Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1ß (IL-1ß), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. RESULTS: Nontoxic concentrations of LL-37 (up to 10 µm) and IL-1ß significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p < 0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p < 0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20 µm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X(7) receptor) and the neutralizing antibody against P2X(7) blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X(7) receptor in HGFs. CONCLUSION: These findings indicate that LL-37 induces IL-8 expression via the P2X(7) receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.

Human beta-defensin-3 up-regulates cyclooxygenase-2 expression and prostaglandin E2 synthesis in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Oral epithelial cells express three antimicrobial peptide human beta-defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in non-immune cells, such as human gingival fibroblasts. MATERIAL AND METHODS: Cultured fibroblasts were treated with different concentrations of hBD-1, -2, -3 or interleukin-1 beta, as a positive control, for various times, in the presence or absence of NS-398, a specific COX-2 inhibitor. The levels of COX-1 and COX-2 mRNA expression were analyzed using RT-PCR and real-time PCR. Whole cell lysates were analyzed for COX-1 and COX-2 protein expression by western blotting. Cell-free culture supernatants were assayed for PGE(2) levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. RESULTS: Ten and 40 microg/mL of hBD-3 up-regulated COX-2 mRNA and protein expression, consistent with COX-2 up-regulation by interleukin-1 beta, whereas hBD-1 and hBD-2 did not. However, COX-1 mRNA and protein were constitutively expressed. The time-course study revealed that hBD-3 up-regulated COX-2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX-2 up-regulation, 10 and 40 microg/mL of hBD-3 significantly increased PGE(2) levels in cell-free culture supernatants (p < 0.05), and this was inhibited by NS-398 in a dose-dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. CONCLUSION: These findings indicate that epithelial human beta-defensin-3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.

Involvement of cytosolic phospholipase A2alpha in MMP-9 up-regulation.

Matrix metalloproteinase-9 (MMP-9) is important in the pathogenesis of periodontitis. Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is involved in MMP-9 up-regulation in human monocytes. We tested the hypothesis that cPLA(2)alpha also regulates MMP-9 induction by Fusobacterium nucleatum and by phorbol 12-myristate-13-acetate (PMA) in gingival epithelial cells. While PMA induced MMP-9 expression considerably, F. nucleatum did so moderately. This time-course study demonstrated that MMP-9 mRNA up-regulation occurred at 3 hours, whereas MMP-9 secretion and activity in cell-free supernatants occurred at 12 hours. cPLA(2)alpha mRNA was constitutively expressed in gingival epithelial cells. Transient activation of cPLA(2) by Ser505 phosphorylation was observed in the nuclei upon stimulation, suggesting its role as a transcription factor, while cPLA(2) protein expression remained unchanged. Induction of MMP-9 expression and activity was significantly inhibited by 1 muM of the specific cPLA(2)alpha inhibitor (P < 0.01). These findings demonstrate the involvement of cPLA(2)alpha in MMP-9 up-regulation.

Anti-inflammatory and analgesic activities of Mallotus spodocarpus Airy shaw.

The chloroform extract from the roots of Mallotus spodocarpus was investigated for anti-inflammatory and analgesic activities in animal models. In acute inflammatory models, the extract significantly inhibited ethyl phenylpropiolate-induced ear edema and carrageenin- and arachidonic acid-induced hind paw edema in rats. In the chronic inflammatory model using the cotton pellet-induced granuloma in rats, the extract exhibited inhibitory activity on the formation of granuloma. The extract also elicited pronounced inhibitory effect on acetic acid-induced writhing response in mice in the analgesic test. The results obtained suggest marked anti-inflammatory and analgesic activity of the extract.

Anti-inflammatory and antipyretic properties of Clerodendrum petasites S. Moore.

