Tubuloside B, a major constituent of Cistanche deserticola, inhibits migration of hepatocellular carcinoma by inhibiting Hippo-YAP pathway.

BACKGROUND: Studies have shown that phenylethanoid glycosides (PhGs) have multiple pharmacological effects such as anti-inflammatory, hepatoprotective or neuroprotective functions, whereas their anti-tumor effects are rarely studied. Tubuloside B (Tub B) is a PhG isolated from Cistanche deserticola, a traditional Chinese medicine. To date, there is a lack of comprehensive research regarding the biological activity of Tub B. PURPOSE: The subject of the current study was to investigate the anti-hepatocellular carcinoma (HCC) cell activity and the underlying mechanism of Tub B. METHODS: We evaluated the in vitro anti-migratory effect of Tub B by scratch and transwell assays. RNA-seq was employed to identify the differential genes by Tub B. Besides, the functional mechanism of Tub B was investigated by distinct molecular biology techniques including immunofluorescent staining, quantitative PCR, as well as western blot analysis. Subsequently, we utilized Hep3B cells for in vivo metastasis assays through spleen injection and evaluated the anti-migratory effect of Tub B in hepatocellular carcinoma (HCC). RESULTS: Tub B exhibited in vitro and in vivo inhibition of HCC cell migration. Tub B decreased the expression of transcriptional target genes downstream of the Hippo pathway, including CTGF, CYR61, and N-cadherin as determined by RNA-seq. Furthermore, mechanistic studies confirmed that Tub B increased phosphorylation of YAP at S127, which contributes to YAP cytoplasmic localization. Additionally, overexpression of YAP abrogated Tub B-induced inhibition of HCC migration and the mRNA levels of CTGF, CYR61, and N-cadherin. CONCLUSIONS: Taken together, these results illustrated that Tub B demonstrated great potential in inhibiting migration of HCC, and a portion of its impact can be attributed to the modulation of the Hippo-YAP pathway.

Classical famous prescription of Jichuan decoction improved loperamide-induced slow transit constipation in rats through the cAMP/PKA/AQPs signaling pathway and maintained inflammatory/intestinal flora homeostasis.

Introduction: Jichuan decoction (JCD) is a well-known traditional Chinese medicinal formula that moistens the intestines and is widely used for the treatment of constipation in China. However, its effects and mechanisms in alleviating slow transit constipation (STC) in vivo remain unclear. We attempted to demonstrate the effect of JCD, with and without essential oil (VO), on intestinal transit and its underlying molecular mechanisms in rats with loperamide-induced STC. Materials and methods: Water consumption, body weight, fecal water content, time to first melena excretion, and intestinal transit ratio of the animals were measured. 5-Hydroxytryptamine (5-HT), substance P (SP), vasoactive intestinal peptide (VIP), and interleukin-6 (IL-6) levels in the sera of rats were evaluated using ELISA. Hematoxylin and eosin and Periodic Acid-Schiff staining were used to determine intestinal tissue histology, while quantitative real-time PCR, western blotting, and immunohistochemical analysis were used to assess the relative expression levels of cAMP/PKA/AQPs pathway- and inflammation-related proteins. 16 S rDNA sequence analysis of rat feces was used to determine the diversity and abundance of the intestinal flora. Results: The JCD groups showed reduced time to first melena excretion and expression of VIP and IL-6. The JCD groups, specifically JCD + VO groups, showed increased fecal water content, intestinal transit rate, and SP expression. Further, these groups showed improved histological characteristics of the colon, with no significant change in the index of immune organs or morphological characteristics of other organs. In addition, a significant decrease in the activation of the cAMP/PKA/AQPs signaling pathway in the colon tissue was observed in these groups, specifically the JCD + VO groups. Moreover, treatment with JCD, with or without VO, downregulated the expression of inflammatory factors and enriched the diversity of intestinal flora as evidenced by polymorphism analysis and the contents of Bacteroides, Lactobacillus, and Erysipelas, with the JCD + VO groups showing better therapeutic outcomes. Conclusion: JCD improved loperamide-induced STC, and co-administration with VO exhibited better activity than sole JCD therapy. JCD may improve STC by inhibiting the cAMP/PKA/AQPs signaling pathway and maintaining inflammatory/intestinal flora homeostasis.

