced Primary Rat Chondrocytes and Organ Explants via NF-κB and MAPK Signaling Inhibition.

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation and inflammation. Interleukin-1ß is the key player in the pathogenesis of OA, which induces the expression of various catabolic factors that contribute to cartilage degradation. Cynaroside (luteolin-7-O-glucoside or luteoloside) is a flavonoid that has various pharmacological properties, such as antitumor, anti-inflammatory, and antioxidant activities. In this study, we investigated the chondroprotective effects of cynaroside on IL-1ß-stimulated chondrocytes and organ explants. The production of nitrite, PGE2, collagen type II, and aggrecan was measured by a Griess reagent and ELISAs, and the production of ROS was measured by H2DCF-DA fluorescence. The protein levels of iNOS, Cox-2, MMP-1, MMP-3, MMP-13, ADAMTS-4, MAPKs, and the NF-κB p65 subunit were measured by western blot. Proteoglycan analysis was performed by Alcian Blue staining (in vitro) and Safranin O staining (ex vivo). Cynaroside inhibited IL-1ß-induced expression of catabolic factors (nitrite, iNOS, ROS, PGE2, Cox-2, MMP-1, MMP-3, MMP-13, and ADAMTS-4) and degradation of anabolic factors (collagen type II and aggrecan). Furthermore, cynaroside suppressed IL-1ß-induced phosphorylation of MAPKs and translocation of the NF-κB p65 subunit into the nucleus. Collectively, these results suggest that cynaroside may be a potential candidate for the development of new therapeutic drugs for the alleviation of OA progression.

Anti-inflammatory potential of Trifolium pratense L. leaf extract in LPS-stimulated RAW264.7 cells and in a rat model of carrageenan-induced inflammation.

This study evaluated the anti-inflammatory potential of a 40% prethanol extract of Trifolium pratense leaves (40% PeTP) using in vitro (RAW264.7 cells) and in vivo (carrageenan-induced inflammation model) experiments. Pretreatment with 40% PeTP significantly inhibited the LPS-induced expression of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6 in RAW264.7 cells, without inducing cytotoxicity. The inhibitory effects of 40% PeTP are mediated through suppression of the nuclear translocation of nuclear factor (NF)-κB and the phosphorylation of mitogen-activated protein kinases (MAPKs). Oral administration of 40% PeTP at 50, 100, and 200 mg/kg of body weight suppressed carrageenan-induced oedema in a dose-dependent manner. Collectively, our results suggested that 40% PeTP exerts potential anti-inflammatory effects by suppressing the activation of the NF-κB and MAPK pathways in vitro, and by reducing carrageenan-induced paw oedema in vivo.

Reversine induces caspase-dependent apoptosis of human osteosarcoma cells through extrinsic and intrinsic apoptotic signaling pathways.

BACKGROUND: The 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) acts as a chemopreventive agent and induces apoptotic cell death in various cancer cells. However, the anticancer effects of reversine on osteosarcoma cells are not clearly established. OBJECTIVE: The purpose of this study was to investigate the effect of reversine on cell proliferation and induction of apoptosis in human osteosarcoma cells. METHODS: Cell viability assay, histological analysis, DAPI staining, caspase activation analysis, flow cytometric analysis and immunoblotting were carried out in MG-63 osteosarcoma cells. RESULTS: Reversine inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Reversine-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was significantly up-regulated by reversine treatment. Moreover, the caspase-8, a part of the extrinsic apoptotic pathway, was activated by reversine treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria dependent intrinsic apoptosis pathway, significantly decreased following reversine treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9 increased by reversine treatments. In addition, reversine activated caspase-3 and Poly (ADP-ribose) polymerase (PARP) to induce cell death. The Z-VAD-fmk significantly inhibited cell death through the suppression of caspase-3 expression in MG-63 cells treated with reversine. CONCLUSION: These results suggest that the reversine may inhibit cell proliferation and induce apoptotic cell death in MG-63 osteosarcoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for the discovery of anti-cancer agents.

Erratum: Adenosine induces intrinsic apoptosis via the PI3K/Akt/mTOR signaling pathway in human pharyngeal squamous carcinoma FaDu cells.

