Commiphora myrrha stimulates insulin secretion from ß-cells through activation of atypical protein kinase C and mitogen-activated protein kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse ß-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. AIM OF THE STUDY: To investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from ß-cells were examined. METHODS: The effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. RESULTS: Ca2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5-2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31-8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCß (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. CONCLUSIONS: Our data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.

Alkaloids Extract from Linum usitatissimum Attenuates 12-OTetradecanoylphorbol- 13-Acetate (TPA)-induced Inflammation and Oxidative Stress in Mouse Skin.

BACKGROUND: In traditional medicine, Linum usitatissimum treats inflammatory, gastrointestinal, and cardiovascular diseases. OBJECTIVES: The present study aims to assess the anti-inflammatory and anti-oxidant effects of total alkaloid extract from Linum usitatissimum seeds (ALU) on the ear histological integrity and oxidant- antioxidant status in a mice model of a sub-chronic inflammation induced by multiapplication of TPA. METHODS: Topical TPA treatment induced various inflammatory changes, including edema formation, epidermal thickness, and the excess production of reactive oxygen species. Tissue samples were used for the measurement of reduced glutathione (GSH) and nitric oxide (NO) levels and Myeloperoxidase (MPO) and Catalase (CAT) activities. RESULTS: Oral administration of ALU (50, 100, and 200 mg/kg) produced anti-inflammatory and anti-oxidant effects. Also, ALU significantly reduced ear edema and inflammatory cell infiltration and restored the integrity of the ear. CONCLUSION: These findings suggest that the total alkaloid extract from Linum usitatissimum seeds presents significant anti-inflammatory and anti-oxidant effects on TPA-induced sub-chronic inflammation model in NMRI mice and can be used as an anti-inflammatory and anti-oxidant agent for the therapeutic management of inflammatory disorders.

Salidroside, 8(E)-Nuezhenide, and Ligustroside from Ligustrum japonicum Fructus Inhibit Expressions of MMP-2 and -9 in HT 1080 Fibrosarcoma.

A phenyl ethanoid, salidroside (SAL), and two secoiridoids, 8(E)-nuezhenide (NZD) and ligustroside (LIG), were isolated from fruits of Ligustrumjaponicum, used as traditional folk medicine, and their chemical structures were elucidated by the comparison of spectral data with published literature. Matrix metalloproteinases (MMPs) are major enzymes that play crucial roles in the metastasis and invasive behavior of tumors. In particular, MMP-2 and MMP-9, regulated by the MAPK signaling pathways, including p38, ERK and JNK, are known to play a key role in the degradation of the basement membrane. In the present study, the effects of SAL, NZD and LIG on the expression of MMP-2 and -9 were examined in phorbol 12-myristate 13-acetate (PMA)-induced HT 1080 cells. All the compounds significantly lowered the amount of MMP-2 and MMP-9 released, as determined by gelatin zymography and ELISA. In addition, the mRNA and protein expression levels of MMP-2 and MMP-9 were significantly suppressed, as measured by RT-PCR and Western blotting. According to the Western blotting assay, SAL and LIG effectively reduced the expression of MMP-2 in a dose-dependent manner. NZD lowered the expression of MMP-9 in a similar way. The phosphorylation of p38, ERK and JNK was also significantly suppressed by these compounds. These findings suggest that all the compounds regulate the release and expression of MMP-2 and MMP-9 via MAPK signaling pathways.

Effect of Fractions from Lycopus lucidus Turcz. Leaves on Genomic DNA Oxidation and Matrix Metalloproteinase Activity.