PLA-HPG based coating enhanced anti-biofilm and wound healing of Shikonin in MRSA-infected burn wound.

Burn wounds are susceptible to bacterial infections, including Methicillin-resistant Staphylococcus aureus (MRSA), which typically form biofilms and exhibit drug resistance. They also have specific feature of abundant exudate, necessitating frequent drug administration. Shikonin (SKN) has been reported to reverse MRSA drug resistance and possesses anti-biofilm and wound healing properties, however, it suffers from drawbacks of low solubility and instability. In this study, we developed PLA-HPG based bioadhesive nanoparticles SKN/BNP, which demonstrated a drug loading capacity of about 3.6%, and exhibited sustained-release behavior of SKN. The aldehyde groups present on the surface of BNP improved the local adhesion of SKN/BNP both in vitro and in vivo, thereby reducing the frequency of drug dosing in exudate-rich burn wounds. BNP alone enhanced proliferation and migration of the fibroblast, while SKN/BNP promoted fibroblast proliferation and migration as well as angiogenesis. Due to its bioadhesive property, BNP directly interacted with biofilm and enhanced the efficacy of SKN against MRSA biofilm in vitro. In a mouse model of MRSA-infected burn wounds, SKN/BNP demonstrated improved anti-biofilm and wound healing efficiency. Overall, our findings suggest that SKN/BNP holds great promise as a novel and effective treatment option for clinical applications in MRSA-infected burn wounds.

Caffeoylquinic acids isolated from Lonicera japonica Thunb. as TAK1 inhibitors protects against LPS plus IFN-γ-stimulated inflammation by interacting with KEAP1-regulated NRF2 activation.

The transforming growth factor-ß-activated kinase 1 (TAK1) phosphorylation promotes inflammation occurrence. Meanwhile, TAK1 directly interacts with KEAP1 and strenghtenes NRF2/HO-1 pathway downregulated-inflammation. Recently, we found that caffeoylquinic acids not only possessed powderful anti-inflammation function, but also attenuated oxidative damage through KEAP1/NRF2 pathway. Whereas it's rarely understood whether the anti-inflammatory activity were regulated by their interaction between TAK1 and NRF2. Herein, 34 caffeoylquinic acids including five new (2, 4-7) were systematically isolated and identified on the basis of spectroscopic evidence from Lonicera japonica Thunb. flower buds. Their inhibitory effects on inflammation induced by LPS plus IFN-γ were exerted substantial NO scavenging activity, and inhibited massive production of inflammatory cytokines and related proteins. Compound 3 (4F5C-QAME) exhibited the best anti-inflammation activity. 4F5C-QAME down-regulated the phosphorylation of TAK1, JNK, and c-JUN, thereby alleviated inflammation stimulated by LPS plus IFN-γ. Meanwhile, 4F5C-QAME could alleviate the interaction between TAK1 and KEAP1, inhibit the ubiquitination degradation of NRF2, activate NRF2/HO-1 signaling pathway, result in the increase in ROS elimination. Furthermore, 4F5C-QAME effectively protected against inflammation through direct inhibition of TAK1 phosphorylation. Based on these findings, 4F5C-QAME directly targeting TAK1 could be represented as a potential drug candidate for preventing/treating inflammatory diseases that regulated NRF2 activation through alleviating the interaction between TAK1 and KEAP1. Moreover, the regulatory mechanism of TAK1 on NRF2 activation under exogenous oxidative stress was revealed for the first time.

Single-cell resolution reveals RalA GTPase expanding hematopoietic stem cells and facilitating of BCR-ABL1-driven leukemogenesis in a CRISPR/Cas9 gene editing mouse model.

