3',4'-Dihydroxyflavone mitigates inflammatory responses by inhibiting LPS and TLR4/MD2 interaction.

BACKGROUND: We previously reported the potential inhibitory activity of 3',4'-dihydroxyflavone (DHF) on nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated macrophages. PURPOSE: We investigated the underlying molecular mechanisms of DHF in LPS-activated macrophages and evaluated its effect on LPS-induced septic shock in mice. METHODS: To explore the anti-inflammatory effect of DHF, nitrite, PGE2, and cytokines were measured in vitro and in vivo experiments. In addition, to verify the molecular signaling pathway, quantitative real time-PCR, luciferase assay, nuclear extraction, electrophoretic mobility shift assay, immunocytochemistry, immunoprecipitation, molecular docking analysis, and myeloid differentiation 2 (MD2)-LPS binding assay were conducted. RESULTS: DHF suppressed the LPS-induced expression of proinflammatory mediators through nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor 3 (IRF3) inactivation pathways in RAW 264.7 macrophages. Importantly, molecular docking analysis and in vitro binding assays showed that DHF interacts with the hydrophobic pocket of MD2 and then interferes with the interaction between LPS and toll-like receptor 4 (TLR4). DHF inhibited LPS-induced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (Nrf2). Treatment of LPS-induced endotoxemia mice with DHF reduced the expression levels of pro-inflammatory mediators via the inactivation of NF-κB, AP-1, and signal transducer and activator of transcription 1 (STAT1) in the lung tissue, thus increasing the survival rate. CONCLUSION: Taken together, our data first time revealed the underlying mechanism of the DHF-dependent anti-inflammatory effect by preventing LPS from binding to the TLR4/MD2 complex. Therefore, DHF may be a possible anti-inflammatory agent for the treatment of LPS-mediated inflammatory diseases.

Beneficial Activities of Alisma orientale Extract in a Western Diet-Induced Murine Non-Alcoholic Steatohepatitis and Related Fibrosis Model via Regulation of the Hepatic Adiponectin and Farnesoid X Receptor Pathways.

The hepatic adiponectin and farnesoid X receptor (FXR) signaling pathways play multiple roles in modulating lipid and glucose metabolism, reducing hepatic inflammation and fibrosis, and altering various metabolic targets for the management of non-alcoholic fatty liver disease (NAFLD). Alisma orientale (AO, Ze xie in Chinese and Taeksa in Korean) is an herbal plant whose tubers are enriched with triterpenoids, which have been reported to exhibit various bioactive properties associated with NAFLD. Here, the present study provides a preclinical evaluation of the biological functions and related signaling pathways of AO extract for the treatment of NAFLD in a Western diet (WD)-induced mouse model. The findings showed that AO extract significantly reversed serum markers (liver function, lipid profile, and glucose) and improved histological features in the liver sections of mice fed WD for 52 weeks. In addition, it also reduced hepatic expression of fibrogenic markers in liver tissue and decreased the extent of collagen-positive areas, as well as inhibited F4/80 macrophage aggregation and inflammatory cytokine secretion. The activation of adiponectin and FXR expression in hepatic tissue may be a major mechanistic signaling cascade supporting the promising role of AO in NAFLD pharmacotherapy. Collectively, our results demonstrated that AO extract improves non-alcoholic steatohepatitis (NASH) resolution, particularly with respect to NASH-related fibrosis, along with the regulation of liver enzymes, postprandial hyperglycemia, hyperlipidemia, and weight loss, probably through the modulation of the hepatic adiponectin and FXR pathways.

Preclinical Evaluation of In Vitro and In Vivo Antiviral Activities of KCT-01, a New Herbal Formula against Hepatitis B Virus.

