A Novel Pyrazolo[3,4-d]pyrimidine Induces Heme Oxygenase-1 and Exerts Anti-Inflammatory and Neuroprotective Effects.

The anti-oxidant enzyme heme oxygenase-1 (HO-1) is known to exert anti-inflammatory effects. From a library of pyrazolo[3,4-d]pyrimidines, we identified a novel compound KKC080096 that upregulated HO-1 at the mRNA and protein levels in microglial BV-2 cells. KKC080096 exhibited anti-inflammatory effects via suppressing nitric oxide, interleukin-1ß (IL-1ß), and iNOS production in lipopolysaccharide (LPS)-challenged cells. It inhibited the phosphorylation of IKK and MAP kinases (p38, JNK, ERK), which trigger inflammatory signaling, and whose activities are inhibited by HO-1. Further, KKC080096 upregulated anti-inflammatory marker (Arg1, YM1, CD206, IL-10, transforming growth factor-ß [TGF-ß]) expression. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, KKC080096 lowered microglial activation, protected the nigral dopaminergic neurons, and nigral damage-associated motor deficits. Next, we elucidated the mechanisms by which KKC080096 upregulated HO-1. KKC080096 induced the phosphorylation of AMPK and its known upstream kinases LKB1 and CaMKKbeta, and pharmacological inhibition of AMPK activity reduced the effects of KKC080096 on HO-1 expression and LPS-induced NO generation, suggesting that KKC080096-induced HO-1 upregulation involves LKB1/AMPK and CaMKKbeta/AMPK pathway activation. Further, KKC080096 caused an increase in cellular Nrf2 level, bound to Keap1 (Nrf2 inhibitor protein) with high affinity, and blocked Keap1-Nrf2 interaction. This Nrf2 activation resulted in concurrent induction of HO-1 and other Nrf2-targeted antioxidant enzymes in BV-2 and in dopaminergic CATH.a cells. These results indicate that KKC080096 is a potential therapeutic for oxidative stress- and inflammation-related neurodegenerative disorders such as Parkinson's disease.

Polyherbal formula SC-E3 inhibits rheumatoid arthritis activity in a mouse model of type-II collagen-induced arthritis.

OBJECTIVE: SC-E3 is a polyherbal formula that contains five medicinal herbs used frequently in traditional herbal medicine. In our previous study, we demonstrated the antioxidant and anti-inflammatory effects of SC-E3. The present study examined the effects of SC-E3 in a mouse model of type-II collagen-induced arthritis (CIA). METHODS: In vivo, male DBA/1J mice were immunized by intradermal injection of bovine type-II collagen and complete or incomplete Freund's adjuvant, to induce arthritis. SC-E3 was orally administered daily for 23 days. In vitro, bone marrow-derived macrophages (BMMs) were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) in the absence or presence of SC-E3. RESULTS: Administrations of SC-E3 were found to have anti-arthritic effects in the joints of CIA mice, as evidenced by reduced paw swelling, bone erosion and deformation, inflammatory cell infiltration, and inflammation in synovial membrane. SC-E3 also reduced serum levels of tumor necrosis factor-α, interleukin-1ß, aspartate aminotransferase and alanine aminotransferase. Furthermore, tartrate-resistant acid phosphatase-positive osteoclast numbers in the joints were significantly lower in SC-E3-treated CIA mice than in CIA mice. In addition, the differentiations of BMMs to multinucleated osteoclasts induced by M-CSF and RANKL stimulation were dose-dependently reduced by SC-E3. CONCLUSION: These results suggest that SC-E3 possesses substantial anti-arthritic activity because it inhibits pro-inflammatory cytokines and osteoclastogenesis, and that SC-E3 has potential therapeutic use for the treatment of rheumatoid arthritis.

Pioglitazone-Primed Mesenchymal Stem Cells Stimulate Cell Proliferation, Collagen Synthesis and Matrix Gene Expression in Tenocytes.