BCR-ABL oncogene-mediated Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) is suggested to originate from leukemic stem cells (LSCs); however, factors regulating self-renewal of LSC and normal hematopoietic stem cells (HSCs) are largely unclear. Here, we show that RalA, a small GTPase in the Ras downstream signaling pathway, has a critical effect on regulating the self-renewal of LSCs and HSCs. A RalA knock-in mouse model (RalARosa26-Tg/+) was initially constructed on the basis of the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) assay to analyze normal hematopoietic differentiation frequency using single-cell resolution and flow cytometry. RalA overexpression promoted cell cycle progression and increased the frequency of granulocyte-monocyte progenitors (GMPs), HSCs and multipotent progenitors (MPPs). The uniform manifold approximation and projection (UMAP) plot revealed heterogeneities in HSCs and progenitor cells (HSPCs) and identified the subclusters of HSCs and GMPs with a distinct molecular signature. RalA also promoted BCR-ABL-induced leukemogenesis and self-renewal of primary LSCs and shortened the survival of leukemic mice. RalA knockdown prolonged survival and promoted sensitivity to imatinib in a patient-derived tumor xenograft model. Immunoprecipitation plus single-cell RNA sequencing of the GMP population confirmed that RalA induced this effect by interacting with RAC1. RAC1 inhibition by azathioprine effectively reduced the self-renewal, colony formation ability of LSCs and prolonged the survival in BCR-ABL1-driven RalA overexpression CML mice. Collectively, RalA was detected to be a vital factor that regulates the abilities of HSCs and LSCs, thus facilitating BCR-ABL-triggered leukemia in mice. RalA inhibition serves as the therapeutic approach to eradicate LSCs in CML.

JFD, a Novel Natural Inhibitor of Keap1 Alkylation, Suppresses Intracellular Mycobacterium Tuberculosis Growth through Keap1/Nrf2/SOD2-Mediated ROS Accumulation.

It is an effective strategy to treat tuberculosis by enhancing reactive oxygen species- (ROS-) mediated killing of Mycobacterium tuberculosis in macrophages, but there are no current therapeutic agents targeting this pathway. Honeysuckle has been used as the traditional medicine for tuberculosis treatment for 1500 years. Japoflavone D (JFD) is a novel biflavonoid isolated from Honeysuckle promoting ROS accumulation by Nrf2 pathway in hepatocarcinoma cells. However, its activity to kill M. tuberculosis in macrophages and molecular mechanism has not been reported. Our results showed that JFD enhances the M. tuberculosis elimination by boosting ROS levels in THP-1 cells. Moreover, the massive ROS accumulation activates p38 to induce apoptosis. Notably, the mechanism revealed that JFD suppresses the nuclear transport of Nrf2, thereby inhibiting SOD2 transcription, leading to a large ROS accumulation. Further studies showed that JFD disrupts the Keap1 alkylation at specific residues Cys14, Cys257, and Cys319, which is crucial for Nrf2 activation, thereby interrupts the nuclear transport of Nrf2. In pharmacokinetic study, JFD can stay as the prototype for 24 h in mice and can be excreted in feces without any toxicity. Our data reveal for the first time that a novel biflavonoid JFD as a potent inhibitor of Keap1 alkylation can suppress the nuclear transport of Nrf2. And it is the first research of the inhibitor of Keap1 alkylation. Furthermore, JFD robustly promotes M. tuberculosis elimination from macrophages by inhibiting Keap1/Nrf2/SOD2 pathway, resulting in the ROS accumulation. This work identified Keap1 alkylation as a new drug target for tuberculosis and provides a preliminary basis for the development of antituberculosis lead compounds based on JFD.

Isoginkgetin, a potential CDK6 inhibitor, suppresses SLC2A1/GLUT1 enhancer activity to induce AMPK-ULK1-mediated cytotoxic autophagy in hepatocellular carcinoma.

