Five New Phenylpropanoyl Phloroglucinol Derivatives from Leptospermum scoparium.

Leptosperols C-G (1-5), five new phenylpropanoyl phloroglucinol derivatives were isolated from the leaves of Leptospermum scoparium. Compounds 1-3 are phenylpropanoyl phloroglucinol-sesquiterpene adducts with new carbon skeletons. Their structures with absolute configurations were elucidated by detailed spectroscopic analyses, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculation. Compounds 2 and 3 exhibited moderate anti-inflammatory activity in zebrafish acute inflammatory models.

Loading a High-Viscous Droplet via the Cone-Shaped Liquid Bridge Induced by an Electrostatic Force.

In transfer printing, the loaded droplet on the probe has a significant influence on the dispensing resolution. A suitable loading approach for a high-viscous liquid is highly required. Herein, a novel electrostatic loading method is presented, in which the main aim is to control precisely the formation and breaking of a cone-shaped liquid bridge. An experimental device is developed. The influence of electrical and geometric parameters on the feature size of the liquid bridge is investigated in detail. In the formation of the liquid bridge, the increase of voltage or the decrease of the air gap can enhance the electric field intensity, thus reducing the formation period and increasing the initial cone tip diameter of the liquid cone. After the liquid bridge is formed, both the circuit current implying the liquid wetted area on the probe surface and the lifting velocity of the probe are utilized to further regulate the volume of the loaded droplet. Loaded droplets ranging from 60 to 600 pL are obtained via the method with a standard deviation of 4 to 30 pL. Moreover, a dot array is transferred with different loaded droplets. The minimum diameter of the printed dots is about 140 µm with a variation less than 5%. The advantages include the reduced risk of contamination, the droplet-size independent of the size of the probe, and the low cost of the device.

Bufadienolides from the Eggs of the Toad Bufo bufo gargarizans and Their Antimelanoma Activities.

Toads produce potent toxins, named bufadienolides, to defend against their predators. Pharmacological research has revealed that bufadienolides are potential anticancer drugs. In this research, we reported nine bufadienolides from the eggs of the toad Bufo bufo gargarizans, including two new compounds (1 and 3). The chemical structures of 1 and 3, as well as of one previously reported semisynthesized compound (2), were elucidated on the basis of extensive spectroscopic data interpretation, chemical methods, and X-ray diffraction analysis. Compound 1 is an unusual 19-norbufadienolide with rearranged A/B rings. A biological test revealed that compounds 2 and 4-8 showed potent cytotoxic activities toward human melanoma cell line SK-MEL-1 with IC50 values less than 1.0 µM. A preliminary mechanism investigation revealed that the most potent compound, 8, could induce apoptosis via PARP cleavage, while 5 and 6 significantly suppressed angiogenesis in zebrafish. Furthermore, an in vivo biological study showed that 5, 6, and 8 inhibit SK-MEL-1 cell growth significantly.

Monoterpenoid indole alkaloids from the fruits of Gelsemium elegans and their anti-inflammatory activities.

Two novel monoterpenoid indole alkaloids (MIAs), gelsechizines A-B (1-2), along with four known ones (3-6) were isolated from the fruits of Gelsemium elegans. Compound 1 features a new carbon skeleton with two additional carbon atoms forming a 4-methylpyridine unit. Their structures with absolute configurations were elucidated by NMR, MS, X-ray diffraction and electronic circular dichroism (ECD) calculations. Compounds 1-3 showed significant anti-inflammatory effects in vivo and in vitro, which may be related to the inhibition of the trecruitment of neutrophils and macrophages as well as the secretion of TNF-α and IL-6. Preliminary structure-activity relationship analysis revealed that the ß-N-acrylate moiety plays an important role in the anti-inflammatory effect.

Asymmetric Total Synthesis of Bufospirostenin A.

The first and asymmetric total synthesis of bioactive bufospirostenin A, an unusual spirostanol with rearranged A/B rings, was accomplished. The synthetically challenging [5-7-6-5] tetracyclic ring system, found in bufospirostenin A and some other natural products, was efficiently constructed by the unique intramolecular rhodium-catalyzed Pauson-Khand reaction of an alkoxyallene-yne. The 11 stereocenters in the final product, including the 10 contiguous stereocenters, were installed diastereoselectively.

Forsythoside A inhibits adhesion and migration of monocytes to type II alveolar epithelial cells in lipopolysaccharide-induced acute lung injury through upregulating miR-124.