[This corrects the article DOI: 10.3892/ol.2018.8089.].

MicroRNA-203 Induces Apoptosis by Targeting Bmi-1 in YD-38 Oral Cancer Cells.

BACKGROUND/AIM: MicroRNAs (miRNAs) are closely associated with a number of cellular processes, including cell development, differentiation, proliferation, carcinogenesis, and apoptosis. The aim of the present study was to elucidate the molecular mechanisms underlying the tumor suppressor activity of miRNA-203 (miR-203) in YD-38 human oral cancer cells. MATERIALS AND METHODS: Polymerase chain reaction analysis, MTT assay, DNA fragmentation assay, fluorescence-activated cell-sorting analysis, gene array, immunoblotting, and luciferase assay were carried out in YD-38 cells. RESULTS: miR-203 expression was significantly down-regulated in YD-38 cells compared to expression levels in normal human oral keratinocytes. miR-203 decreased the viability of YD-38 cells in a time- and dose-dependent manner. In addition, over-expression of miR-203 significantly increased not only DNA segmentation, but also the apoptotic population of YD-38 cells. These results indicate that miR-203 overexpression induces apoptosis in YD-38 cells. Target gene array analysis revealed that the expression of the polycomb complex protein gene Bmi-1, a representative oncogene, was significantly down-regulated by miR-203 in YD-38 cells. Moreover, both mRNA and protein levels of Bmi-1 were significantly reduced in YD-38 cells transfected with miR-203. These results indicate that Bmi-1 is a target gene of miR-203. A luciferase reporter assay confirmed that miR-203 suppressed Bmi-1 expression by directly targeting the 3'-untranslated region. CONCLUSION: miR-203 induces apoptosis in YD-38 cells by directly targeting Bmi-1, which suggests its possible application as an anti-cancer therapeutic.

Chondroprotective effects of aqueous extract of Anthriscus sylvestris leaves on osteoarthritis in vitro and in vivo through MAPKs and NF-κB signaling inhibition.

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage degradation and inflammation, in the elderly population. Anthriscus sylvestris has been used in Korean traditional medicine and contains many polyphenolic compounds such as cynaroside and chlorogenic acid, which are major active components responsible for its antioxidant effect. In this study, we aimed to evaluate the chondroprotective effect of an aqueous extract of A. sylvestris leaves (AE-ASL) on OA, both in vitro and in vivo. Rat primary chondrocytes were pretreated with AE-ASL for 1 h before interleukin-1ß (20 ng/mL) stimulation. The production of nitrite, PGE2, aggrecan, and collagen type II were detected by Griess reagent and ELISAs. The mRNA levels of iNOS, COX-2, MMP-3, and MMP-13 were measured by RT-PCR. In addition, protein levels of iNOS, COX-2, MMP-3, MMP-13, ADAMTS-4, MAPKs, and NF-κB p65 subunit were measured by western blot analysis. Sulfated glycosaminoglycan (sGAGs) were detected by dimethylmethylene blue (DMMB) assay. During in vivo study, the effects of AE-ASL were evaluated for 8 weeks in a rat model of destabilization of the medial meniscus (DMM) surgery-induced OA. AE-ASL significantly inhibited expression of nitrite, iNOS, PGE2, COX-2, MMP-3, MMP-13, and ADAMTS-4 in IL-1ß-stimulated chondrocytes. Moreover, it decreased the IL-1ß-induced degradation of aggrecan, collagen type II, and proteoglycan. In addition, AE-ASL suppressed IL-1ß-induced phosphorylation of MAPKs and NF-κB p65 subunit translocation to nucleus. In vivo, AE-ASL inhibited DMM surgery-induced cartilage destruction and proteoglycan loss. Taken together, these results suggest that AE-ASL may be a potential therapeutic agent for the alleviation of OA progression.

Adenosine induces intrinsic apoptosis via the PI3K/Akt/mTOR signaling pathway in human pharyngeal squamous carcinoma FaDu cells.