Isoginkgetin (ISO), a natural biflavonoid, exhibited cytotoxic activity against several types of cancer cells. However, its effects on hepatocellular carcinoma (HCC) cells and mechanism remain unclear. Here, we revealed that ISO effectively inhibited HCC cell proliferation and migration in vitro. LC3-II expression and autophagosomes were increased under ISO treatment. In addition, ISO-induced cell death was attenuated by treatment with chloroquine or knockdown of autophagy-related genes (ATG5 or ULK1). ISO significantly suppressed SLC2A1/GLUT1 (solute carrier family 2 member 1) expression and glucose uptake, leading to activation of the AMPK-ULK1 axis in HepG2 cells. Overexpression of SLC2A1/GLUT1 abrogated ISO-induced autophagy. Combining molecular docking with thermal shift analysis, we confirmed that ISO directly bound to the N terminus of CDK6 (cyclin-dependent kinase 6) and promoted its degradation. Overexpression of CDK6 abrogated ISO-induced inhibition of SLC2A1/GLUT1 transcription and induction of autophagy. Furthermore, ISO treatment significantly decreased the H3K27ac, H4K8ac and H3K4me1 levels on the SLC2A1/GLUT1 enhancer in HepG2 cells. Finally, ISO suppressed the hepatocarcinogenesis in the HepG2 xenograft mice and the diethylnitrosamine+carbon tetrachloride (DEN+CCl4)-induced primary HCC mice and we confirmed SLC2A1/GLUT1 and CDK6 as promising oncogenes in HCC by analysis of TCGA data and human HCC tissues. Our results provide a new molecular mechanism by which ISO treatment or CDK6 deletion promotes autophagy; that is, ISO targeting the N terminus of CDK6 for degradation inhibits the expression of SLC2A1/GLUT1 by decreasing the enhancer activity of SLC2A1/GLUT1, resulting in decreased glucose levels and inducing the AMPK-ULK1 pathway.

Tubuloside B, isolated from Cistanche tubulosa, a promising agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1/2.

Targeting macrophage M1 polarization is a promising strategy with fewer detrimental effects in COVID-19 curation. Phenylethanoid glycosides (PhGs) of Cistanche tubulosa are a botanical drug to possess various anti-inflammation-related functions, such as immunomodulating, hepatoprotective or neuroprotective functions, whereas their anti-inflammatory activity is rarely understood. A search into their anti-inflammatory characteristics led to the isolation of 49 PhGs along with 15 new PhGs. Their inhibitory effects against M1 polarization induced by LPS plus IFN-γ were explored in RAW264.7 macrophages. Of these PhGs, tubuloside B (Tub B) exerted substantial NO scavenging effect both in chemical- and cell-based assays, and it inhibited massive production of cytokines and chemokines. Tub B decreased ERK1/2 phosphorylation via direct binding and inhibited the MAPK signaling pathway. Tub B also directly binded to Mob1 protein, thereby increased the stability and level of Mob1 protein by inhibiting ubiquitinated degradation. Mob1 was pivotal for the anti-inflammatory activity of Tub B, and it acted independently of the canonical Hippo-YAP pathway. Moreover, ERK1/2 and Mob1 also had a synergic effect on modulating the inflammatory response. Finally, these effects of Tub B were verified in mice with LPS-induced systemic inflammatory response syndrome. Taken together, these results indicated that Tub B acted as a promising agent against M1 macrophage activation by synergistically targeting ERK1/2 and Mob1, and that it may potentially be a drug candidate to prevent/treat inflammatory diseases, especially in COVID-19.

Isoginkgetin synergizes with doxorubicin for robust co-delivery to induce autophagic cell death in hepatocellular carcinoma.

Doxorubicin (DOX) widely used in hepatocellular carcinoma (HCC) can induce serious side effects and drug resistance. Herein, we aimed to seek a strategy to improve the efficacy and reduce the side effects of DOX in HCC based on an autophagy inducer drug called isoginkgetin (ISO). The design of multifunctional nanocarriers based on hyaluronic acid-conjugated and manganese-doped mesoporous silica nanoparticles (HM) for the co-delivery of antitumor drugs against HCC provided an effective and promising antitumor strategy. Our results showed that HM@ISO@DOX could efficiently inhibit HCC cell proliferation through activating autophagy through AMPKa-ULK1 pathway. Moreover, intravenous injection of HM@ISO@DOX significantly suppressed HCC tumor progression in nude mouse HCC model. Collectively, our findings revealed an anti-HCC mechanism of HM@ISO@DOX through autophagy and provide an effective therapeutic strategy for HCC. STATEMENT OF SIGNIFICANCE: In our study, we constructed a co-delivery system by loading ISO and DOX in the mesoporous channels of manganese-doped mesoporous silica nanoparticles, which could be further conjugated with hyaluronic acid to obtain HM@ISO@DOX. The nanocarriers had been demonstrated to be biodegradable under the acidic and reducing tumor microenvironment, as well as to possess the tumor targeting capability via the conjugated hyaluronic acid. In addition, HM@ISO@DOX enhanced the therapeutic efficacy against human HCC tumor through the combinatorial therapies of chemotherapeutics, Mn2+-mediated chemodynamic therapeutics and autophagic cell death, which might be achieved through AMPK-ULK1 signaling. This work revealed that such a nanomedicine exhibited superior tumor accumulation and antitumor efficiency against HCC with extremely low systemic toxicity in an autophagy-boosted manner.