Hepatitis B virus (HBV) infectious diseases currently remain incurable due to limitations of conventional antivirals such as incapability of eradicating HBV DNA, prolonged use, drug resistance, and virological relapse. KCT-01, a 30% ethanol extract consisting of Artemisia capillaris, Sanguisorba officinalis, and Curcuma longa, was newly developed. The objective of this study was to investigate pharmacological activities of KCT-01 against HBV using HepG2.2.15 cells and a hydrodynamic injection model. KCT-01 significantly lowered antigen secretion, virion production, and pgRNA synthesis in HepG2.2.15 cells without affecting cell viability. KCT-01 administration also resulted in significant decrease of serum virion production, liver covalently closed circular (ccc) DNA levels, and mRNA synthesis of cytokines in the liver of mice injected with HBV DNA hydrodynamically. Interestingly, coadministration of KCT-01 with entecavir enhanced its in vitro and in vivo antiviral activities. Moreover, safety of KCT-01 was assured up to 5000 mg/kg in rats in both single and repeated-dose preclinical studies. Taken together, our findings demonstrate that KCT-01 is capable of suppressing HBV replication and inflammatory cytokine production in in vitro and in vivo models without showing toxicity, suggesting the potential of using KCT-01 alone or in combination with entecavir as antiviral agent.

Phytotherapeutic Activities of Sanguisorba officinalis and its Chemical Constituents: A Review.

Sanguisorba officinalis Linne (S. officinalis, Rosaceae) has been used as a medicinal plant for the treatment of burns, hematemesis, melena, intestinal infections, and dermatitis for a long time in China, Korea, and Japan. The therapeutic efficacy of this herb is intimately associated with its anti-oxidant, anti-inflammatory, antiviral, antifungal, hemostatic, and anticancer activities. Its root contains triterpenoid saponins (zigyuglycoside I: C[Formula: see text]H[Formula: see text]O[Formula: see text] and ziyuglycoside II: C[Formula: see text]H[Formula: see text]O8) and tannins (sanguiin H-6: C[Formula: see text]H[Formula: see text]O[Formula: see text]). It has been recently revealed that these active constituents of S. officinalis possess antiwrinkle properties without cytotoxicity. They also have anticancer effects by inducing apoptosis and cell cycle arrest. Moreover, they can inhibit proliferative tumorigenesis. The underlying mechanism involved in the pharmacological actions of these active constituents is mainly related to p38 MAPK signaling. Although various studies have reported its therapeutic activities and major chemical components, review articles that extensively organize various properties of S. officinalis and its major constituents are still scarce. Taken together, the objective of this paper is to provide overall pharmacological and phytochemical profiles of S. officinalis and its constituents (including ziyuglycoside I, ziyuglycoside II, and sanguiin H-6), and their potential roles in clinical applications for the treatment of inflammatory diseases, bleeding disorders, and cancer.

Kyungheechunggan-Tang-01, a New Herbal Medication, Suppresses LPS-Induced Inflammatory Responses through JAK/STAT Signaling Pathway in RAW 264.7 Macrophages.

Medicinal plants have been used as alternative therapeutic tools to alleviate inflammatory diseases. The objective of this study was to evaluate anti-inflammatory properties of Kyungheechunggan-tang- (KCT-) 01, KCT-02, and Injinchunggan-tang (IJCGT) as newly developed decoctions containing 3-11 herbs in LPS-induced macrophages. KCT-01 showed the most potent inhibitory effects on LPS-induced NO, PGE2, TNF-α, and IL-6 production among those three herbal formulas. In addition, KCT-01 significantly inhibited LPS-induced iNOS and COX-2 at protein levels and expression of iNOS, COX-2, TNF-α, and IL-6 at mRNA levels. Molecular data revealed that KCT-01 attenuated the activation of JAK/STAT signaling cascade without affecting NF-κB or AP-1 activation. In ear inflammation induced by croton oil, KCT-01 significantly reduced edema, MPO activity, expression levels of iNOS and COX-2, and STAT3 phosphorylation in ear tissues. Taken together, our findings suggest that KCT-01 can downregulate the expression of proinflammatory genes by inhibiting JAK/STAT signaling pathway under inflammatory conditions. This study provides useful data for further exploration and application of KCT-01 as a potential anti-inflammatory medicine.

Evaluation of antitumor activity of Artemisia capillaris extract against hepatocellular carcinoma through the inhibition of IL-6/STAT3 signaling axis.

Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays critical roles in the development and progression of hepatocellular carcinoma (HCC). Artemisia capillaris (AC) has been widely used to treat various liver diseases including HCC as a herbal medicine. The effects of AC on IL-6/STAT3 signaling axis in HCC cells and subsequent anticancer activity of AC against HCC were analyzed using HCC cell lines and HBV W4P-LHB-expressing NIH3T3 cell line, which has been shown to gain tumorigenicity by activating IL-6/STAT3 signaling in our previous study. AC extract significantly suppressed the growth and colony formation of HCC cells. In addition, it inhibited the activation of STAT3 by IL-6 and subsequent synthesis of downstream molecules in HCC and W4P-NIH3T3 cells. Consequently, migration of cells was significantly suppressed by the AC extract. Collectively, the findings suggest that AC extract is capable of conferring various antitumor effects against HCC through the modulation of the IL-6/STAT3 pathway. The results provide a basis for the therapeutic use of AC in the treatment of HCC. Identification of the compound responsible for the effect may lead to the development of a novel anticancer agent against HCC.

A Survey of Therapeutic Effects of Artemisia capillaris in Liver Diseases.

Artemisia capillaris has been recognized as an herb with therapeutic efficacy in liver diseases and widely used as an alternative therapy in Asia. Numerous studies have reported the antisteatotic, antioxidant, anti-inflammatory, choleretic, antiviral, antifibrotic, and antitumor activities of A. capillaris. These reports support its therapeutic potential in various liver diseases such as chronic hepatitis B virus (HBV) infection, cirrhosis, and hepatocellular carcinoma. In addition, several properties of its various constituents, which provide clues to the underlying mechanisms of its therapeutic effects, have been studied. This review describes the scientific evidence supporting the therapeutic potential of A. capillaris and its constituents in various liver diseases.

Synthesis and biological evaluation of N-cyclopropylbenzamide-benzophenone hybrids as novel and selective p38 mitogen activated protein kinase (MAPK) inhibitors.

A series of hybrid molecules consisting of benzophenones and N-cyclopropyl-3-methylbenzamides were synthesized and biologically evaluated as novel p38 mitogen activated protein kinase (MAPK) inhibitors. In particular, we found that compound 10g displayed potent p38α MAPK inhibitory activity (IC50=0.027 µM), high kinase selectivity, and significant anti-inflammatory activity in THP-1 monocyte cells.

Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina.

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.

Antiviral activity of carnosic acid against respiratory syncytial virus.

BACKGROUND: Human respiratory syncytial virus (hRSV) is a leading cause of severe lower respiratory infection and a major public health threat worldwide. To date, no vaccine or effective therapeutic agent has been developed. In a screen for potential therapeutic agents against hRSV, we discovered that an extract of Rosmarinus officinalis exerted a strong inhibitory effect against hRSV infection. Subsequent studies identified carnosic acid as a bioactive constituent responsible for anti-hRSV activity. Carnosic acid has been shown to exhibit potent antioxidant and anti-cancer activities. Anti-RSV activity of carnosic acid was further investigated in this study. METHODS: Effects of extracts from various plants and subfractions from R. officinalis on hRSV replication were determined by microneutralization assay and plaque assay. Several constituents were isolated from ethyl acetate fraction of R. officinalis and their anti-RSV activities were assessed by plaque assay as well as reverse-transcription quantitative PCR to determine the synthesis of viral RNAs. RESULTS: Among the tested bioactive constituents of R. officinalis, carnosic acid displayed the most potent anti-hRSV activity and was effective against both A- and B-type viruses. Carnosic acid efficiently suppressed the replication of hRSV in a concentration-dependent manner. Carnosic acid effectively suppressed viral gene expression without inducing type-I interferon production or affecting cell viability, suggesting that it may directly affect viral factors. A time course analysis showed that addition of carnosic acid 8 hours after infection still effectively blocked the expression of hRSV genes, further suggesting that carnosic acid directly inhibited the replication of hRSV. CONCLUSIONS: The current study demonstrates that carnosic acid, a natural compound that has already been shown to be safe for human consumption, has anti-viral activity against hRSV, efficiently blocking the replication of this virus. Carnosic acid inhibited both A- and B- type hRSV, while it did not affect the replication of influenza A virus, suggesting that its antiviral activity is hRSV-specific. Collectively, this study suggests the need for further evaluation of carnosic acid as a potential treatment for hRSV.

The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors.

Innate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.