Nepetaefuran and leonotinin isolated from Leonotis nepetaefolia R. Br. potently inhibit the LPS signaling pathway by suppressing the transactivation of NF-κB.

Leonotis nepetaefolia R. Br., also known as Klip Dagga or Lion's Ear, has traditionally been used as a folk medicine to treat inflammatory diseases such as rheumatism, bronchitis, and asthma; however, the components that exhibit its anti-inflammatory activity have not yet been identified. In the present study, we investigated the effects of three types of diterpenoids, nepetaefuran, leonotinin, and leonotin, which were isolated from L. nepetaefolia R. Br., on the LPS signaling pathway in order to elucidate the anti-inflammatory mechanism involved. Nepetaefuran more potently inhibited the LPS-induced production of NO and CCL2 than leonotinin by suppressing the expression of iNOS mRNA and CCL2 mRNA. On the other hand, leonotin failed to inhibit the production of NO and CCL2 induced by LPS. Although nepetaefuran and leonotinin had no effect on the LPS-induced degradation of IκBα or nuclear translocation of NF-κB p65, they markedly inhibited the transcriptional activity of NF-κB. Nepetaefuran and leonotinin also inhibited the transcriptional activity of the GAL4-NF-κB p65 fusion protein. On the other hand, nepetaefuran, leonotinin and leonotin did not affect the LPS-induced activation of MAP kinase family members such as ERK, p38, and JNK. In addition, inhibitory effect of nepetaefuran and leonotinin on NF-κB activation is well correlated with their ability to induce activation of Nrf2 and ER stress. Taken together, these results demonstrated that nepetaefuran and leonotinin could be the components responsible for the anti-inflammatory activity of L. nepetaefolia R. Br. by specifically inhibiting the LPS-induced activation of NF-κB.

Anti-inflammatory activity of flavonoids in Nepalese propolis is attributed to inhibition of the IL-33 signaling pathway.

Propolis has been used in folk medicine to improve health and prevent inflammatory diseases; however, the components that exhibit its anti-inflammatory activity remain unknown. We herein investigated the effects of flavonoids isolated from Nepalese propolis on the IL-33 signaling pathway to clarify the anti-inflammatory mechanism involved. Of the 8 types of flavonoids isolated from Nepalese propolis, 4 types of compounds, such as 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin, markedly inhibited the IL-33-induced mRNA expression of inflammatory genes including IL-6, TNFα and IL-13 in bone marrow-derived mast cells (BMMC). These four flavonoids also inhibited the IL-33-induced activation of nuclear factor κB (NF-κB), which was consistent with their inhibitory effects on cytokine expression. The effects of these flavonoids are attributed to inhibition of IL-33-induced activation of IKK, which leads to the degradation of IκBα and nuclear localization of NF-κB. On the other hand, other flavonoids isolated from Nepalese propolis, such as isoliquiritigenin, plathymenin, 7-hydroxyflavanone, and (+)-medicarpin, had no effect on the IL-33 signaling pathway or cytokine expression. Therefore, these results indicate that 3',4'-dihydroxy-4-methoxydalbergione, 4-methoxydalbergion, cearoin, and chrysin are the substances responsible for the anti-inflammatory activity of Nepalese propolis.

Licochalcones suppress degranulation by decreasing the intracellular Ca2+ level and tyrosine phosphorylation of ERK in RBL-2H3 cells.

Mast cells play a key role in allergic inflammation by releasing various mediators, such as histamine, serotonin, leukotrienes and cytokines. A signaling cascade of events activated by stimulation with antigens contributes to the regulation of mast cell degranulation. While various anti-inflammatory and anti-allergic drugs have been developed that inhibit degranulation of mast cells, the inhibitory mechanism has been poorly understood. Licochalcone A (Lico A) is a retrochalcone isolated from the root of Xinjiang liquorice and has been reported to exhibit various biological activities such as anti-inflammatory activity. We examined the effects of Lico A and related chalcones on degranulation in a rat basophilic leukemia cell line, RBL-2H3. Whereas Lico A and licochalcone C (Lico C) exhibited inhibitory activity with cytotoxicity, licochalcone D (Lico D) significantly inhibited the degranulation in RBL-2H3 cells with low cytotoxicity. Moreover, Lico D significantly inhibited the Ca2+ influx and phosphorylation of extracellular signal regulated kinase (ERK) and MEK. These results suggest that Lico D inhibits mast cell degranulation via the inhibition of both extracellular Ca2+ influx and activation of the MEK-ERK pathway.