Acute lung injury (ALI) is a severe disease for which effective drugs are still lacking at present. Forsythia suspensa is a traditional Chinese medicine commonly used to relieve respiratory symptoms in China, but its functional mechanisms remain unclear. Therefore, forsythoside A (FA), the active constituent of F. suspensa, was studied in the present study. Inflammation models of type II alveolar epithelial MLE-12 cells and BALB/c mice stimulated by lipopolysaccharide (LPS) were established to explore the effects of FA on ALI and the underlying mechanisms. We found that FA inhibited the production of monocyte chemoattractant protein-1 (MCP-1/CCL2) in LPS-stimulated MLE-12 cells in a dose-dependent manner. Moreover, FA decreased the adhesion and migration of monocytes to MLE-12 cells. Furthermore, miR-124 expression was upregulated after FA treatment. The luciferase report assay showed that miR-124 mimic reduced the activity of CCL2 in MLE-12 cells. However, the inhibitory effects of FA on CCL2 expression and monocyte adhesion and migration to MLE-12 cells were counteracted by treatment with a miR-124 inhibitor. Critically, FA ameliorated LPS-induced pathological damage, decreased the serum levels of tumor necrosis factor-α and interleukin-6, and inhibited CCL2 secretion and macrophage infiltration in lungs in ALI mice. Meanwhile, administration of miR-124 inhibitor attenuated the protective effects of FA. The present study suggests that FA attenuates LPS-induced adhesion and migration of monocytes to type II alveolar epithelial cells though upregulating miR-124, thereby inhibiting the expression of CCL2. These findings indicate that the potential application of FA is promising and that miR-124 mimics could also be used in the treatment of ALI.

[Determination of Aromatic Compounds Metabolites in Human Urine by Solid Phase Extraction-liquid Chromatography-Tandem Mass Spectrometry].

OBJECTIVE: To establish the method based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with solid phase extraction (SPE) for simultaneous determination of the biological metabolites of aromatic compounds, including N-acetyl-S-phenyl-L-cysteine (SPMA), N-acetyl-S-benzyl-cysteine (SBMA), p-nitrophenol (PNP), methylhippuric acids (MHA), p-Aminophenol (PAP), mandelic acid (MA), phenylglyoxylic acid (PGA) and 1-hydroxypyrene (1-OHP) in urine. METHODS: After adding 20 µL of ß-glucuronidase and 1 mL ammonium acetate buffer solution in 1 mL of urine, the sample was digested in a 37 ℃ incubator for 20 h. After digestion, the enzymatic hydrolysate was purified by PRIME HLB solid phase extraction column. The target compounds were eluted with 4 mL of acetonitrile and blown to dryness with nitrogen, reconstituted with 0.20 mL of methanol. Injected the sample solution into LC-MS/MS system for analysis after filtering with 0.22 µm filter membrane. LC separation was carried out on a reversed-phase C18 column (2.1 mm×150 mm, 3.5 µm); gradient eluting was performed at a flow rate of 0.2 mL/min. The water containing 0.1% formic acid was used as mobile phase A and methanol was used as mobile phase B. The mass spectrometry was performed with multiple reaction monitoring (MRM) mode, using alternating positive and negative ions, and internal standard curves were used for quantification. RESULTS: The eight metabolites showed good linearity within the range of 1-100 ng/mL, with a correlation coefficients greater than 0.995, and the relative precision deviation (RSDs) was 0.050%-9.95%. The method detection limits (MDLs) of the eight target metabolites were 0.041-0.12 ng/mL. The proposed method was used for urine sample analysis and the spiked recoveries were 80.1%-114.0%. CONCLUSION: The established method is quick, sensitive and accurate; it meets the requirementof the biological monitoring of aromatic compounds for the general population and occupational population.

Alopecuroides A-E, Matrine-Type Alkaloid Dimers from the Aerial Parts of Sophora alopecuroides.

Five new matrine-type alkaloid dimers, alopecuroides A-E, were isolated from the aerial parts of Sophora alopecuroides. Alopecuroides A and B represent the first dimeric matrine-type alkaloids possessing a cyano group and an epoxy moiety. Alopecuroides C and D are dimeric matrine-type alkaloids connected via C-2-C-9' and C-10-C-3' bonds, respectively. The chemical structures of alopecuroides A-E were elucidated by spectroscopic methods combined with single-crystal X-ray diffraction analysis. The anti-inflammatory effects of alopecuroides A-E were evaluated, and alopecuroide B exhibited the most significant activity, better than that of matrine, the representative compound from S. alopecuroides.