AIMS AND OBJECTIVE: We investigated the inhibitory effects of fractions from Lycopus lucidus Turcz. leaves on genomic DNA oxidation, Nitric Oxide (NO) production, and Matrix Metalloproteinase (MMP) activity. MATERIALS AND METHODS: Oxidative damage of genomic DNA was detected after Fenton reaction with H2O2 using DNA electrophoresis. Western blotting was performed to compare the expression levels of MMP-2 in phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Lipopolysacchride (LPS)-induced NO production in RAW 264.7 cells was measured using Griess reagent. RESULTS: All fractions (n-Hexane, 85% aq. MeOH, n-BuOH, and water fractions) from the leaves of L. lucidus Turcz. significantly inhibited intracellular production of reactive oxygen species (ROS) (p<0.05). Particularly, 85% aq. MeOH and n-BuOH fractions showed higher ROS inhibitory activity than the other fractions. n-Hexane, 85% aq. MeOH, n-BuOH and water (0.05 mg/mL) fractions significantly inhibited oxidative DNA damage by 57.97%, 68.48%, 58.97%, and 68.39%, respectively (p <0.05). Treatment of RAW 264.7 cells with each fraction reduced LPS-induced NO production in a dose-dependent manner (p<0.05). n-Hexane and 85% aq. MeOH fractions notably reduced MMP-2 secretion levels in the culture supernatants from HT-1080 cells. CONCLUSION: Overall, these results indicated that L. lucidus Turcz. leaves can be exploited as plant based sources of antioxidants in the pharmaceutical, cosmetic, nutraceutical, and food industries.

The Trace Element Selenium Is Important for Redox Signaling in Phorbol Ester-Differentiated THP-1 Macrophages.

Physiological selenium (Se) levels counteract excessive inflammation, with selenoproteins shaping the immunoregulatory cytokine and lipid mediator profile. How exactly differentiation of monocytes into macrophages influences the expression of the selenoproteome in concert with the Se supply remains obscure. THP-1 monocytes were differentiated with phorbol 12-myristate 13-acetate (PMA) into macrophages and (i) the expression of selenoproteins, (ii) differentiation markers, (iii) the activity of NF-κB and NRF2, as well as (iv) lipid mediator profiles were analyzed. Se and differentiation affected the expression of selenoproteins in a heterogeneous manner. GPX4 expression was substantially decreased during differentiation, whereas GPX1 was not affected. Moreover, Se increased the expression of selenoproteins H and F, which was further enhanced by differentiation for selenoprotein F and diminished for selenoprotein H. Notably, LPS-induced expression of NF-κB target genes was facilitated by Se, as was the release of COX- and LOX-derived lipid mediators and substrates required for lipid mediator biosynthesis. This included TXB2, TXB3, 15-HETE, and 12-HEPE, as well as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Our results indicate that Se enables macrophages to accurately adjust redox-dependent signaling and thereby modulate downstream lipid mediator profiles.

Flavonoids and saponins from Passiflora edulis f. edulis leaves (purple passion fruit) and its potential anti-inflammatory activity.

OBJECTIVES: The objective of this work was to evaluate the anti-inflammatory activity of the aqueous extract, fractions and major compounds, which are isolated and identified from Passiflora edulis f. edulis (purple passion fruit) leaves extract. METHODS: For the isolation of the major compounds, reversed-phase chromatography and normal phase countercurrent chromatography were used. The separation was followed by thin layer chromatography and HPLC-DAD-ELSD. One-dimensional and two-dimensional NMR and ESI-TOF-MS/MS were used for structural elucidation. The anti-inflammatory activity was evaluated on a TPA multiple dose model of skin chronic inflammation in mice. Additionally, myeloperoxidase (MPO) and nitric oxide synthase (NOS) activity assays were performed as possible mechanisms of action studies. KEY FINDINGS AND CONCLUSIONS: The study of the butanolic fraction mainly showed the presence of saponins and flavonoids. Three minor flavonoids were detected; and three known saponins, cyclopassiflosides IX, XI and III were isolated and identified. This is the first unequivocal report of the presence of these compounds in P. edulis f. edulis leaves. The most favourable results of anti-inflammatory activity were obtained for the flavonoid-rich fraction. All the fractions and isolated compounds evaluated, presented high percentages of inhibition of nitric oxide synthase activity.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by Induction of the Affinity Receptor for IgE Internalization.

PURPOSE: Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. METHODS: Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. RESULTS: Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. CONCLUSIONS: Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

Bamboo leave extract ameliorated 12-O-tetradecanoylphorbol-13-acetate (TPA) induced ear inflammation by reducing MAP kinase levels and NF-κB activation in mice model.