The fixed structure of Licochalcone A by alpha, beta-unsaturated ketone is necessary for anti-inflammatory activity through the inhibition of NF-kappaB activation.

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity. Previously, we reported that a major component, Licochalcone A, potently inhibited TNFalpha-induced NF-kappaB activation by inhibiting IKKbeta activation. In this study, we investigated whether the fixed structure of Licochalcone A by alpha, beta-unsaturated ketone is required for its inhibitory effect of NF-kappaB activation. Interestingly, reduced Licochalcone A, which lacks a double bond, failed to inhibit TNFalpha-induced NF-kappaB activation. Whereas Licochalcone A potently inhibited TNFalpha-induced IKK activation, IkappaBalpha degradation, nuclear localization of NF-kappaB and its DNA binding activity, no inhibitory effect was observed by reduced Licochalcone A. In addition, TNFalpha-induced expression of inflammatory cytokines, CCL2/MCP-1 and CXCL1/KC, was clearly inhibited by Licochalcone A but not reduced Licochalcone A. As a result, culture media pretreated with Licochalcone A but not reduced Licochalcone A following TNFalpha stimulation significantly inhibited the chemotactic activity of neutrophils. Furthermore, acute carrageenan-induced paw edema in mice was markedly inhibited by administration of Licochalcone A but not reduced Licochalcone A. Taken together, it is suggested that Licochalcone A is a promising anti-inflammatory drug in vivo and its fixed structure is critical for anti-inflammatory activity.

Licochalcone A potently inhibits tumor necrosis factor alpha-induced nuclear factor-kappaB activation through the direct inhibition of IkappaB kinase complex activation.

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity; however, its mechanism has not been fully understood. Licochalcone A is a major and biogenetically characteristic chalcone isolated from G. inflata. Here, we found that licochalcone A strongly inhibited tumor necrosis (TNF)-alpha-induced nuclear localization, DNA binding activity, and the transcriptional activity of nuclear factor-kappaB (NF-kappaB). Whereas licochalcone A had no effect on the recruitment of receptor-interacting protein 1 and IkappaB kinase beta (IKKbeta) to TNF receptor I by TNF-alpha, it significantly inhibited TNF-alpha-induced IkappaB kinase complex (IKK) activation and inhibitor of nuclear factor-kappaB degradation. It is interesting that we found that the cysteine residue at position 179 of IKKbeta is essential for licochalcone A-induced IKK inhibition, because licochalcone A failed to affect the kinase activity of the IKKbeta (C179A) mutant. In contrast, a structurally related compound, echinatin, failed to inhibit TNF-alpha-induced IKK activation and NF-kappaB activation, suggesting that the 1,1-dimethy-2-propenyl group in licochalcone A is important for the inhibition of NF-kappaB. In addition, TNF-alpha-induced expression of inflammatory cytokines CCL2/monocyte chemotactic protein-1and CXCL1/KC was clearly inhibited by licochalcone A but not echinatin. Taken together, licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the inhibition of IKK activation.

Glycyrrhiza inflata-derived chalcones, Licochalcone A, Licochalcone B and Licochalcone D, inhibit phosphorylation of NF-kappaB p65 in LPS signaling pathway.

Licorice root has been used as a traditional medicine for the treatment of gastric ulcer, bronchial asthma and inflammation. Licochalcone A is a major component of Xinjiang licorice, Glycyrrhiza inflata. Previously we showed that Licochalcone A significantly inhibited LPS-induced NF-kappaB transcriptional activation by abrogating the phosphorylation of NF-kappaB p65 at serine 276. Glycyrrhiza inflata contains not only Licochalcone A but also Licochalcone B, Licochalcone C, Licochalcone D, Echinatin and Isoliquiritigenin, harboring the common structure of chalcones. No chalcones had any effect on LPS-induced IkappaB degradation, nuclear translocation and DNA binding activity of NF-kappaB p65; however, we observed that Licochalcone B and Licochalcone D significantly inhibited LPS-induced phosphorylation at serine 276 and transcriptional activation of NF-kappaB, the same as Licochalcone A. Interestingly, we also found that Licochalcone A, Licochalcone B and Licochalcone D effectively inhibited LPS-induced activation of PKA, which is required for the phosphorylation of NF-kappaB p65 at serine 276. Consequently, Licochalcone B and Licochalcone D significantly reduced the LPS-induced production of NO, TNFalpha and MCP-1. On the other hand, Licochalcone C, Echinatin and Isoliquitigenin failed to inhibit LPS-induced NF-kappaB activation. These findings suggest that the anti-inflammatory effect of Glycyrrhiza inflata is ascribable to the potent inhibition of NF-kappaB by Licochalcone A, Licochalcone B and Licochalcone D.