Arenobufagin induces MCF-7 cell apoptosis by promoting JNK-mediated multisite phosphorylation of Yes-associated protein.

BACKGROUND: It has been demonstrated that bufadienolides exert potent anti-cancer activity in various tumor types. However, the mechanisms that underlie their anti-cancer properties remain unclear. Yes-associated protein, a key effector of Hippo signaling, functions as a transcription coactivator, plays oncogenic and tumor suppressor roles under different conditions. Here, we report that arenobufagin (ABF), a representative bufadienolide, induced breast cancer MCF-7 cells to undergo apoptosis, which occurred through the JNK-mediated multisite phosphorylation of YAP. METHODS: Cytotoxicity was examined using an MTT assay. ABF-induced apoptosis was measured with a TUNEL assay and Annexin V-FITC/PI double staining assay. Western blotting, immunofluorescence, qRT-PCR and coimmunoprecipitation were employed to assess the expression levels of the indicated molecules. Lose-of-function experiments were carried out with siRNA transfection and pharmacological inhibitors. ABF-induced phosphopeptides were enriched with Ti4+-IMAC chromatography and further subjected to reverse-phase nano-LC-MS/MS analysis. RESULTS: ABF significantly reduced the viability of MCF-7 cells and increased the percentage of early and late apoptotic cells in a concentration- and time-dependent manner. Following ABF treatment, YAP accumulated in the nucleus and bound to p73, which enhanced the transcription of the pro-apoptotic genes Bax and p53AIP1. YAP knock-down significantly attenuated ABF-induced apoptotic cell death. Importantly, we found that the mobility shift of YAP was derived from its phosphorylation at multiple sites, including Tyr357. Moreover, mass spectrometry analysis identified 19 potential phosphorylation sites in YAP, with a distribution of 14 phosphoserine and 5 phosphothreonine residues. Furthermore, we found that the JNK inhibitor SP600125 completely diminished the mobility shift of YAP and its phosphorylation at Tyr357, the binding of YAP and p73, the transcription of Bax and p53AIP1 as well as the apoptosis induced by ABF. These data indicate that ABF induced YAP multisite phosphorylation, which was associated with p73 binding, and that apoptosis was mediated by the JNK signaling pathway. CONCLUSIONS: Our data demonstrate that ABF suppresses MCF-7 breast cancer proliferation by triggering the pro-apoptotic activity of YAP, which is mediated by JNK signaling-induced YAP multisite phosphorylation as well as its association with p73. The present work not only provides additional information on the use of ABF as an anti-breast cancer drug, but also offers evidence that the induction of the tumor suppressor role of YAP may be a therapeutic strategy.

Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway.

Hepatocellular carcinoma (HCC) is a deadly form of cancer without effective chemotherapy so far. Currently, only sorafenib, a multitargeted tyrosine kinase inhibitor, slightly improves survival in HCC patients. In searching for natural anti-HCC components from toad venom, which is frequently used in the treatment of liver cancer in traditional Chinese medicine, we discovered that arenobufagin, a bufadienolide from toad venom, had potent antineoplastic activity against HCC HepG2 cells as well as corresponding multidrug-resistant HepG2/ADM cells. We found that arenobufagin induced mitochondria-mediated apoptosis in HCC cells, with decreasing mitochondrial potential, as well as increasing Bax/Bcl-2 expression ratio, Bax translocation from cytosol to mitochondria. Arenobufagin also induced autophagy in HepG2/ADM cells. Autophagy-specific inhibitors (3-methyladenine, chloroquine and bafilomycin A1) or Beclin1 and Atg 5 small interfering RNAs (siRNAs) enhanced arenobufagin-induced apoptosis, indicating that arenobufagin-mediated autophagy may protect HepG2/ADM cells from undergoing apoptotic cell death. In addition, we observed the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway by arenobufagin. Interestingly, inhibition of mTOR by rapamycin or siRNA duplexes augmented arenobufagin-induced apoptosis and autophagy. Finally, arenobufagin inhibited the growth of HepG2/ADM xenograft tumors, which were associated with poly (ADP-ribose) polymerase cleavage, light chain 3-II activation and mTOR inhibition. In summary, we first demonstrated the antineoplastic effect of arenobufagin on HCC cells both in vitro and in vivo. We elucidated the underlying antineoplastic mechanisms of arenobufagin that involve cross talk between apoptosis and autophagy via inhibition of the PI3K/Akt/mTOR pathway. This study may provide a rationale for future clinical application using arenobufagin as a chemotherapeutic agent for HCC.