The methanol extract from Clerodendrum petasites S. Moore (CP extract) was assessed for anti-inflammatory and antipyretic activities on the experimental animal models. It was found that CP extract possessed moderate inhibitory activity on acute phase of inflammation in a dose-related manner as seen in ethyl phenylpropiolate-induced ear edema (ED(50)=2.34 mg/ear) as well as carrageenin-induced hind paw edema (ED(30)=420.41 mg/kg) in rats. However, CP extract did not elicit any inhibitory effect on arachidonic acid-induced hind paw edema in rats. In subchronic inflammatory model, CP extract provoked a significant reduction of transudation but had no effect on proliferative phase when tested in cotton pellet-induced granuloma model. CP extract also reduced the alkaline phosphatase activity in serum of rats in this animal model. Moreover, CP extract possessed an excellent antipyretic effect when tested in yeast-induced hyperthermic rats. It is postulated that the anti-inflammatory and antipyretic effects of CP extract are caused by the inhibition of the prostaglandin synthesis. Anyhow, CP extract did not possess any analgesic activity in acetic acid-induced writhing response in mice. The results obtained show that C. petasites has beneficial properties since it possesses potent antipyretic and moderate anti-inflammatory activities without ulcerogenic effect.

2-substituted furans from Polyalthia suberosa.

Two new 2-substituted furans, 1-(2-furyl)pentacosa-16,18-diyne and 23-(2-furyl)tricosa-5,7-diynoic acid, have been isolated from stems of Polyalthia suberosa. The structures were assigned by spectroscopic methods. The two compounds together with the previously reported kalasinamide, N-trans-feruloyltyramine and N-trans-coumaroyltyramine showed anti-HIV activities.

Long-term effects of triptolide on spermatogenesis, epididymal sperm function, and fertility in male rats.

Prior studies had suggested that triptolide, a diterpene triepoxide isolated from a Chinese medicinal plant, might be an attractive candidate as a post-testicular male contraceptive agent. Despite the promise that triptolide would not affect testis function, nagging concerns remained that a delayed onset of testicular effect might exist. The objectives of this study were to assess the effects of relatively longer treatment duration of triptolide on fertility, spermatogenesis, and epididymal sperm pathophysiology; and to evaluate the reversibility of these effects after the cessation of treatment. Adult male Sprague-Dawley rats were fed daily with either 30% gum acacia as a vehicle control (n = 12) or 100 microg/kg body weight (BW) of triptolide for 82 days (n = 12) followed by a recovery period of up to 14 weeks (n = 6). At the end of the treatment period, all rats treated with triptolide were sterile. Cauda epididymal sperm content decreased by 84.8% and sperm motility was reduced to zero. In addition, virtually all cauda epididymal sperm in the triptolide-treated group exhibited severe structural abnormalities. The most striking changes observed were head-tail separation, premature chromatin decondensation of sperm nuclei, a complete absence of the plasma membrane of the entire middle and principle pieces, disorganization of the mitochondrial sheath, and aggregation of many sperm tails. Longer treatment duration of triptolide also affected spermatogenesis, with marked variability in the response of individual animals. The degree of damage ranged from apparently normal-looking seminiferous tubules to flattened seminiferous epithelium lined by a single layer of cells consisting of Sertoli cells and a few spermatogonia. Affected tubules exhibited intraepithelial vacuoles of varying sizes, multinucleated giant cells, germ cell exfoliation, and tubular atrophy. Recovery occurred as early as 6 weeks after cessation of treatment. By 14 weeks, 4 out of 6 triptolide-treated males were fertile and the females that were impregnated by 3 out of 4 triptolide-treated male rats produced apparently normal litters. These results suggest that triptolide has 2 phenotypic effects on mature and maturing germ cells. The first action appears earlier and manifests mainly in epididymal sperm. The second action presumably is directly on germ cells in testis and causes a variable impairment of spermatogenesis that may not be completely reversible. It is unclear if the earlier effect is a delayed manifestation of subtle testicular injury or post-testicular action.

Posttesticular antifertility action of triptolide in the male rat: evidence for severe impairment of cauda epididymal sperm ultrastructure.

