Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Effect of safflower seeds supplementation on stimulation of the proliferation, differentiation and mineralization of osteoblastic MC3T3-E1 cells.

Anti-bone resorption properties of the Korean herbal formulation, Gami-Honghwain (HJ), which comprises Carthamus tinctorius L. seed and hominis placenta, were investigated. We demonstrate that the production of PGE2 is inhibited by 20-100 microg/ml HJ in nontransformed osteoblastic cells (MC3T3-E1 cells), indicating that HJ inhibits PGE2 production. The effect of HJ on the proliferation and osteoblastic differentiation in MC3T3-E1 was also studied. HJ dose-dependently increased DNA synthesis (significant at 20-100 microg/ml), and increased alkaline phosphatase (ALP) and prolyl hydroxylase activities of MC3T3-E1 cells (20-100 microg/ml), while anti-estrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1 which was induced by HJ. These results indicate that HJ directly stimulates cell proliferation and differentiation of osteoblasts. Also, when we assessed the effects of HJ on osteoblastic differentiation in MC3T3-E1, HJ enhanced ALP activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the HJ was observed at relatively low doses (significant at 20-100 microg/ml and maximal at 100 microg/ml). Northern blot analysis showed that the HJ (60 microg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. HJ (100 microg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that HJ has anabolic effect on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases.

Prevention of collagen-induced arthritis in mice by Cervus korean TEMMINCK var. mantchuricus Swinhoe.

The effect of water extract of deer antler (DAA) prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe (Nokyong) on collagen-induced mouse rheumatoid arthritis (RA) model was studied. Identification of common DAA capable of affording protection or modulating the onset and severity of arthritis may have important human health implications. DAA has been shown to possess anti-inflammatory and anti-carcinogenic properties in experimental animals. In this study, we determined the effect of DAA-injection on collagen-induced arthritis in mice. In three independent experiments, mice given DAA in water exhibited significantly reduced incidence of arthritis (30-45%) as compared with mice not given DAA in water (86-98%). The arthritis index also was significantly lower in DAA-injected animals. Western blot analysis showed a marked reduction in the expression of inflammatory mediators such as cyclooxygenase 2, IFN-γ, and tumor necrosis factor α in arthritic joints of DAA-injected mice. The neutral endopeptidase activity was approximately six-fold higher in arthritic joints of non-DAA-injected mice in comparison to non-arthritic joints of unimmunized mice, whereas it was only two-fold higher in the arthritic joints of DAA-injected mice. Additionally, total IgG and type II collagen-specific IgG levels were lower in serum and arthritic joints of DAA-injected mice. Taken together our studies suggest that DAA may be useful in the prevention of onset and severity of arthritis.

Inhibitory effect of Salvia miltiorrhia BGE on matrix metalloproteinase-9 activity and migration of TNF-alpha-induced human aortic smooth muscle cells.

The migration and matrix metalloproteinases (MMPs) production of vascular smooth muscle cells (VSMC) may play a key role in the development of atherosclerosis. The Radix of Salvia miltiorrhiza Bunge (Labiatae) (SM), an eminent herb, is often included as an ingredient in various herbal remedies recommended for vascular therapies and has been used to treat vascular diseases for many centuries. In this study, we investigated the inhibitory effect of SM on TNF-alpha induced human aortic smooth muscle cells (HASMC) migration and MMP-9 activity. Various extracts prepared from stems of SM were tested for cytotoxic activity on HASMC using the XTT assay method. The ethanol extract (IC50 > 100 microg/ml), water extract (IC50 > 100 microg/ml) and chloroform (IC50 = 90 microg/ml) fraction exhibited weak cytotoxic activity. However, buthanol (IC50 = 80 microg/ml) and ethyl acetate (EtOAc; IC50 = 70 microg/ml) fraction exhibited strongly cytotoxic activity. Also, the extracts and fractions were investigated the inhibitory effects on MMP-9 activity using gelatin zymography. Gelatin zymography showed that the TNF-alpha-treated HASMC secreted MMP, probably including MMP-9, which may be involved in HASMC migration. The EtOAc fraction showed stronger inhibitory effect of proteolytic activity than other fractions. The EtOAc fraction was able to decrease the proteolytic activity of MMP-9 in a concentration-dependent manner on zymography. The Matrigel migration assay showed that SM effectively inhibited the TNF-alpha induced migration of HASMC as compared with the control group in a dose-dependent manner (IC50 = 65 microg/ml) and that the EtOAc fraction effectively inhibited the migration of HASMC, as compared with the control group in a dose-dependent manner. These results suggest that SM could be used as potential anti-atherosclerotic agent for anti-migration in TNF-alpha treated HASMC.

Effects and mechanisms of Clematis mandshurica Maxim. as a dual inhibitor of proinflammatory cytokines on adjuvant arthritis in rats.

Effect and mechanism of Clematis mandshurica Maxim. water extract (CMA), a dual inhibitor of interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α), on rat adjuvant arthritis (AA) were investigated. Complete Freund's adjuvant (CFA) was used to induce AA in rats. The extents of inflammation and treatment response were evaluated with regard to lymphocyte proliferation. Serial evaluation was carried out on days 1, 7, 14, 21 and 28 after creation of inflammation. The lymphocyte proliferation study revealed cellular immunosuppression during the early phase of the disease. Administration of CMA on the same day or 5 days prior to inflammatory insult into the joint significantly reduced the inflammation as compared to the untreated animals in a dose dependent manner. The administration of CMA (2, 5 and 10mg/kg, subcutaneously (s.c.)) inhibited the inflammatory response and restored the weight of body and immune organs of AA rats. Synoviocytes proliferation of AA rats significantly increased, and the levels of TNF-α and IL-1 in supernatants of synoviocytes in AA rats were also elevated compared with the nonimmunized rats group. The administration of CMA (2, 5 and 10mg/kg, s.c.) reduced the above changes significantly. In contrast to TNF-α and IL-1, IL-10 production and the level of its mRNA of synoviocytes in AA rats were apparently decreased. CMA (2, 5 and 10mg/kg, s.c.) markedly increased IL-10 in synoviocytes at protein and transcription level. The results indicated that CMA had a beneficial effect on rats AA due to modulating inflammatory cytokines production of synoviocytes, which played a crucial role in pathogenesis of this disease.