Various therapeutic effects of mesenchymal stem cells (MSCs) have been reported. However, the rapid clearance of these cells in vivo, difficulties in identifying their therapeutic mechanism of action, and insufficient production levels remain to be resolved. We investigated whether a pioglitazone pre-treatment of MSCs (Pio-MSCs) would stimulate the proliferation of co-cultured tenocytes. Pioglitazone increased the proliferation of MSCs and enhanced the secretion of VEGF (vascular endothelial growth factor) and collagen in these cells. We then examined the effects of Pio-MSCs on tenocytes using an indirect transwell culture system. A significant increase in tenocyte proliferation and cell cycle progression was observed in these co-cultures. Significant increases were observed in wound scratch closure by tenocytes from a Pio-MSC co-culture. Pio-MSCs also enhanced the secretion of collagen from tenocytes. A higher mRNA level of collagen type 1 (Col 1) and type 3 (Col 3), scleraxis (Scx), and tenascin C (TnC) was found in the tenocytes in Pio-MSC co-cultures compared with monocultured cells or tenocytes cultured with non-treated MSCs. Our results indicate that pioglitazone enhances the therapeutic effects of MSCs on tendon repair.

Neuroprotective Effects of Gagam-Sipjeondaebo-Tang, a Novel Herbal Formula, against MPTP-Induced Parkinsonian Mice and MPP+-Induced Cell Death in SH-SY5Y Cells.

Parkinson's disease is a neurodegenerative disease characterized by progressive cell death of dopaminergic neuron and following neurological disorders. Gagam-Sipjeondaebo-Tang (GST) is a novel herbal formula made of twelve medicinal herbs derived from Sipjeondaebo-Tang, which has been broadly used in a traditional herbal medicine. In the present study, we investigated the effects of GST against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor abnormalities in mice and 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in SH-SY5Y cell. First, we found that GST alleviated motor dysfunction induced by MPTP, and the result showed dopaminergic neurons recovery in substantia nigra. In the cell experiment, pretreatment with GST increased the cell viability and attenuated apoptotic cell death in MPP+-treated SH-SY5Y cells. GST also inhibited reactive oxygen species production and restored the mitochondrial membrane potential loss, which were induced by MPP+. Furthermore, GST extract significantly activated ERK and Akt, cell survival-related proteins, in SH-SY5Y cells. The effect of GST preventing mitochondrial dysfunction was antagonized by pretreatment of PD98059 and LY294002, selective inhibitors of ERK and Akt, respectively. Taken together, GST alleviated abnormal motor functions and recovered neuronal cell death, mitochondrial dysfunction, possibly via ERK and Akt activation. Therefore, we suggest that GST may be a candidate for the treatment and prevention of Parkinson's disease.

Antioxidant and Anti-Inflammatory Effects of Herbal Formula SC-E3 in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

SC-E3 is a novel herbal formula composed of five oriental medicinal herbs that are used to treat a wide range of inflammatory diseases in Korean traditional medicine. In this study, we sought to determine the effects of SC-E3 on free radical generation and inflammatory response in lipopolysaccharide- (LPS-) treated RAW 264.7 macrophages and the molecular mechanism involved. The ethanol extract of SC-E3 showed good free radical scavenging activity and inhibited LPS-induced reactive oxygen species generation. SC-E3 significantly inhibited the production of the LPS-induced inflammatory mediators, nitric oxide and prostaglandin E2, by suppressing the expressions of inducible nitric oxide synthase and cyclooxygenase-2, respectively. SC-E3 also prevented the secretion of the proinflammatory cytokines, IL-1ß, TNF-α, and IL-6, and inhibited LPS-induced NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Furthermore, SC-E3 induced the expression of heme oxygenase-1 (HO-1) by promoting the nuclear translocation and transactivation of Nrf2. Taken together, these results suggest that SC-E3 has potent antioxidant and anti-inflammatory effects and that these effects are due to the inhibitions of NF-κB and MAPK and the induction of Nrf2-mediated HO-1 expression in macrophages. These findings provide scientific evidence supporting the potential use of SC-E3 for the treatment and prevention of various inflammatory diseases.

Anticancer Effects of a Korean Herbal Medicine Formula (H9) via AMPK and HER2-PI3K/Akt Signaling in Breast Cancer Cells.