C23 steroids from the venom of Bufo bufo gargarizans.

Five new C23 steroids (1-5) together with five known bufadienolides (6-10) were isolated from the venom of Bufo bufo gargarizans (ChanSu in Chinese). The structures of the new steroids were elucidated by extensive spectroscopic methods in combination with X-ray diffraction analysis. Among these C23 steroids, only compound 3 showed cytotoxicities against HepG2 and A549 cancer cells, with respective IC50 values of 26.8 ± 8.3 and 45.6 ± 2.5 µM. In contrast, the bufadienolides (7-10) displayed potent inhibitory activities against these cancer cells, with respective IC50 values in the ranges 0.5-5.5 and 0.6-6.5 µM, but relatively less cytotoxicity on normal mouse spleen cells. In addition, the Na(+)/K(+)-ATPase inhibitory activities of 2, 5, and 7 revealed that the lactone moiety of a bufadienolide was important for the inhibitory activity.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION: Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

N-Butanol Extract of Glycyrrhizae Radix et Rhizoma Inhibits Dengue Virus through Targeting Envelope Protein.

BACKGROUND: At present, about half of the world's population is at risk of being infected with dengue virus (DENV). However, there are no specific drugs to prevent or treat DENV infection. Glycyrrhizae Radix et Rhizome, a well-known traditional Chinese medicine, performs multiple pharmacological activities, including exerting antiviral effects. The aim of this study was to investigate the anti-DENV effects of n-butanol extract from Glycyrrhizae Radix et Rhizome (GRE). METHODS: Compounds analysis of GRE was conducted via ultra-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The antiviral activities of GRE were determined by the CCK-8 assay, plaque assay, qRT-PCR, Western blotting, and the immunofluorescence assay. The DENV-infected suckling mice model was constructed to explore the antiviral effects of GRE in vivo. RESULTS: Four components in GRE were analyzed by UHPLC-MS/MS, including glycyrrhizic acid, glycyrrhetnic acid, liquiritigenin, and isoliquiritigenin. GRE inhibited the attachment process of the virus replication cycle and reduced the expression of the E protein in cell models. In the in vivo study, GRE significantly relieved clinical symptoms and prolong survival duration. GRE also significantly decreased viremia, reduced the viral load in multiple organs, and inhibited the release of pro-inflammatory cytokines in DENV-infected suckling mice. CONCLUSIONS: GRE exhibited significant inhibitory activities in the adsorption stage of the DENV-2 replication cycle by targeting the envelope protein. Thus, GRE might be a promising candidate for the treatment of DENV infection.

Periplocin inhibits hepatocellular carcinoma progression and reduces the recruitment of MDSCs through AKT/NF-κB pathway.

AIMS: We aimed to evaluate the effect of periplocin on inhibiting hepatocellular carcinoma (HCC) and further determine its mechanisms. MAIN METHODS: Cytotoxic activity of periplocin against HCC cells was tested by CCK-8 and colony formation assays. The antitumor effects of periplocin were evaluated in human HCC SK-HEP-1 xenograft and murine HCC Hepa 1-6 allograft mouse models. Flow cytometry was used to measure cell cycle distribution, apopotosis, and the number of myeloid-derived suppressor cells (MDSCs). Hoechst 33258 dye was applied to observe the nuclear morphology. Network pharmacology was performed to predict possible signaling pathways. Drug affinity responsive target stability assay (DARTS) was used to evaluate AKT binding of periplocin. Western blotting, immunohistochemistry, and immunofluorescence were used to examine the protein expression levels. KEY FINDING: Periplocin inhibited cell viability with IC50 values from 50 nM to 300 nM in human HCC cells. Periplocin disrupted cell cycle distribution and promoted cell apoptosis. Moreover, AKT was predicted as the target of periplocin by network pharmacology, which was confirmed by that AKT/NF-κB signaling was inhibited in periplocin-treated HCC cells. Periplocin also inhibited the expression of CXCL1 and CXCL3, leading to decreased accumulation of MDSCs in HCC tumors. SIGNIFICANCE: These findings reveal the function of periplocin in inhibiting HCC progression by G2/M arrest, apoptosis and suppression of MDSCs accumulation through blockade of the AKT/NF-κB pathway. Our study further suggests that periplocin has the potential to be developed as an effective therapeutic agent for HCC.