Licochalcone A significantly suppresses LPS signaling pathway through the inhibition of NF-kappaB p65 phosphorylation at serine 276.

Licorice root, Glycyrrhiza inflata, has been used as a traditional medicine for the treatment of bronchial asthma and inflammation; however, the mechanism of its anti-inflammatory activity has not been clarified. Here, we investigated the effect of Licochalcone A, a major component of G. inflata, on the LPS signaling pathway. We found that Licochalcone A remarkably inhibited LPS-induced NO production, and TNFalpha expression and MCP-1 expression in both RAW264.7 cells and primary macrophages. Furthermore, when injected with Licochalcone A prior to injection of LPS, the serum level of TNFalpha and MCP-1 in C57BL/6 mice was clearly decreased, indicating that Licochalcone A has a potent anti-inflammatory effect both in vitro and in vivo. Strikingly, Licochalcone A significantly inhibited LPS-induced NF-kappaB transcriptional activation; however, it had no effect on not only the phosphorylation and degradation of IkappaBalpha but also nuclear translocation and DNA binding activity of NF-kappaB p65. Interestingly, Licochalcone A markedly inhibited the phosphorylation of p65 at serine 276. As a result, it reduced NF-kappaB transactivation by preventing the interaction of p65 with p300. Taken together, Licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the unique mechanism of NF-kappaB inhibition.

Licochalcone A is a potent inhibitor of TEL-Jak2-mediated transformation through the specific inhibition of Stat3 activation.

Aberrant activation of Jak/Stat signaling causes a number of hematopoietic disorders and oncogenesis, and therefore the effective inhibitors of the Jak/Stat signaling pathway may be therapeutically useful. TEL-Jak2 gene fusion, which has been identified in human leukemia, encodes a chimeric protein endowed with constitutive tyrosine kinase activity. Expression of TEL-Jak2 protects Ba/F3 cells from IL-3 withdrawal-induced apoptotic cell death and leads to IL-3-independent growth. However, its mechanisms remain to be only partially understood. Here, we first found that Licochalcone A, one of the flavonoids isolated from the root of Glycyrrhiza inflate, inhibited TEL-Jak2-mediated cell proliferation and survival in the absence of IL-3. Licochalcone A failed to inhibit the activity of TEL-Jak2, however, this induced apoptosis of TEL-Jak2-transformed cells with a much lower concentration in the absence of IL-3 than in the presence of IL-3. Interestingly, Licochalcone A significantly inhibited the phosphorylation and nuclear localization of Stat3, which is essential for TEL-Jak2-induced cell transformation. These data suggest that Licochalcone A is a specific inhibitor for Stat3 and would be employed for the treatment of various diseases caused by disorders of the Jak/Stat pathway.

Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells.

Indirubin, a purple vegetable dye, is a traditional Chinese medicine for myelocytic leukaemia. Indirubin inhibits cyclin-dependent protein kinases (CDKs) and is present in human urine and serum. When indirubin was present during the neutrophilic differentiation of human myelocytic leukaemia HL-60 cells, it augmented superoxide production triggered by opsonized zymosan (OZ) by the terminally differentiated HL-60 cells. It also augmented the calcium response to OZ stimulation, and HL-60 cell chemotaxis evoked by interleukin-8 (IL-8, CXCL8) and formylpeptide. In addition, indirubin induced marked IL-8 release by the cells during differentiation and the cells differentiated with indirubin had typical neutrophilic properties, deformed nuclei and granules. Use of stable cloned HL-60 cells that contained a reporter vector for monitoring the activity of the transcription factor PU.1, which acts specifically at the stage of promyelocyte differentiation into neutrophils and monocytes, revealed that indirubin has a potent promoting activity on intracellular PU.1. Indirubin enhanced the expression of typical neutrophil proteins, including granulocyte-colony stimulating factor receptor, the beta2-integrin subunit CD18, the NADPH-oxidase subunit p47phox, and the IL-8 receptor CXCR1, all are controlled by PU.1. Indirubin also inhibited CDK2-dependent phosphorylation of retinoblastoma protein during neutrophilic differentiation. These results suggest that indirubin augments the neutrophilic differentiation of human myelocytic leukaemia HL-60 cells through inhibition of CDK2 and activation of PU.1.