Sasa coreana Nakai (SCN) is a medicinal plant commonly used against inflammation. However, the underlined mechanisms against skin inflammation is poorly understood. The present study investigated the effects of SCN leave extract on ear inflammation. To this aim, six-week-old male ICR mice was subjected to 12-O-tetradecanoyl-phorbol-13-acetate induce ear edema, which were then topically treated with the leave extract of SCN. Ear thickness, weight, and morphological changes were recorded to ensure the induction of ear edema. Further, histological analysis and protein expression for inflammatory markers were also recorded to validate the study. Topical treatment with SCN repressed TPA-induced ear edema in a dose-dependent manner. Further, SCN treatment significantly antagonized the protein expression of MAP kinase signaling pathway and reduced the effect of TPA-induced NF-κB activation, sequentially, deactivated its transcriptional targets in a dose-dependent manner. Collectively, the study suggested that SCN could be a useful therapeutic agent against skin inflammation.

Dietary Antioxidants Significantly Reduced Phorbol Myristate Acetate Induced Oxidative Stress of Peripheral Blood Mononuclear Cells of Patients with Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease responsible for maximum human morbidity in modern life, whereas oxidative stress is the ultimate potential biomarker for determining disease activity in patients with RA. OBJECTIVE: The present study scientifically validated the effectiveness of antioxidants commonly present in different food supplements to neutralize the free radicals mediated oxidative stress in isolated peripheral blood mononuclear lymphocytes (PBML) of patients with RA. METHODS: The study population included patients with Rheumatoid arthritis, RA (n =15), who fulfilled the American College of Rheumatology criteria for RA. Peripheral blood was collected, and isolated mononuclear lymphocyte cells (PBML) were pretreated with phorbol myristate acetate (PMS) and furthermore, incubated with different concentrations of Naringenin, ß carotene and Nacetyl cysteine (NAC) in an ex vivo condition. The resultant cell lysate was used for further studies for the determination of other oxidative biomarkers. The increase of superoxide and nitric oxide production was observed when PBML was treated PMS. RESULTS: Importantly, the increased oxidative stress was effectively decreased by the selected plantderived compounds ß-carotene and naringenin. CONCLUSION: The study scientifically evaluated the efficacy of the molecules validated by one-way ANOVA, followed by Dunnett's post hoc test of significance. Collectively, our results indicate that both ß carotene and naringenin may be a promising non-toxic food supplement in attenuating the oxidative stress associated pathology in RA, meriting further pharmacological studies on other inflammatory cells like neutrophils.

3'-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis.

BACKGROUND: A natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3'-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear. PURPOSE: The aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism. STUDY DESIGN AND METHODS: This study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated. RESULTS: The results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes. CONCLUSION: This is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.

Tripterine: A Potential Anti-Allergic Compound.

BACKGROUND: Tripterine (TRI), an active monomer in Tripterygium wilfordii, has significant pharmacological activities, such as anti-inflammatory, immunosuppressive and anti-tumor activities. TRI may be used to treat allergic diseases because of its characteristics of immunosuppression. OBJECTIVE: This study aims to explore the anti-allergic effect of TRI. METHODS: It was tested in vivo and in vitro in this study. RESULTS: The results showed that TRI could significantly inhibit histamine release from rat peritoneal mast cells; the inhibitory effect of TRI on histamine release was stronger than that of other known histamine inhibitors such as disodium cromoglyceride. TRI also significantly inhibited systemic anaphylactic shock induced by compound 48/80 and skin allergy induced by IgE, and inhibited the expression of inflammatory factors secreted by Human Mast Cells (HMC-1) induced by Phorbol 12-Myristate 13- Acetate (PMA) and calcium carrier A23187. In the animal model of allergic rhinitis induced by Ovalbumin (OA), the scores of friction, histamine, IgE, inflammatory factors and inflammatory cells decreased after TRI was administered orally or nasally. CONCLUSION: TRI, as an active immunoregulatory factor, has great potential in the treatment of mast cell-mediated allergic diseases.

Phorbol 12-Myristate 13-Acetate Induced Toxicity Study and the Role of Tangeretin in Abrogating HIF-1α-NF-κB Crosstalk In Vitro and In Vivo.