A variety of active diterpene epoxides, including the triptolide (isolated from Tripterygium wilfordii) have been reported to cause infertility in male rats. Previously, we showed that oral administration of triptolide at a dosage of 100 microg/kg per body weight for 70 days completely inhibited fertility in male rats, with little or no demonstrable detrimental effect on spermatogenesis and Leydig cell function as determined by testicular light microscopic appearance and serum and intratesticular testosterone levels. Despite the apparent absence of effects on the testes, cauda epididymal sperm were abnormal, with complete cessation of sperm motility and some reduction in sperm numbers. This study was undertaken to provide additional insight into the subcellular sites and possible mechanisms of action of this compound using ultrastructural analysis of the testes and epididymidis. The most striking effect of triptolide treatment was observed in sperm in the epididymis. In rats rendered infertile with 100 microg/kg per body weight of triptolide daily for 70 days, virtually all cauda epididymal sperm exhibited complete absence of plasma membrane over the entire middle and principal piece, premature decondensation of the nuclei, and disorganization of the mitochondrial sheath with many vacuolated mitochondria. No ultrastructural differences in the epididymal epithelium were observed between control and triptolide-treated rats. The testes appeared to be mildly affected after triptolide treatment but exhibited only subtle ultrastructural defects in the germ cells. The findings of severe impairment of cauda epididymal sperm ultrastructure, along with minimal discernible abnormalities in the fine structural cytology of the testes, further suggest that the site of action of this compound is posttesticular and may be confined to the cauda epididymal sperm. However, we cannot rule out an effect of triptolide that occurs during germ cell maturation but is delayed in its manifestation or triggered at the rete testis and epididymal level.

An azaanthracene alkaloid from Polyalthia suberosa.

An azaanthracene alkaloid, 1-aza-9,10-dimethoxy-4-methyl-2-oxo-1,2-dihydroanthracene (kalasinamide) has been isolated from the stems of Polyalthia suberosa. In addition, the known N-trans-feruloyltyramine and N-trans-coumaroyltyramine are also reported from the same source. The structures were elucidated by spectroscopic methods.

Evaluation of anti-HSV-2 activities of Barleria lupulina and Clinacanthus nutans.

Barleria lupulina Lindl. and Clinacanthus nutans (Burm. f.) Lindau, both belonging to the family Acantaceae, are well-known medicinal plants used in Thai folklore medicine. Virucidal effects of organic extracts of these two plants against herpes simplex virus type 2 strain G, HSV-2 (G), the standard HSV-2 strain were noted. The extracts were assessed for intracellular activities against HSV-2 (G) and five clinical HSV-2 isolates. B. lupulina extract exhibited activity against all five isolates but not the standard strain while that of C. nutans did not show any activity against these viruses as determined by plaque inhibition assay. When the activities were verified by yield reduction assay, anti-HSV-2 activities of B. lupulina extract were observed against HSV-2 (G) as well. The results suggest a therapeutic potential of B. lupulina but not C. nutans against HSV-2.

Novel cytotoxic acylated oligorhamnosides from Mezzettia leptopoda.

Activity-guided fractionation of a stem extract of Mezzettia leptopoda using human oral epidermoid carcinoma (KB) cells led to the isolation of seven highly acylated oligorhamnosides. Four of these constituents are novel, namely, n-octyl 2-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-2, 4-di-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-4-O-hexanoyl-alpha-L-rh amnopyranoside (mezzettiaside 8) (1); n-octyl 2, 3-di-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-4-O-hexanoyl-alpha-L-rh amnopyranoside (mezzettiaside 9) (2); n-octyl 2, 4-di-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-4-O-hexanoyl-alpha-L-rh amnopyranoside (mezzettiaside 10) (3); and n-octyl 2,3, 4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-4-O-hexanoyl-alpha-L-r hamnopyranoside (mezzettiaside 11) (4). Three known compounds were identified as mezzettiasides 2 (5), 3 (6), and 4 (7), respectively, previously isolated from this same plant. The structures of novel compounds 1-4 were determined by spectroscopic methods. All the isolates were evaluated against a panel of human cancer cell lines in this study, and compounds 1-2 and 4-7 were found to be weakly cytotoxic toward KB and/or human colon and lung cancer cell lines.