Adenosine has been identified to occur abundantly intra-and extracellularly, and to exert diverse biological functions, including the suppression of cell proliferation and the induction of apoptosis. Adenosine has been reported to induce apoptosis in several cancer cell lines; however, to the best of our knowledge, the effect of adenosine on head and neck cancer cells has not been investigated. Therefore, the purpose of the present study was to evaluate whether adenosine exerts any anticancer effect via induction of apoptosis in human pharyngeal squamous carcinoma FaDu cells. An MTT assay demonstrated that adenosine-treated FaDu cells inhibited a dose-dependent rate of cell growth, whereas human oral keratinocytes cells were unaffected by adenosine treatment. In addition, A1 and A2a adenosine receptor mRNA was detected in FaDu cells by reverse transcription-polymerase chain reaction, and adenosine-induced FaDu cell death was significantly suppressed by treatment with ATL-444, an antagonist of these receptors. Furthermore, adenosine-induced cell growth inhibition was exerted via apoptosis, as confirmed by the analysis of DNA fragmentation, Hoechst nuclear staining and flow cytometry with Annexin V-fluorescein isothiocyanate and propidium iodide staining. Adenosine was also demonstrated to induce an increase in Bcl-associated X expression, a decrease in B-cell lymphoma 2 expression, the release of cytochrome c from mitochondria, and the activation of caspase-3, -9 and poly(ADP-ribose) polymerase in FaDu cells. Finally, phosphoinositide 3-kinase (PI3K), RAC serine/threonine-protein kinase (Akt) and mechanistic target of rapamycin (mTOR) phosphorylation was found to be significantly inhibited in adenosine-treated FaDu cells, as was phosphorylation of the mTOR downregulators, S6 kinase ß1, eukaryotic translation initiation factor 4E-binding protein 1, and eukaryotic translation initiation factor 4 γ1. Taken together, these results indicate that adenosine induces apoptosis via the mitochondrial intrinsic pathway, and activates caspase-3 and -9 activity via the PI3K/Akt/mTOR signaling pathway.

Anthricin­induced caspase­dependent apoptosis through IGF1R/PI3K/AKT pathway inhibition in A549 human non­small lung cancer cells.

Anthricin (deoxypodophyllotoxin) is a major lignan in Anthriscus sylvestris and possesses many bioactivities such as antiproliferative, antitumor, anti­platelet aggregation, antiviral and anti­inflammatory actions. However, the anticancer effects of anthricin on A549 human non­small cell lung cancer cells and potential molecular mechanisms remain unknown. Therefore, we investigated the anticancer effect of anthricin and the underlying mechanism in A549 cells. Anthricin (10­200 nM) inhibited the viability of A549 cells in a dose­ and time­dependent manner. Moreover, anthricin­induced apoptosis was confirmed by live and dead assay, 4,6­dianmidino­2­phenylindole staining, and flow cytometric analysis. In addition, anthricin induced cell cycle arrest at the G2/M phase through suppression of the expression of cell cycle cascade proteins, Cdc2 and Cdc25C. Furthermore, it induced the expression of caspase­related proteins and significantly suppressed the phosphorylation of insulin­like growth factor 1 receptor (IGF1R), PI3K and Akt. Anthricin significantly inhibited tumor growth without any significant change in the body weight of mice in A549 tumor xenograft BALB/c nude mice. Anthricin induced caspase­dependent apoptosis through the IGF1R/PI3K/Akt signaling pathway in A549 cells.

Oleamide suppresses inflammatory responses in LPS-induced RAW264.7 murine macrophages and alleviates paw edema in a carrageenan-induced inflammatory rat model.

Oleamide compounds purified from green algae have been used for the prevention and treatment of atherosclerosis, thrombosis, arthritis, and cancer. They function through their metabolic conversion into prostaglandins, thromboxanes, and leukotrienes. However, the actual mechanism of action has not been well characterized. To investigate the underlying anti-inflammatory activity and associated mechanisms, oleamide purified from Codium fragile was studied using RAW264.7 murine macrophages and a carrageenan-induced inflammatory rat model. Our results indicate that pre-treatment of RAW264.7 cells with oleamide significantly suppressed LPS-induced nitrite production and PGE2 secretion. Oleamide inhibited LPS-induced iNOS and COX-2 mRNA and protein expression. It also inhibited the LPS-induced production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6. In addition, oleamide prevented the nuclear translocation of NF-κB by suppressing the phosphorylation of the inhibitor of kappa B (IκB)-α. Oleamide also suppressed the phosphorylation of mitogen-activated protein kinases such as ERK1/2 and JNK. Furthermore, inhibition of paw swelling (%) was suppressed 2 h after the intraperitoneal injection of oleamide (20 mg/kg, body weight) in a carrageen-induced rat model. Therefore, our results suggest that oleamide can be used as a single ingredient treatment for inflammatory diseases.