Phorbol 12-myristate 13-acetate (PMA) is a potent tumor promoter and highly inflammatory in nature. Here, we investigated the toxic effects of PMA on different model system. PMA (10 µg) caused chromosomal aberrations on the Allium cepa root tip and induced mitotic dysfunction. Similarly, PMA caused embryonic and larval deformities and a plummeted survivability rate on zebrafish embryo in a dose-dependent manner. Persistently, PMA treatment on immortalized human keratinocyte human keratinocyte (HaCaT) cells caused massive inflammatory rush at 4 h and a drop in cell survivability at 24 h. Concomitantly, we replicated a cutaneous inflammation similar to human psoriasis induced by PMA. Herein, we used tangeretin (TAN), as an antagonist to counteract the inflammatory response. Results from an in vivo experiment indicated that TAN (10 and 30 mg/kg) significantly inhibited PMA stimulated epidermal hyperplasia and intra-epidermal neutrophilic abscesses. In addition, its treatment effectively neutralized PMA induced elevated reactive oxygen species (ROS) generation on in vitro and in vivo systems, promoting antioxidant response. The association of hypoxia-inducible factor 1-alpha (HIF-1α)-nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB) crosstalk triggered by PMA enhanced PKCα-ERK1/2-NF-κB pathway; its activation was also significantly counteracted after TAN treatment. Conclusively, we demonstrated TAN inhibited the nuclear translocation of HIF-1α and NF-κB p65. Collectively, TAN treatment ameliorated PMA incited malignant inflammatory response by remodeling the cutaneous microenvironment.

In vivo and in vitro anti­allergic and anti­inflammatory effects of Dryopteris crassirhizoma through the modulation of the NF­Ä¸B signaling pathway in an ovalbumin­induced allergic asthma mouse model.

Dryopteris crassirhizoma (DC) has a wide range of pharmacological effects, including antibacterial, anti­influenza virus, anti­tumor, anti­reverse transcriptase and antioxidant effects. However, the inhibitory effect of DC on allergic inflammatory response remains unclear; therefore, the current study used an experimental ovalbumin (OVA)­induced allergic asthma mouse model and phorbol myristate acetate (PMA)­ and A23187­stimulated HMC­1 cells to reveal the effects of DC in regulating airway inflammation and its possible mechanism. Allergic asthma was initiated in BALB/c mice via exposure to OVA emulsified in aluminum, on days 1 and 14. Thereafter, the mice were treated with DC or dexamethasone (Dex) orally, before being challenged, from days 15 to 26. Subsequently, the mice were challenged with OVA on days 27, 28 and 29. The results of histological analysis indicated that the administration of DC decreased the number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and suppressed eosinophilic infiltration, mucus production and collagen deposition in the lung tissue. DC treatment increased the level of T helper type 1 (Th1) cytokines (IL­10 and interferon (IFN)­Î³) and decreased the levels Th2 cytokines (IL­4, IL­5 and IL­13) and proinflammatory cytokines (IL­6 and TNF­α). Furthermore, DC treatment inhibited the activation of NF­κB signaling (NF­κB, p­NF­κB, IκB and p­IκB), both in BALF and lung homogenates. Serum levels of total IgE and OVA­specific IgE and IgG1 were significantly lower after DC treatment compared with after OVA treatment. However, the anti­inflammatory effect of OVA­specific IgG2a was higher after DC treatment. In addition, DC treatment attenuated the production of proinflammatory cytokines, including IL­6 and TNF­α, and the activation of NF­κB signaling (NF­κB and p­NF­κB), in PMA and calcium ionophore A23187­stimulated HMC­1 cells. In summary, the current study demonstrated that DC acts a potent anti­allergic and anti­inflammatory drug by modulating the Th1 and Th2 response and reducing the allergic inflammatory reaction in PMA and A23187­stimulated HMC­1 cells via NF­κB signaling in an OVA­induced allergic asthma model.

Molecular Targets of Natural Products for Chondroprotection in Destructive Joint Diseases.

Osteoarthritis (OA) is the most common type of arthritis that occurs in an aged population. It affects any joints in the body and degenerates the articular cartilage and the subchondral bone. Despite the pathophysiology of OA being different, cartilage resorption is still a symbol of osteoarthritis. Matrix metalloproteinases (MMPs) are important proteolytic enzymes that degrade extra-cellular matrix proteins (ECM) in the body. MMPs contribute to the turnover of cartilage and its break down; their levels have increased in the joint tissues of OA patients. Application of chondroprotective drugs neutralize the activities of MMPs. Natural products derived from herbs and plants developed as traditional medicine have been paid attention to, due to their potential biological effects. The therapeutic value of natural products in OA has increased in reputation due to their clinical impact and insignificant side effects. Several MMPs inhibitor have been used as therapeutic drugs, for a long time. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize numerous natural products for the development of MMPs inhibitors in arthritic diseases and describe the major signaling targets that were involved for the treatments of these destructive joint diseases.

