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.

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.

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.

Aqueous extract of Codium fragile alleviates osteoarthritis through the MAPK/NF-κB pathways in IL-1ß-induced rat primary chondrocytes and a rat osteoarthritis model.

BACKGROUND: Codium fragile (Suringar) Hariot has been used as a potential remedy in traditional medicine because of its anti-inflammatory and anti-oxidant effects. Osteoarthritis is a chronic progressive joint disease, characterized by complex mechanisms related to inflammation and degeneration of articular cartilage. In this study, we aimed to evaluate the cartilage protective effect of an aqueous extract of Codium fragile (AECF) using rat primary chondrocytes and the osteoarthritis animal model induced by destabilization of the medial meniscus (DMM). METHODS: In vitro, rat primary cultured chondrocytes were pre-treated with AECF (0.5, 1, and 2mg/mL) for 1h and then incubated with interleukin-1ß (10ng/mL) for 24h. Nitrite production was detected by the Griess reagent. Alteration of the protein levels of iNOS, MMP-13, ADAMTS-4, ADAMTS-5, mitogen-activated protein kinases (MAPKs), and nuclear factor-κB (NF-κB) was detected by western blotting. In vivo, osteoarthritis was induced by DMM of Sprague Dawley (SD) rats. The rats subjected to destabilization of the medial meniscus (DMM) surgery were orally administered with AECF (50, 100, and 200mg/kg bodyweight) or distilled water for 8w. The severity of cartilage lesions was evaluated by safranin O staining and the Osteoarthritis Research Society International (OARSI) score. RESULTS: These results demonstrated that AECF significantly inhibited nitrite production and inhibited the levels of iNOS, MMP-13, ADAMTS-4, and ADAMTS-5 in interleukin-1ß-induced rat primary cultured chondrocytes. Moreover, AECF suppressed interleukin-1ß-induced NF-κB activation in the nucleus and phosphorylation of ERK1/2 and JNK in the cytosol. In vivo, the cartilage lesions in AECF-treated osteoarthritis rats exhibited less proteoglycan loss and lower OARSI scores. CONCLUSIONS: These results suggested that AECF is a potential therapeutic agent for the alleviation of osteoarthritis progression.

Aqueous extract of Codium fragile suppressed inflammatory responses in lipopolysaccharide-stimulated RAW264.7 cells and carrageenan-induced rats.

Codium fragile (Suringar) Hariot has been used in Oriental medicine for the treatment of enterobiasis, dropsy, and dysuria and has been shown to have various biological effects. In this study, we evaluated the anti-inflammatory effects of aqueous extract of C. fragile (AECF) using in vitro and in vivo models. Nitric oxide (NO), prostaglandin E2 (PGE2), inflammatory-related mRNAs, and proteins were determined using the Griess assay, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and western blotting, respectively. Our results indicate that pretreatment of cells with AECF (50, 100 and 200µg/mL) significantly inhibited LPS-induced secretion of NO and PGE2 in RAW264.7 cells without cytotoxicity. We also found that AECF (100 and 200µg/mL) inhibited LPS-induced inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression in a dose-dependent manner. Additionally, pretreatment of cells with AECF (100 and 200µg/mL) inhibited LPS-induced production of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. It also prevented the nuclear translocation of nuclear factor (NF)-κB by suppressing the phosphorylation and degradation of inhibitor of NF-κB (IκB)-α. Furthermore, AECF (100 and 200µg/mL) inhibited the phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and p38. In addition, orally administered 50, 100, and 200mg/kg body weight of AECF dose-dependently suppressed carrageenan-induced rat paw edema thickness by 6%, 31%, and 50% respectively, after 4h. Furthermore, the anti-inflammatory effect was comparable to that observed in animals treated with the standard drug diclofenac sodium (56%) in vivo. Collectively, our results suggest that AECF exerts potential anti-inflammatory effects by suppressing NF-κB activation and MAPKs pathways in vitro, as well as inhibiting carrageenan-induced rat paw edema thickness in vivo. These findings indicate that AECF could be further developed as an anti-inflammatory drug.

Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells.

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer.

Licochalcone-A induces intrinsic and extrinsic apoptosis via ERK1/2 and p38 phosphorylation-mediated TRAIL expression in head and neck squamous carcinoma FaDu cells.

We investigated Licochalcone-A (Lico-A)-induced apoptosis and the pathway underlying its activity in a pharyngeal squamous carcinoma FaDu cell line. Lico-A purified from root of Glycyrrhiza inflata had cytotoxic effects, significantly increasing cell death in FaDu cells. Using a cell viability assay, we determined that the IC50 value of Lico-A in FaDu cells was approximately 100 µM. Chromatin condensation was observed in FaDu cells treated with Lico-A for 24 h. Consistent with this finding, the number of apoptotic cells increased in a time-dependent manner when FaDu cells were treated with Lico-A. TRAIL was significantly up-regulated in Lico-A-treated FaDu cells in a dose-dependent manner. Apoptotic factors such as caspases and PARP were subsequently activated in a caspase-dependent manner. In addition, levels of pro-apoptotic factors increased significantly in response to Lico-A treatment, while levels of anti-apoptotic factors decreased. Lico-A-induced TRAIL expression was mediated in part by a MAPK signaling pathway involving ERK1/2 and p38. In xenograft mouse model, Lico-A treatment effectively suppressed the growth of FaDu cell xenografts by activating caspase-3, without affecting the body weight of mice. Taken together, these data suggest that Lico-A has potential chemopreventive effects and should therefore be developed as a chemotherapeutic agent for pharyngeal squamous carcinoma.

Anticancer activity of Saussurea lappa extract by apoptotic pathway in KB human oral cancer cells.

CONTEXT: Saussurea lappa Dence (Compositae) is used as a traditional herbal medicine to treat abdominal pain and tenesmus in East Asia. Current studies have shown that S. lappa has anticancer activity in divergent of cancer cells. However, the effects of S. lappa on oral cancer and its mechanisms of action have yet to be elucidated. OBJECTIVE: To explore its potential chemotherapeutic effects and mechanism of cell growth inhibition on human oral cancer cells. MATERIALS AND METHODS: The dried roots of S. lappa were used in this study. Cell viability of KB cells was evaluated by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay after treatment with 30 µg/ml of methanol extract from the dried roots of S. lappa. To understand whether its effect on cell death is related with apoptosis pathway, we performed DNA fragmentation assay, western blot, caspase activity assay and fluorescence-activated cell sorting (FACS) analysis. RESULTS: Treatment of S. lappa extract onto KB cells reduced cell viability significantly with an IC50 value of 30 µg/ml. The formation of a DNA ladder was observed starting at the 24 h treatment. In western blotting analysis, the S. lappa extract induced the proteolytic processing of caspase-3, -9 and poly (ADP-ribose) polymerase, a significant increase of Bax and marked reduction of Bcl-2. We also confirmed the activation of caspase-3/-7 in living KB cells by fluorescence microscopy. CONCLUSION: These results suggested that S. lappa extract inhibited cell proliferation through the apoptosis pathway in KB human oral cancer cells.