Anti-inflammatory, anti-angiogenetic and antiviral activities of dammarane-type triterpenoid saponins from the roots of Panax notoginseng.

Panax notoginseng has been used both as a traditional medicine and as a functional food for hundreds of years in Asia. However, the active constituents from P. notoginseng and their pharmacologic properties still need to be further explored. In this study, one new dammarane-type triterpenoid saponin (1), along with fourteen known analogs (2-15) were isolated and identified from the roots of P. notoginseng. The anti-inflammatory, anti-angiogenetic and anti-dengue virus effects of these isolated compounds were further evaluated. Compounds 1, 3, 5-7 and 10-12 exerted anti-inflammatory effects in two different zebrafish inflammatory models. Among them, 11, with the most significant activities, alleviated the inflammatory response by blocking the MyD88/NF-κB and STAT3 pathways. Moreover, compound 15 showed anti-angiogenetic activities in Tg(fli1:EGFP) and Tg(flk1:GFP) zebrafish, while 3 and 5 only inhibited angiogenesis in Tg(fli1:EGFP) zebrafish. Additionally, compounds 1, 3, 6, 8, 9 and 12 suppressed the replication of dengue virus either at the viral adsorption and entry stages or at the intracellular replication step. In conclusion, these findings enrich knowledge of the diversity of saponins in P. notoginseng and suggest that the dammarane-type triterpenoid saponins from P. notoginseng may be developed as potential functional foods to treat inflammation, angiogenesis or dengue-related diseases.

Emodin Protects Against Lipopolysaccharide-Induced Acute Lung Injury via the JNK/Nur77/c-Jun Signaling Pathway.

Background: Acute lung injury (ALI) is a serious inflammatory disease with clinical manifestations of hypoxemia and respiratory failure. Presently, there is no effective treatment of ALI. Although emodin from Rheum palmatum L. exerts anti-ALI properties, the underlying mechanisms have not been fully explored. Purpose: This study aimed to investigate the therapeutic effect and mechanism of emodin on LPS-induced ALI in mice. Methods: RAW264.7 cells and zebrafish larvae were stimulated by LPS to establish inflammatory models. The anti-inflammatory effect of emodin was assessed by ELISA, flow cytometric analysis, and survival analysis. In vitro mechanisms were explored by using Western blotting, luciferase assay, electrophoretic mobility shift assay (EMSA), and small interfering RNA (siRNA) approach. The acute lung injury model in mice was established by the intratracheal administration of LPS, and the underlying mechanisms were assessed by detecting changes in histopathological and inflammatory markers and Western blotting in lung tissues. Results: Emodin inhibited the inflammatory factor production and oxidative stress in RAW264.7 cells, and prolonged the survival of zebrafish larvae after LPS stimulation. Emodin suppressed the expression levels of phosphorylated JNK at Thr183/tyr182 and phosphorylated Nur77 at Ser351 and c-Jun, and increased the expression level of Nur77 in LPS-stimulated RAW264.7 cells, while these regulatory effects of emodin on Nur77/c-Jun were counteracted by JNK activators. The overexpression of JNK dampened the emodin-mediated increase in Nur77 luciferase activity and Nur77 expression. Moreover, the inhibitory effect of emodin on c-Jun can be attenuated by Nur77 siRNA. Furthermore, emodin alleviated LPS-induced ALI in mice through the regulation of the JNK/Nur77/c-Jun pathway. Conclusions: Emodin protects against LPS-induced ALI through regulation on JNK/Nur77/c-Jun signaling. Our results indicate the potential of emodin in the treatment of ALI.

[Anti-angiogenetic effect of arenobufagin in vitro and in vivo].

This study is to investigate the anti-angiogenetic effect of arenobufagin in vitro and in vivo. The anti-proliferation effect of arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay. Cell morphological changes of LOVO and HUVECs after arenobufagin treatment were observed by microscopy. Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner. Furthermore, it was obviously observed that the subcytotoxic concentration of arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs. Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of arenobufagin in vivo. Arenobufagin significantly suppressed the angiogenesis of CAM. Cell cycle analysis demonstrated that G2/M phase was arrested and the sub-G1 peak appeared with the increase of arenobufagin concentration. PI/Annexin V double staining assay further demonstrated that arenobufagin could induce apoptosis in a dose- and time-dependent manner. Mitochondrial potential collapse detected by flow cytometric analysis was increased after arenobufagin treatment. It also observed that PARP was cleaved to p85 active form by Western blotting. Taken together, arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.