Role of PKC in the Regulation of the Human Kidney Chloride Channel ClC-Ka.

The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 µM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.

Dioscorea nipponica Makino suppresses TPA-induced migration and invasion through inhibition of matrix metalloproteinase-9 in human cervical cancer cells.

Dioscorea nipponica Makino has been used for the treatment of chronic bronchitis, rheumatoid arthritis, cough, and asthma. Several studies have established the antitumor effect of D. nipponica Makino extract (DNE). However, no investigations have considered the antimetastatic potential of DNE in cervical cancer cells. The present study examined the effects of DNE on cervical cancer cells treated with 12-O-tetradecanoylphorbol-13-acetate and characterized the possible molecular mechanisms. MTT assay results indicated that DNE exhibited very low cytotoxicity, and DNE significantly reduced the invasion and migration abilities of cervical cancer cells. Gelatin zymography analysis revealed that DNE significantly inhibited matrix metalloproteinase-9 (MMP-9) activity. Reverse transcription-polymerase chain reaction assay results revealed that DNE treatment inhibited the MMP-9 mRNA levels of HeLa and SiHa cells. Western blot results revealed that DNE significantly diminished the ERK1/2 phosphorylation. In conclusion, we revealed that the antimetastatic effects of DNE on cervical cancer cells are due to its inhibition of MMP-9 expression through the ERK1/2 pathway.

Phytochemical and Biological Screening of Oenothera Biennis L. Hydroalcoholic Extract.

Oenothera biennis L. (OB), also commonly known as evening primrose, belongs to the Onagraceae family and has the best studied biological activity of all the members in the family. In therapy, the most frequently used type of extracts are from the aerial part, which are the fatty oils obtained from the seeds and have a wide range of medicinal properties. The aim of this study was to evaluate the phytochemical composition and biological activity of OB hydroalcoholic extract and to provide directions for the antimicrobial effect, antiproliferative and pro-apoptotic potential against A375 melanoma cell line, and anti-angiogenic and anti-inflammatory capacity. The main polyphenols and flavonoids identified were gallic acid, caffeic acid, epicatechin, coumaric acid, ferulic acid, rutin and rosmarinic acid. The total phenolic content was 631.496 µgGAE/mL of extract and the antioxidant activity was 7258.67 µmolTrolox/g of extract. The tested extract had a mild bacteriostatic effect on the tested bacterial strains. It was bactericidal only against Candida spp. and S. aureus. In the set of experimental conditions, the OB extract only manifested significant antiproliferative and pro-apoptotic activity against the A375 human melanoma cell line at the highest tested concentration, namely 60 µg/mL. The migration potential of A375 cells was hampered by the OB extract in a concentration-dependent manner. Furthermore, at the highest tested concentration, the OB extract altered the mitochondrial function in vitro, while reducing the angiogenic reaction, hindering compact tumor formation in the chorioallantoic membrane assay. Moreover, the OB extract elicited an anti-inflammatory effect on the experimental animal model of ear inflammation.

Expanding the anti-inflammatory potential of Moringa oleifera: topical effect of seed oil on skin inflammation and hyperproliferation.

ETHNOPHARMACOLOGICAL RELEVANCE: Popularly used in India and sub-Hymalaian region, Moringa oleifera (Moringaceae) is associated with healing properties demonstrated in its use as treatment of acute and chronic skin diseases. Our study aimed at investigating the effects of M. oleifera seed oil (MOSO) in animal models for inflammatory and hyperproliferative skin conditions. MATERIALS AND METHODS: MOSO was analyzed using gas chromatography/mass spectrometry. The anti-inflammatory and anti-hyperproliferative effects of treatment with either MOSO or oleic acid (OA), its main constituent, was evaluated. Acute and chronic inflammation was induced by applying 12-O-Tetradecanoylphorbol-13-acetate (TPA) and acute inflammation with either Arachidonic Acid (AA) or Phenol onto the ear of Swiss mice. Systemic activity and the influence of glucocorticoid receptors (GC) was also evaluated. RESULTS: Topical application of MOSO and OA inhibited ear edema caused by TPA, and Phenol. Only MOSO inhibited ear edema induced by AA. Neutrophil migration was also inhibited by treatment with MOSO. Topical application of MOSO, but not OA, significantly reduced chronic skin inflammation and epidermal hypertrophy induced by multiple TPA applications. Pre-treatment with GC antagonist mifepristone reversed the anti-inflammatory effect of MOSO and OA on the TPA model. Repeated administration of MOSO show a similar effect to dexamethasone on thymus weight, though MOSO did not present any influence on skin thickness, as well as in the weight of the spleen, adrenal gland and lymph node. CONCLUSION: The results suggest that MOSO is effective as a treatment for skin diseases that rely on keratinocyte hyperproliferation. OA is also effective in acute inflammation. Both MOSO and OA depend on GC activation for anti-inflammatory effect but do not exhibit the same adverse effects seen in topical treatment with dexamethasone. We hereby evidence the use of MOSO as a topical anti-inflammatory agent in inflammatory skin diseases, thus, expanding its therapeutic potential.

