Altered TNF-α response by Aconibal® and methotrexate in a lipopolysaccharide-induced setting of inflammatory conditions: Potential on a synergistic combination.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum carmichaelii (AC) is a common herbal medicine used as anti-inflammatory and analgesic agent in Eastern Asia. In Korea, a commercial processed AC (Aconibal®) is traditionally used to treat the symptoms of spondylosis deformans and rheumatic pain. AIM OF STUDY: Rheumatoid arthritis (RA) is systemic and autoimmune disease characterized by chronic inflammation. Methotrexate (MTX) is often the first-line therapy for RA. If MTX monotherapy is ineffective or RA is initially severe, adding a tumor necrosis factor alpha (TNF-α) inhibitor to the treatment can be beneficial. However, its inhibitory effects on RA when combined with MTX are unknown. Therefore, we investigated the stable modulation of and synergistic to additive effect on TNF-α using AC combined with MTX (AMC). MATERIALS AND METHODS: An inflammatory response mimicking RA was induced in the mouse macrophage cell line Raw 264.7 using interferon-γ or lipopolysaccharide (LPS). We predicted that AC and MTX at a 3:1 ratio would have synergistic therapeutic effects and this was determined using the Chou-Talalay method of median effect analysis and CalcuSyn software. We analyzed the profiles of various inflammatory cytokine-related proteins using Search tool for retrieval of interacting genes and Kyoto Encyclopedia of Genes and Genomes. RESULTS: The expression levels of selected inflammatory immune mediators such as interleukin (IL)-6, IL-1α, chemokine ligand 5, granulocyte-colony stimulating factor, nitric oxide synthase, and cyclooxygenase were reduced via regulation of the mitogen-activated protein kinase signaling pathway. AMC inhibited the levels of matrix metalloproteinases-1 and -3 in the human synovial cell line SW982. CONCLUSIONS: Our data show for the first time the potential beneficial effects of AMC in RA management.

Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections.

Alteration of macrophage function has an important regulatory impact on the survival of intracellular mycobacteria. We found that macrophages infected with attenuated Mycobacterium tuberculosis (Mtb) strain H37Ra had elevated expression of M1-related molecules, whereas the M2 phenotype was dominant in macrophages infected with virulent Mtb H37Rv. Further, the TLR signalling pathway played an important role in modulating macrophage polarization against Mtb infection. Interestingly, endoplasmic reticulum (ER) stress was significantly increased in M1 polarized macrophages and these macrophages effectively removed intracellular Mtb, indicating that ER stress may be an important component of the host immune response to Mtb in M1 macrophages. This improved understanding of the mechanisms that regulate macrophage polarization could provide new therapeutic strategies for tuberculosis.

The Role of Prostate Apoptosis Response-4 (Par-4) in Mycobacterium tuberculosis Infected Macrophages.

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that forms a complex with glucose-regulated protein 78 (GRP78) to induce apoptosis. Previously, we reported that ER stress-induced apoptosis is a critical host defense mechanism against Mycobacterium tuberculosis (Mtb). We sought to understand the role of Par-4 during ER stress-induced apoptosis in response to mycobacterial infection. Par-4 and GRP78 protein levels increased in response Mtb (strain: H37Ra) infection. Furthermore, Par-4 and GRP78 translocate to the surface of Mtb H37Ra-infected macrophages and induce apoptosis via caspase activation. NF-κB activation, Mtb-mediated ER stress, and Par-4 production were significantly diminished in macrophages with inhibited ROS production. To test Par-4 function during mycobacterial infection, we analyzed intracellular survival of Mtb H37Ra in macrophages with Par-4 overexpression or knockdown. Mtb H37Ra growth was significantly reduced in Par-4 overexpressing macrophages and increased in knockdown macrophages. We also observed increased Par-4, GRP78, and caspases activation in Bacillus Calmette-Guérin (BCG)-infected prostate cancer cells. Our data demonstrate that Par-4 is associated with ER stress-induced apoptosis resulting in reduced intracellular survival of mycobacteria. BCG treatment increases Par-4-dependent caspase activation in prostate cancer cells. These results suggest ER stress-induced Par-4 acts as an important defense mechanism against mycobacterial infection and regulates cancer.

Synthesis and immunosuppressive activity of new artemisinin derivatives. 1. [12(beta or alpha)-Dihydroartemisininoxy]phen(ox)yl aliphatic acids and esters.

A series of novel dihydroartemisinin derivatives were synthesized and evaluated on their immunosuppressive activity in the search for potential immunosuppressive agents with high efficacy and low toxicity. These compounds were assayed in their cytotoxicity of lymphocyte, inhibition activity on concanavalin A (ConA) induced T cell proliferation and lipopolysaccharide (LPS) induced B cell proliferation. Among them, 11b, 13b, 14d, 15b, 16, and 17 remarkably exhibited lower cytotoxicity and higher inhibition activity on the mitogen-induced T cell and B cell proliferation in comparison with artemisinin, artesunate, and artemether in vitro. More significantly, compound 11b displayed reduced cytotoxicity by over 100-fold compared with cyclosporin A (CsA) and comparable inhibition activity (SI = 848) on ConA-induced T cell proliferation to CsA (SI = 963) and more than 4000 times the inhibitory effect (SI = 28473) on LPS-induced B cell proliferation compared with CsA (SI = 7) in vitro. The in vivo experimental results showed that compound 16 could inhibit 2,4-dinitrofluorobenzene (DNFB)-induced delayed-type hypersensitivity (DTH) reaction and sheep red blood cell (SRBC) induced antibody production, respectively. The structure and activity relationships (SAR) of these compounds were also discussed.