Genus Lonicera: New drug discovery from traditional usage to modern chemical and pharmacological research.

BACKGROUND: Lonicera Linn. belonging to the family Caprifoliaceae, the largest genus in the plant family, includes about more than 200 species, which are mainly distributed in northern Africa, North America, Europe and Asia. Some species of this genus have been usually used in traditional Chinese medicine as well as functional foods, cosmetics and other applications, such as L. japonica Thunb. Bioactive components and pharmacological activities of the genus Lonicera plants have received an increasing interest from the scientific community. Thus, a comprehensive and systematic review on their traditional usage in China, chemical components, and their pharmacological properties of their whole plants, bioactive extracts, and bioactive isolates including partial structure-activity relationships from the genus is indispensable. METHODS: Information on genus Lonicera of this systematic electronic literature search was gathered via the published articles, patents, clinical trials website (https://clinicaltrials.gov/) and several online bibliographic databases (PubMed, Sci Finder, Research Gate, Science Direct, CNKI, Web of Science and Google Scholar). The following keywords were used for the online search: Lonicera, phytochemical composition, Lonicerae japonica, Lonicera review articles, bioactivities of Lonicera, anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, anti-diabetic, and clinical trials. This review paper consists of a total of 225 papers covering the Lonicera genus from 1800 to 2021, including research articles, reviews, patents, and book chapters. RESULTS: In this review (1800s-2021), about 420 components from the genus of Lonicera Linn. including 87 flavonoids, 222 terpenoids, 51 organic acids, and other compounds, together with their pharmacological activities including anti-inflammatory, antiviral, antimicrobial, anticancer, hepatoprotective, antioxidant, neuroprotective, antidiabetic, anti-allergic, immunomodulatory effects, and toxicity were summarized. CONCLUSION: The relationship is discussed among their traditional usage, their pharmacological properties, and their chemical components, which indicate the genus Lonicera have a large prospect in terms of new drug exploitation, especially in COVID-19 treatment.

Coumaroyl and feruloyl flavonoid glycosides from the male flowers of Ginkgo biloba L. and their inhibitory activity against α-glucosidase.

Four flavonoid glycosides containing coumaroyl or feruloyl groups were isolated from the male flowers of Ginkgo biloba L., and compounds 3 and 4 were identified as novel compounds. The inhibitory activities against α-glucosidase were investigated by docking studies, in vitro assays and kinetic studies. The docking results showed that all compounds mainly formed hydrogen-bond and π-π-stacking interactions with α-glucosidase. Compound 4 had the lowest binding energy and maximum number of hydrogen bonds. Subsequently, the in vitro assays showed that compound 4 exhibited the strongest inhibitory potency. Finally, the kinetic studies indicated the inhibitory mode of compounds 1-4 against α-glucosidase were mixed types of competitive and non-competitive. Together, these findings suggested that the isolated flavonoid glycosides in this study, especially compound 4, have potential as α-glucosidase inhibitors.

Capturing functional two-dimensional nanosheets from sandwich-structure vermiculite for cancer theranostics.