Preclinical study of the topical anti-inflammatory activity of Cyperus rotundus L. extract (Cyperaceae) in models of skin inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae) is considered one of the most widely distributed plant species in the world, especially in tropical and subtropical regions. In addition, it is commonly used in India, China and Japan in traditional medicine to treat different diseases, including dermatitis and other skin disorders. AIM OF THE STUDY: To investigate the topical anti-inflammatory activity of C. rotundus rhizome ethanolic extract in models of acute and chronic dermatitis. MATERIALS AND METHODS: Phytochemical analysis was carried out using High-performance liquid chromatography-ultraviolet detection (HPLC/UV) to determine the presence of quercetin and chlorogenic acid in C. rotundus extract. Topical anti-inflammmatory effects of C. rotundus extract were evaluated on arachidonic acid (AA) and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice. Skin biopsies were collected and submitted to histological and enzymatic analysis to evaluate the C. rotundus effect in leukocyte migration into inflamed tissue. Antiproliferative activity of C. rotundus was confirmed by PCNA immunostained cell analysis. Systemic and possible adverse effects of topical treatment with C. rotundus were evaluated by the skin atrophy and same organ weights. In addition, the glucocorticoid receptor (GR) antagonist mifepristone was used to investigate possible GR-mediated mechanisms of action. RESULTS: The phytochemical analysis show that C. rotundus ethanol extract contains 45 µg/g of chlorogenic acid. Topical treatment with C. rotundus extract reduced ear edema and cellular infiltrate in acute and chronic skin inflammation models. Moreover, mice topically treated with C. rotundus exhibited decrease in TPA-induced keratinocyte hyperproliferation. Relevantly, topical treatment with C. rotundus did not caused skin atrophy or changes in lymphoid organ weight. The anti-inflammatory effect of C. rotundus was not influenced by the GR antagonist. CONCLUSION: The results here demonstrate for the first time the topical anti-inflammatory and antiproliferative efficacy of C. rotundus extract, suggesting that the extract could be a potential new therapeutic tool for the treatment of inflammatory skin disorders.

Methanol Extract of Dicranopteris linearis Leaves Attenuate Pain via the Modulation of Opioid/NO-Mediated Pathway.

Dicranopteris linearis leaf has been reported to exert antinociceptive activity. The present study elucidates the possible mechanisms of antinociception modulated by the methanol extract of D. linearis leaves (MEDL) using various mouse models. The extract (25, 150, and 300 mg/kg) was administered orally to mice for 30 min priot to subjection to the acetic acid-induced writhing-, hot plate- or formalin-test to establish the antinociceptive profile of MEDL. The most effective dose was then used in the elucidation of possible mechanisms of action stage. The extract was also subjected to the phytochemical analyses. The results confirmed that MEDL exerted significant (p < 0.05) antinociceptive activity in those pain models as well as the capsaicin-, glutamate-, bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced paw licking model. Pretreatment with naloxone (a non-selective opioid antagonist) significantly (p < 0.05) reversed MEDL effect on thermal nociception. Only l-arginine (a nitric oxide (NO) donor) but not N(ω)-nitro-l-arginine methyl ester (l-NAME; a NO inhibitor) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a specific soluble guanylyl cyclase inhibitor) significantly (p < 0.05) modified MEDL effect on the writhing test. Several polyphenolics and volatile antinociceptive compounds were detected in MEDL. In conclusion, MEDL exerted the opioid/NO-mediated antinociceptive activity, thus, justify D. linearis as a potential source for new analgesic agents development.