Discovery of diarylheptanoids that activate α7 nAchR-JAK2-STAT3 signaling in macrophages with anti-inflammatory activity in vitro and in vivo.

Acute inflammatory diseases, such as sepsis, are life-threatening illnesses. Regulating the α7 nicotinic acetylcholine receptor (α7 nAchR)-mediated signaling may be a promising strategy to treat sepsis. Diarylheptanoids have long been found to exhibit anti-inflammatory properties. However, the possible mechanism of diarylheptanoids has rarely been investigated. In this study, we isolated and synthesized 49 diarylheptanoids and analogues and evaluated their anti-inflammatory activities. Among them, compounds 28 and 40 markedly blocked lipopolysaccharide (LPS)-induced production of nitric oxide (NO), interleukin-1ß (IL-1ß) and interleukin-6 in murine RAW264.7 cells. Furthermore, compounds 28 and 40 also effectively attenuated LPS-induced sepsis, acute lung injury, and cytokines release in vivo. Mechanistically, compounds 28 and 40 significantly induced phosphorylation of janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling and suppression of nuclear factor-κB (NF-κB) pathway. Furthermore, blocking α7 nAchR could effectively abolish compounds 28 and 40-mediated activation of JAK2-STAT3 signaling as well as inhibition of NF-κB activation and NO production in LPS-exposed RAW264.7 cells. Collectively, our findings have identified a new diarylheptanoid, compound 28, as an agonist of α7 nAchR-JAK2-STAT3 signaling, which can be potentially developed as a valuable candidate for the treatment of sepsis, and provide a new lead structure for the development of anti-inflammatory agents targeting α7 nAchR-JAK2-STAT3 signaling.

6-Hydroxy-3-O-methyl-kaempferol 6-O-glucopyranoside potentiates the anti-proliferative effect of interferon α/ß by promoting activation of the JAK/STAT signaling by inhibiting SOCS3 in hepatocellular carcinoma cells.

Suppressor of cytokine signaling 3 (SOCS3) is a key negative regulator of type I interferon (IFN α/ß) signaling. Inhibition of SOCS3 by small molecules may be a new strategy to enhance the efficacy of type I IFN and reduce its side effects. We established a cell-based screening assay using human hepatoma HepG2 cells stably transfected with a plasmid wherein the luciferase reporter activity was propelled by interferon α-stimulated response element (ISRE), which is a motif specifically recognized by type I IFN-induced activation of Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. After screening our chemical library, 6-hydroxy-3-O-methyl-kaempferol 6-O-glucopyranoside (K6G) was identified to be a potent activator of type I IFN with EC50 value of 3.33±0.04µM. K6G enhanced the phosphorylation of JAK1, Tyk2, and STAT1/2 but decreased the phosphorylation of STAT3. K6G also promoted endogenous IFN-α-regulated genes expression. More interestingly, K6G significantly decreased the expression of SOCS3 without affecting the expression of SOCS1. Furthermore, K6G enhanced the anti-proliferative effect of IFN-α on hepatocellular carcinoma (HCC) cells. These results suggested that K6G potentiated the inhibitory effect of IFN-α on HCC cell proliferation through activation of the JAK/STAT signaling pathway by inhibiting SOCS3 expression. K6G warrants further investigation as a novel therapeutic method to enhance the efficacy of IFN-α/ß.

Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway.

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3', 5'-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Methyl jasmonate and cyclodextrin-mediated defense mechanism and protective effect in response to paraquat-induced stress in peanut hairy root.

Plant cells have a variety of defense mechanisms to alleviate the deleterious effects of oxidative stress. The present work elucidated a schematic diagram of the proposed pathway of peanut hairy root tissue treated with different elicitors; paraquat (PQ), methyl jasmonate (MeJA), and cyclodextrin (CD). The different elicitation approaches could provoke intrinsic stress in plant cells and might activate a distinct response pathway, allowing plants to overcome the deleterious effects of oxidative stress. Among all strategies, hairy root culture pretreated with PQ followed by application of MeJA plus CD showed an extensive induction of antioxidant defense mechanisms. The expression of the antioxidant enzyme genes and stilbene-synthesized enzyme genes were up-regulated in accordance with the dramatic increase in the production of stilbene compounds. The non-enzymatic antioxidant substances exhibited a highly enhanced capability. The pathogenesis-related protein (PR) genes were also highly up-regulated. In summary, we demonstrated that the interplay among MeJA plus CD and PQ may activate a complex signaling network to regulate plant defense mechanisms involving the up-regulation of detoxifying enzymes, induction of free-radical scavengers and overexpression of genes associated with plant defense pathways.

Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa: Isolation, chemical modification and estrogenic activity evaluation.

Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10-12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute stereochemistry of the isolated compounds has also been determined using the modified Mosher's method. The isolated compounds and the chemically modified analogues were evaluated for their estrogenic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phytoestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERbeta gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the 1-position of the heptyl chain.

Flavonoid glycosides isolated from Epimedium brevicornum and their estrogen biosynthesis-promoting effects.

Epimedium brevicornum Maxim has a long history of use in the treatment of estrogen deficiency-related diseases. However, the chemical constituents and mechanism of action of this medicinal plant are not fully understood. In the present study, we isolated four new isoprenylated flavonoid glycosides, as well as 16 known flavonoids (13 isoprenylated flavonoids), from this plant. The chemical structures of the new flavonoid glycosides were elucidated by extensive spectroscopic analysis. The new compounds 1-4 were potent promoters of estrogen biosynthesis in human ovarian granulosa-like KGN cells. ZW1, an isoprenylated flavonoid analogue and a specific inhibitor of phosphodiesterase 5 (PDE5), was synthesized and used to explore the mechanism of the isoprenylated analogues on estrogen biosynthesis. ZW1 treatment increased estrogen production by upregulation of aromatase mRNA and protein expression. ZW1 increased the phosphorylation of cAMP response element-binding protein (CREB). Further study showed that the inhibition of PDE5 by ZW1 increased estrogen biosynthesis partly through suppression of phosphodiesterase 3 (PDE3). Our results suggested that the isoprenylated flavonoids from E. brevicornum may produce beneficial health effects through the promotion of estrogen biosynthesis. PDE5 warrants further investigation as a new therapeutic target for estrogen biosynthesis in the prevention and treatment of estrogen-deficiency related diseases.

Diarylheptanoids of Curcuma comosa with inhibitory effects on nitric oxide production in macrophage RAW 264.7 cells.

Eight new diarylheptanoids, a 1.2:1 mixture of (3S)- and (3R)-1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-ol (1a and 1b), a racemic mixture of (3S)- and (3R)-1-(4-hydroxyphenyl)-3-methoxy-7-phenyl-(4E,6E)-4,6-heptadiene (2a and 2b), a ca. 1:1 mixture of (3S)- and (3R)-1-(4-hydroxy-3- methoxyphenyl)-3-methoxy-7-phenyl)-(4E,6E)-4,6-heptadiene (3a and 3b), 3-acetoxy-1-(3,4-dihydroxyphenyl)-7-phenylheptan-5-ol (4), (3R)-1-(4,5- dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3,2'-epoxide (5), and thirteen known diarylheptanoids, 6-12, a 3:1 mixture of 13a and 13b, and 14-17, were isolated from the rhizomes of Curcuma comosa from Sakon Nakhon, northeastern part of Thailand. The isolated compounds were evaluated for their anti- inflammatory activities on the inhibition of lipopolysaccharide-induced nitric oxide production in macrophage RAW 264.7 cells and the diarylheptanoids 1a and 1b mixture and 14 exhibited potent inhibitory activity.

Iridoid glucosides from the flowers of Barleria lupulina.

A new iridoid diglucoside, lupulinoside, and eight known iridoid glucosides, acetylbarlerin, ipolamiidoside ( 3), 6-O-acetylshanzhiside methyl ester, barlerin, shanzhiside methyl ester, mussaenosidic acid, 8-O-acetylshanzhiside, and shanzhiside have been isolated from the flowers of Barleria lupulina. The structure of the new compound was established as 8-O-acetyl-2'- O-(beta-glucopyranosyl)mussaenoside by spectroscopic, especially 2D NMR, techniques. When tested for anti-herpes simplex type 1 activity, only compound 3 exhibited antiviral properties. None of the compounds showed cytotoxic effects to the vero cells and none of them inhibited cyclooxygenase-2 enzyme.