Clay-based nanomaterials, especially 2:1 aluminosilicates such as vermiculite, biotite, and illite, have demonstrated great potential in various fields. However, their characteristic sandwiched structures and the lack of effective methods to exfoliate two-dimensional (2D) functional core layers (FCLs) greatly limit their future applications. Herein, we present a universal wet-chemical exfoliation method based on alkali etching that can intelligently "capture" the ultrathin and biocompatible FCLs (MgO and Fe2O3) sandwiched between two identical tetrahedral layers (SiO2 and Al2O3) from vermiculite. Without the sandwich structures that shielded their active sites, the obtained FCL nanosheets (NSs) exhibit a tunable and appropriate electron band structure (with the bandgap decreased from 2.0 eV to 1.4 eV), a conductive band that increased from -0.4 eV to -0.6 eV, and excellent light response characteristics. The great properties of 2D FCL NSs endow them with exciting potential in diverse applications including energy, photocatalysis, and biomedical engineering. This study specifically highlights their application in cancer theranostics as an example, potentially serving as a prelude to future extensive studies of 2D FCL NSs.

The Anti-inflammatory Effects of Lignan Glycosides from Cistanche tubulosa stems on LPS/IFN-γ-induced RAW264.7 Macrophage Cells via PI3K/ AKT Pathway.

BACKGROUND: Cistanche tubulosa is a tonic in traditional Chinese medicines and has a broad spectrum of biological activity, including anti-inflammatory. However, the anti-inflammatory major constituents of C. tubulosa and their underlying mechanisms are still unknown. OBJECTIVE: The aim of the current study was to explore the separation and structural characterization of lignan glycosides from C. tubulosa (Schenk) Wight., their anti-inflammatory activity and the underlying mechanism. MATERIALS AND METHODS: Fractionation and isolation of the 85% EtOH extract of C. tubulosa (Schenk) Wight. were carried out and the primary ingredients lignan glycosides (1-6) were structurally characterized. CCK8 methods were used to evaluate the cytotoxic effect of lignan glycosides (1-6). Effects of lignan glycosides (1-6) on NO production in LPS/IFN-γ-induced RAW264.7 macrophages cells were measured using Griess reagent by reaction with nitrite. The mRNA expression levels of iNOS, COX-2, IL-1ß, IL-6, TNF-a, and TGF-ß treated RAW264.7 cells with various concentrations (0, 25 and 50 µg/ml) of lignan glycosides (1, 4) in the presence of LPS (10 ng/ml) and IFN-γ (20 ng/ml) for 24 h were analyzed by quantitative RT-PCR. Also, the protein expressions of iNOS, COX-2, PI3K, AKT, p-AKT and ß -actin were determined using Western blot analysis. A molecular docking study was performed to investigate the interactions between the lignan glycosides and the PI3K using Autodock vina 1.1.2 package. RESULTS: Six lignan glycosides (1-6) were isolated from stems of C. tubulosa. Among them, (+)- pinoresinol-4-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranoside (5) and eleutheroside E (6) were firstly isolated from C. tubulosa. Of these lignans, 1 and 4 exhibited pronounced inhibitions on NO production with the values of 33.63 ± 4.78 and 39.28 ± 5.52 % at 50 µg/ml, respectively. Additionally, LPS/IFN-γ-induced expression of inducible Nitric Oxide Synthase (iNOS), Cyclooxygenase-2 (COX-2), Interleukin-1ß (IL-1ß), IL-6, and Tumor Necrosis Factor-a (TNF-a) was significantly suppressed by pre-treatment of 1 and 4 in a dose-dependent manner. While 1 and 4 increased the mRNA levels of anti-inflammatory cytokines (TGF-ß). Furthermore, 1 and 4 significantly inhibited the protein levels of PI3K and p-AKT in a dose-dependent manner. CONCLUSION: Taken together, these results suggest that 1 and 4 play an important role in the attenuation of LPS/IFN-γ-induced inflammatory responses in RAW264.7 cells and that the mechanisms involve down-regulation of the PI3K/AKT pathway.

Si-Wei-Qing-Gan-Tang Improves Non-Alcoholic Steatohepatitis by Modulating the Nuclear Factor-κB Signal Pathway and Autophagy in Methionine and Choline Deficient Diet-Fed Rats.