Triptolide: a potential male contraceptive.

The antifertility effect of triptolide and other related compounds, isolated from Tripterygium wilfordii, has been demonstrated in male rats. The exact sites and mechanism of action of triptolide remain unknown. Our objectives were to determine whether triptolide at selected dose levels that induce infertility has any detrimental effects on the testes and to determine the sites and the possible mechanisms of its action. Groups of six adult male Sprague-Dawley rats were given oral administration of either vehicle (control group) or triptolide (50 or 100 microg/kg body weight) daily for 35 or 70 days. Body weight gain was normal in all treated groups. All six rats treated with a high dosage of triptolide were infertile during the second (63-70 days) mating trial. A lower dose (50 microg) of triptolide gave intermediate fertility values. Plasma levels of luteinizing hormone, follicle-stimulating hormone, testosterone, and intratesticular testosterone were not significantly different between control and triptolide-treated groups. Cauda epididymal sperm content was decreased by 68% and the motility, which averaged 58.2% in the control rat, was reduced to almost zero. No effects of triptolide were observed on testis and accessory organs weight, volumes of tubular lumen and the total Leydig cells, tubule diameter, and the number of Sertoli cells, spermatogonia, preleptotene (PL), and pachytene (P) spermatocytes. There were, however, modest but significant decreases in tubule volume and the number of round spermatids at stages VII-VIII. No changes in the germ cell apoptotic index measured at stages VII-VIII and XIV-I were noted between controls and rats rendered infertile with a high dose of triptolide. Thus, triptolide, at a dose level that induces complete infertility in the adult rats, has minimal adverse effects on the testes and acts primarily on the epididymal sperm making triptolide an attractive lead as a post-testicular male contraceptive.

Xanthones from the twigs of Mammea siamensis.

1,2-Dimethoxy-5-hydroxyxanthone, a new xanthone, was isolated from the twigs of Mammea siamensis, in addition to six known xanthones (5-hydroxy-1-methoxy-, 1,3-dimethoxy-5-hydroxy-, 2,5-dihydroxy-1-methoxy-, 1,7-dihydroxy-, 1,3,7-trihydroxy- and 3,5-dihydroxy-1-methoxyxanthone). Structures for these compounds were deduced from their spectral data.

Limonoids from Azadirachta excelsa.

Activity-directed fractionation of a stem extract of Azadirachta excelsa using KB (human oral epidermoid carcinoma) cells led to the isolation of four meliacin-type limonoids. Two of these constituents were novel, namely, 2,3-dihydronimbolide and 3-deoxymethylnimbidate, and these were purified along with the known compounds, nimbolide and 28-deoxonimbolide. The structures of the new compounds were determined by spectroscopic methods. Nimbolide and 28-deoxonimbolide were broadly cytotoxic when evaluated against a panel of human cancer cell lines, while the two novel compounds were inactive in this regard. The defection of nimbolide and 28-deoxonimbolide as cytotoxic constituents was facilitated by an electrospray LC/MS dereplication procedure.

Flavonol glycosides from Dasymaschalon sootepense.

Two novel flavonol glycosides have been isolated from a methanolic extract of Dasymaschalon sootepense leaves. They are the 3'-neohesperidoside and 3'(6G-alpha-rhamnosylneohesperidoside) of quercetin 3,7-dimethyl ether.

New compounds with DNA strand-scission activity from the combined leaf and stem of Uvaria hamiltonii.

Two flavanones, hamiltones A (1) and B (2), an aurone, hamiltrone (3), a chalcone, hamilcone (4), and a tetrahydroxanthene, hamilxanthene (5), were isolated from Uvaria hamiltonii extracts guided initially by fractionation based on DNA strand-scission and/or 9KB cytotoxicity assays. Compounds 2-5 have not been reported previously, while 1 is new as a natural product. Structural assignments were made based on extensive spectroscopic measurements. Compounds 1-3 were inactive in the 9KB cytotoxicity assay, with compounds 4 and 5 having weak activity. In the DNA strand-scission assay, 3 was the most active compound found in the DNA strand-scission assay, being 10 times more potent than 1 or 2. Compound 4 was only weakly active, and 5 was inactive.