In Vivo and In Vitro Anti-Inflammatory Effects of Aqueous Extract of Anthriscus sylvestris Leaves.

Anthriscus sylvestris (L.) Hoffm. is a common perennial herb that is widely distributed in Europe, Korea, and New Zealand. The root of A. sylvestris has been used in Korean traditional medicine as an antitussive and cough remedy. However, the physiologically active function of A. sylvestris leaves is not yet known. In this study, we evaluated the anti-inflammatory effects, as well as the underlying molecular mechanisms of an aqueous extract of A. sylvestris leaves (AE-ASL) in vitro and in vivo. Our results indicated that pretreatment with AE-ASL significantly inhibited the lipopolysaccharide (LPS)-induced secretion of nitric oxide (NO) and prostaglandin E2 in RAW264.7 cells, without showing cytotoxicity. In addition, the LPS-induced mRNA and protein expression of inducible NO synthase, cyclooxygenase-2, and inflammatory mediators such as tumor necrosis factor alpha interleukin (IL)-1ß, and IL-6 was attenuated by pretreatment with AE-ASL in a dose-dependent manner. Therefore, we investigated the activation of nuclear factor (NF)-κB, a transcription factor regulating the expression of inflammation-related genes. AE-ASL inhibited the nuclear translocation of the NF-κB p65 subunit by suppressing the phosphorylation and degradation of the inhibitor of NF-κB (IκBα). Further, AE-ASL inhibited the LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) in RAW264.7 cells. Orally administered AE-ASL (50, 100, and 200 mg/kg of body weight [BW]) suppressed the development of carrageenan-induced rat paw edema by 15%, 31%, and 40%, respectively, after 4 h. Altogether, our results suggest that AE-ASL possesses anti-inflammatory activity, based on the suppression of NF-κB and MAPK pathways in vitro and inhibition of the carrageenan-induced paw edema in vivo.

The Effect of the Prethanol Extract of Trifolium pratense Leaves on Interleukin-1ß-Induced Cartilage Matrix Degradation in Primary Rat Chondrocytes.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease, characterized by cartilage degradation and inflammation. The proinflammatory cytokine, interleukin (IL)-1ß, plays a crucial role in the pathogenesis of OA by inducing the release of other catabolic factors that contribute to cartilage degradation. Trifolium pratense L. (red clover) has been used as a medicinal plant in many countries and as a source of nutraceuticals to alleviate the symptoms of menopause. Ob-jectives: In this study, we aimed to evaluate the anticatabolic effect of 40% prethanol extract of T. pratense (40% PeTP) on IL-1ß-stimulated chondrocytes. METHODS: Primary rat chondrocytes were pretreated with 40% PeTP for 1 h before stimulation with IL-1ß (20 ng/mL). The production of nitrite, prostaglandin E2 (PGE2), and aggrecan was measured by using Griess reagent and ELISA. Protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-4, mitogen-activated protein kinase (MAPK), and the nuclear factor (NF)-κB p65 subunit was measured by using Western blotting. RESULTS: PeTP (40%) significantly inhibited the IL-1ß-induced expression of nitrite, iNOS, PGE2, COX-2, MMP-1, MMP-3, MMP-13, and ADAMTS-4 in isolated primary rat chondrocytes. Furthermore, 40% PeTP decreased the IL-1ß-induced degradation of aggrecan, the phosphorylation of MAPKs, and the nuclear translocation of the NF-κB p65 subunit. CONCLUSION: These results suggested that 40% PeTP has a chondroprotective effect on inflammation and may be a potential preventative agent for OA progression.