Si-Wei-Qing-Gan-Tang (SWQGT) is a Chinese medicine formula that is widely used as a folk remedy of herbal tea for the treatment of chronic hepatitis, like non-alcoholic steatohepatitis (NASH), around Ganzhou City (Jiangxi province, China). However, the underlying mechanisms of this formula against NASH are still unknown. This study aimed to explore the effect and mechanisms of SWQGT against NASH. A network pharmacology approach was used to predict the potential mechanisms of SWQGT against NASH. Then a rat model of NASH established by feeding the methionine and choline deficient (MCD) diet was used to verify the effect and mechanisms of SWQGT on NASH in vivo. SWQGT (1 g/kg/d and 3 g/kg/d) were given by intragastric administration. Body weight, liver weight, serum biochemical indicators, liver triglyceride and total cholesterol were all measured. Tumor necrosis factor-α (TNF-α), Interleukin (IL)-1ß, IL-6 levels in the livers were evaluated using ELISA. Hematoxylin and eosin (HE) and Oil Red O staining were used to determine histology, while western blot was used to assess the relative expression levels of the nuclear factor-κB (NF-κB) pathway- and autophagy-related proteins. Functional and pathway enrichment analyses revealed that SWQGT obviously influenced inflammation-related signal pathways in NASH. Furthermore, in vivo experiment showed that SWQGT caused a reduction in liver weight and liver index of MCD diet-fed rats. The formula also helped to reduce hepatomegaly and improve pathological liver changes and hepatic steatosis. SWQGT likewise reduced liver TNF-α, IL-1ß, and IL-6 levels and down-regulated p-NF-κB p65, p-p38 MAPK, p-MEK1/2, p-ERK1/2, p-mTOR, and p62, while up-regulating p-ULK1 and LC3II protein expression levels. SWQGT could improve NASH in MCD diet-fed rats, and this effect may be associated with its down-regulation of NF-κB and activation of autophagy.

4,5-di-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. targets the Keap1/Nrf2 pathway to attenuate H2O2-induced liver oxidative damage in HepG2 cells.

BACKGROUND: 4,5-di-O-caffeoylquinic acid methyl ester (4,5-CQME) is a caffeoylquinic acid (CQA) isolated from Lonicera japonica Thunb., a traditional Chinese medicine. To date, the biological activity of 4,5-CQME has not been fully investigated. PURPOSE: The aim of the current study was to explore the anti-oxidative activity and the underlying mechanism of 4,5-CQME. METHODS: MTT assay was used to evaluate the cytoprotective effect of 4,5-CQME. DCFH-DA was used as a fluorescence probe to detect intracellular ROS. The mitochondrial membrane potential was detected using the fluorescent probe JC-1. MDA and GSH levels were measured using MDA and GSH commercial kits, respectively. Apoptosis assay was performed using the Annexin V-FITC/PI method. The functional mechanism of 4,5-CQME was investigated by analyzing relative signaling pathways through immunofluorescent staining, quantitative PCR and western blot analysis. RESULTS: HepG2 cells were incubated with different concentrations of 4,5-CQME for 12 h before exposure to 500 µM H2O2 for 3 h. 4,5-CQME attenuated H2O2-induced oxidative damage and had a higher cytoprotective effect than 3-caffeoylquinic acid, 3-caffeoylquinic acid methyl ester, or 4,5-di-O-caffeoylquinic acid. 4,5-CQME also reduced ROS and MDA levels and rescued GSH depletion. Western blots demonstrated that 4,5-CQME decreased Bax/Bcl-2 and Bak levels. A mechanistic study confirmed that 4,5-CQME significantly suppressed H2O2-induced MAPKs phosphorylation but had little effect on MAPKs phosphorylation under normal conditions. By contrast, 4,5-CQME induced AKT phosphorylation in the presence or absence of H2O2. 4,5-CQME also regulated the Keap1/Nrf2 signaling pathway and enhanced both the mRNA and protein expressions of HO-1 and NQO1. The anti-oxidative effect of 4,5-CQME was greatly abolished by co-incubation with the Nrf2 inhibitor ML385 or PI3K inhibitor wortmannin. CONCLUSIONS: Taken together, these results showed that 4,5-CQME offered significant protection against H2O2-induced oxidative stress, and its effect was in part due to the modulation of the Keap1/Nrf2 pathway.