A sensitive assay for anti-HIV-1 drug discovery in a biological safety level-2 laboratory.

Studies involving infectious, wild type HIV-1 must be performed under strict BSL-3 practice. We have employed a defective (deltaTat/Rev)MC99 and cloned 1A2 line, ie, mutated HIV-1 and Tat/Rev transfected cells to verify anti-HIV-1 activity in a BSL-2 laboratory. A number of extracts from various parts of 11 species of plants were studied. Results were correlated with those of an anti-HIV-1 reverse transcriptase (RT) assay.

Antitumor activity of triptolide against cholangiocarcinoma growth in vitro and in hamsters.

One of the diverse biological activities of triptolide, a diterpene from Tripterygium wilfordii, is its antitumor effect. We recently reported its in vitro cytotoxicity against several cultured tumor cell lines. Limited availability of purified fraction has prevented detailed investigation on its antitumor activity. In the present study, we showed by in vitro cytotoxicity assay and in vivo inhibition of tumor growth in hamsters that the triptolide was also highly effective against cholangiocarcinoma, a highly fatal tumor predominantly occurring in developing countries. Its ED50 for these hamster cholangiocarcinoma cell lines was found to be as low as 0.05 microg/ml. The compound was highly potent in the induction of apoptotic death in these tumor cells. DNA fragmentation and disintegrating apoptotic cells could be observed within 24 h of exposure to 0.5 microg/ml triptolide. The compound was tested against the growth of cholangiocarcinoma in a hamster model. A significant growth inhibition (P < 0.05) was noted in triptolide-treated hamsters (each of the 10 animals received 10 injections for a total of 1.2 mg/animal). At the time of sacrifice 1 month after the initial injection, the mean tumor mass of the treated group was only 20-25% of that of the control group.

Evaluation of the mutagenic, cytotoxic, and antitumor potential of triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii.

Triptolide, a highly oxygenated diterpene isolated from Tripterygium wilfordii Hook f. (Celastraceae), has been shown to demonstrate potent antileukemic activity in rodent models at remarkably low treatment doses. A variety of other physiological responses are known to be mediated by this compound, including immunosuppressive and antifertility effects. We currently report that triptolide was not mutagenic toward Salmonella typhimurium strain TM677, either in the presence or absence of a metabolic activating system. Relatively potent but non-specific cytotoxicity was observed with a panel of cultured mammalian cell lines, and modest antitumor activity was observed when an i.p. dose of 25 microg was administered three times weekly to athymic mice carrying human breast tumors. Treatment regimens involving higher doses of triptolide (e.g. 50 microg/mouse three times weekly) were lethal.

Topical antiinflammatory activity of the major lipophilic constituents of the rhizome of Zingiber cassumunar. Part II: Hexane extractives.

A hexane extract of the rhizome of Zingiber cassumunar was found to exhibit topical antiinflammatory activity, when tested in the model of 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in rats (ID(50) = 854 µg/ear). Bioassay-guided fractionation (by MPLC on silica gel) of the hexane extract led to the isolation and identification of (E)-4-(3',4'-dimethoxyphenyl)but-3-enyl acetate (1), cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3,‴,4‴- dimethoxystyryl]cyclohex-l-ene (2), cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-2‴,4‴,5‴- trimethoxystyryl]cyclohex-1-ene (3), cis-3-(2',4',5'-trimethoxyphenyl)-4-[(E)-2‴,4‴,5‴- trimethoxystyryl]cyclohex-l-ene (4) and (E)-4-(3'-4'-dime-thoxyphenyl)but-3-en-l-ol (5). Compounds 1-5 exerted potent topical antiinflammatory activities with ID(50)-values of 62, 21, 20, 2 and 47µg/ear, respectively. The ID(50) of the reference drug diclofenac was determined to be 61 µg/ear.