An H9 is a formula of nine medicinal herbs derived from Osuyubujaijung-tang, a traditional Korean prescription for Soeumin constitution. In our previous study, H9 showed anticancer effects against breast cancer and non-small-cell lung cancer. However, the underlying mechanisms of these effects have not yet been elucidated. In this study, we investigated the effects of H9, both alone and in combination with trastuzumab, on breast cancer cells and sought to elucidate the mechanisms involved. H9 suppressed the proliferation of human breast cancer cells, induced arrest of the cell cycle at the G0/G1 phase, and caused mitochondrial dysfunction and apoptosis. In addition, H9 induced the activation of AMPK and inhibited the HER2-PI3K/Akt signaling pathway. Furthermore, H9 attenuated hypoxia-induced HIF-1α and VEGF, resulting in decreased migration and invasion of breast cancer cells. Compared with treatment with either drug alone, co-treatment with H9 and trastuzumab significantly inhibited the growth of BT-474 cells through induction of apoptosis. These results suggest that H9 should be considered as a potent anticancer agent that targets the HER2-PI3K/Akt pathway, and the combination of H9 with trastuzumab should be considered as a new therapeutic regimen for treating breast cancer. Copyright © 2017 John Wiley & Sons, Ltd.

Herbal formula SC-E1 suppresses lipopolysaccharide-stimulated inflammatory responses through activation of Nrf2/HO-1 signaling pathway in RAW 264.7 macrophages.

BACKGROUND: SC-E1 is a novel herbal formula consisting of five oriental medicinal herbs used frequently in traditional herbal medicine for the treatment of inflammatory diseases in Korea. This study examined the effects of SC-E1 on lipopolysaccharide (LPS)-stimulated macrophages and the molecular mechanism involved. METHODS: The cytotoxic effect of the SC-E1 extract was evaluated in RAW 264.7 cells by MTT assay. The effects of SC-E1 on the free radical scavenging and generation of intracellular reactive oxygen species were measured using DPPH and DCFH-DA, respectively. The effects of SC-E1 on the production of pro-inflammatory cytokines, inflammatory mediators, and related products were determined by ELISA and western blotting. The molecular mechanism and the nuclear translocation of nuclear factor-kappa B (NF-κB) and NF-E2-related factor 2 (Nrf2) were examined by western blot analysis and immunocytochemistry. RESULTS: SC-E1 exhibited strong anti-oxidant activity and inhibited LPS-induced NO secretion as well as iNOS expression and the production of pro-inflammatory cytokines, without affecting the cell viability. SC-E1 also suppressed the LPS-induced NF-κB activation and the mitogen-activated protein kinase (MAPK) pathway. Moreover, SC-E1 induced heme oxygenase-1 (HO-1) expression via the nuclear translocation of Nrf2. The inhibitory effects of SC-E1 on the production of pro-inflammatory cytokines were abrogated by treatment with SnPP, an HO-1 inhibitor. CONCLUSION: These results suggest that SC-E1 exerts its anti-oxidant and anti-inflammatory effects through the inhibition of NF-κB and MAPK as well as Nrf2-mediated HO-1 induction in macrophages. These findings provide evidences for SC-E1 to be considered as a new prescription for treating inflammatory diseases.

Tryptanthrin Suppresses the Activation of the LPS-Treated BV2 Microglial Cell Line via Nrf2/HO-1 Antioxidant Signaling.

Microglia are the resident macrophages in the central nervous system (CNS) and play essential roles in neuronal homeostasis and neuroinflammatory pathologies. Recently, microglia have been shown to contribute decisively to neuropathologic processes after ischemic stroke. Furthermore, natural compounds have been reported to attenuate inflammation and pathologies associated with neuroinflammation. Tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione) is a phytoalkaloid with known anti-inflammatory effects in cells. In present study, the authors confirmed middle cerebral artery occlusion (MCAO) injury triggers the activation of microglia in brain tissue, and investigated whether tryptanthrin influences the function of mouse murine BV2 microglia under LPS-induced inflammatory conditions in vitro. It was found tryptanthrin protected BV2 microglia cells against LPS-induced inflammation and inhibited the induction of M1 phenotype microglia under inflammatory conditions. In addition, tryptanthrin reduced the production of pro-inflammatory cytokines in BV2 microglia cells via nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling and NF-κB signaling. The authors suggest that tryptanthrin might alleviate the progress of neuropathologies by controlling microglial functions under neuroinflammatory conditions.