3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells.

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

Chemical constituents from Lonicera japonica flower buds and their anti-hepatoma and anti-HBV activities.

Three new naturally occurring monoterpenoids, japopenoid A (1), japopenoid B (23) japopenoid C (24), and one new caffeoylquinic acid derivative (28), together with thirty-one known compounds (2-22, 25-27, 29-35), were isolated and identified from the flower buds of Lonicera japonica Thunb. Their structures were determined by extensive 1D and 2D NMR spectroscopic methods, high-resolution mass spectrometry, and the absolute configurations of 1, 23, 24 were determined by comparison of their electronic circular dichroism (ECD) spectrum with literature and theoretical calculation. Structurally, compound 1 is a monoterpenoid featured with an unusual tricyclic skeleton. All compounds (1-35) were evaluated for their cytotoxicities against human liver cancer cell lines (HepG 2 and SMMC-7721). Compound 12 exhibited the most potent activity with IC50 values of 26.54 ±â€¯1.95 and 8.72 ±â€¯1.57 µg/ml against HepG 2 and SMMC-7721, and the IC50 values of compound 13 were 26.54 ±â€¯1.95 and 12.35 ±â€¯1.43 µg/ml, respectively. Western blot results further proved that compound 13 induces hepatoma cell apoptosis via the intrinsic apoptosis pathway. In addition, most terpenoids showed inhibitory activity against HBsAg and HBeAg secretion, and HBV DNA replication. In particular, 25 µg/mlof compound 11 inhibits HBsAg and HBeAg secretion, and HBV DNA replication by 39.39 ±â€¯5.25, 15.64 ±â€¯1.25, and 16.13 ±â€¯4.10% compared to the control (p < 0.05). These results indicated that L. japonica flower buds could be served as functional food for anti-hepatoma and anti-HBV activities.

1H and 13C-NMR data for novel meroterpenoids isolated from Arnebia euchroma (Royle) Johnst.

The data presented in this article are associated with the research article entitled " Meroterpenoids isolated from Arnebia euchroma (Royle) Johnst. and their cytotoxic activity in human hepatocellular carcinoma cells " [1]. The aim of this data was to provide the 1D-NMR spectrum of novel meroterpenoids from Arnebia euchroma (Royle) Johnst.

Effects of a novel biflavonoid of Lonicera japonica flower buds on modulating apoptosis under different oxidative conditions in hepatoma cells.

BACKGROUND: In our previous work, we purified a novel biflavonoid named Japoflavone D (JFD) from Lonicera japonica flower buds. Biflavonoids are chemical compounds characterized by their high levels of antioxidative activity. PURPOSE: The present study aimed to investigate the function and molecular mechanism of JFD under different oxidative conditions in hepatoma cells. METHODS: MTT assay and apoptosis assay were used to evaluate the cytotoxic effect of JFD. The activities of SOD and CAT were detected to evaluate the oxidative level. Oxidative stress was induced by H2O2 stimulation. The molecular mechanism of JFD was investigated by analyzing relative signaling pathway. RESULTS: JFD inhibited cell viability in all hepatoma cell lines we examined. Under quiescent conditions, JFD treatment of SMMC-7721 cells resulted in upregulation of AKT/mTOR signal pathway and ERK activities and downregulation of KEAP1/NRF2/ARE signaling axis, together with apoptosis. However, under oxidative stress, JFD played a quite different role. Treatment of JFD suppressed the activation of ERK and mTOR and activated the KEAP1/NRF2/ARE signaling axis, which is a predominant regulator of cytoprotective responses to oxidative stress, thereby lessening the damage caused by excess reactive oxygen species (ROS). A molecular docking analysis suggested that JFD may interrupt the interaction between KEAP1 and NRF2 by competitively anchoring to the NRF2 binding site on KEAP1. CONCLUSION: The results indicate that JFD functions as a potent antioxidant and plays dual roles in modulating apoptosis under different oxidative conditions. JFD has the potential to be developed as a protective drug for diseases related with excess ROS.