Alismol Purified from the Tuber of Alisma orientale Relieves Acute Lung Injury in Mice via Nrf2 Activation.

Since the ethanol extract of Alisma orientale Juzepzuk (EEAO) suppresses lung inflammation by suppressing Nuclear Factor-kappa B (NF-κB) and activating Nuclear Factor Erythroid 2-related Factor 2 (Nrf2), we set out to identify chemicals constituting EEAO that suppress lung inflammation. Here, we provide evidence that among the five most abundant chemical constituents identified by Ultra Performance Liquid Chromatography (UPLC) and Nuclear Magnetic Resonance (NMR), alismol is one of the candidate constituents that suppresses lung inflammation in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model and protects mice from ALI-like symptoms. Alismol did not induce cytotoxicity or reactive oxygen species (ROS). When administered to the lung of LPS-induced ALI mice (n = 5/group), alismol decreased the level of neutrophils and of the pro-inflammatory molecules, including Tumor Necrosis Factor-alpha (TNF-α), Interleukin-1 beta (IL-1ß), Interleukin-6 (IL-6), Monocyte Chemoattractant Protein-1 (MCP-1), Interferon-gamma (IFN-γ), and Cyclooxygenase-2 (COX-2), suggesting an anti-inflammatory activity of alismol. Consistent with these findings, alismol ameliorated the key features of the inflamed lung of ALI, such as high cellularity due to infiltrated inflammatory cells, the development of hyaline membrane structure, and capillary destruction. Unlike EEAO, alismol did not suppress NF-κB activity but rather activated Nrf2. Consequently, alismol induced the expression of prototypic genes regulated by Nrf2, including Heme Oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO-1), and glutamyl cysteine ligase catalytic units (GCLC). Alismol activating Nrf2 appears to be associated with a decrease in the ubiquitination of Nrf2, a key suppressive mechanism for Nrf2 activity. Together, our results suggest that alismol is a chemical constituent of EEAO that contributes at least in part to suppressing some of the key features of ALI by activating Nrf2.

The ethanol extract of Garcinia subelliptica Merr. induces autophagy.

BACKGROUND: Garcinia subelliptica Merr. is a multipurpose coastal tree, the potential medicinal effects of which have been studied, including cancer suppression. Here, we present evidence that the ethanol extract of G. subelliptica Merr. (eGSM) induces autophagy in human lung adenocarcinoma cells. METHODS: Two different human lung adenocarcinoma cell lines, A549 and SNU2292, were treated with varying amounts of eGSM. Cytotoxicity elicited by eGSM was assessed by MTT assay and PARP degradation. Autophagy in A549 and SNU2292 was determined by western blotting for AMPK, mTOR, ULK1, and LC3. Genetic deletion of AMPKα in HEK293 cells was carried out by CRISPR. RESULTS: eGSM elicited cytotoxicity, but not apoptosis, in A549 and SNU2292 cells. eGSM increased LC3-II production in both A549 and, more extensively, SNU2292, suggesting that eGSM induces autophagy. In A549, eGSM activated AMPK, an essential autophagy activator, but not suppressed mTOR, an autophagy blocker, suggesting that eGSM induces autophagy by primarily activating the AMPK pathway in A549. By contrast, eGSM suppressed mTOR activity without activating AMPK in SNU2292, suggesting that eGSM induces autophagy by mainly suppressing mTOR in SNU2292. In HEK293 cells lacking AMPKα expression, eGSM increased LC3-II production, confirming that the autophagy induced by eGSM can occur without the AMPK pathway. CONCLUSION: Our findings suggest that eGSM induces autophagy by activating AMPK or suppressing mTOR pathways, depending on cell types.

Herbal medicines for anorexia in lung cancer: A protocol for systematic review and meta-analysis.

INTRODUCTION: Lung cancer is the leading cause of cancer-related death worldwide. Anorexia is the most common cause of malnutrition in lung cancer patients as well as an independent prognostic factor for cancer survival. This review will deal with the clinical evidence of herbal medicine use for reducing anorexia in lung cancer patients. METHODS AND ANALYSIS: Fourteen electronic databases will be searched from inception until October 2020. We will include randomized controlled trials (RCTs) assessing herbal medicines for anorexia in lung cancer patients. Interventions of any herbal medicines will be included. The methodological qualities of the included RCTs will be assessed via the Cochrane Collaboration tool for assessing the risk of bias. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) instrument will be used to evaluate the confidence in the cumulative evidence. ETHICS AND DISSEMINATION: This systematic literature review does not require an ethics review. This review will be published in a peer-reviewed journal and disseminated electronically and in print. The review will be updated to inform and guide healthcare practices. REGISTRATION NUMBER: reviewregistry1038.

Sikyungbanha-Tang Suppressing Acute Lung Injury in Mice Is Related to the Activation of Nrf2 and TNFAIP3.

Sikyungbanha-Tang (SKBHT) is a Chinese traditional medicine popularly prescribed to patients with respiratory inflammatory symptoms in Korea. Although the Korea Food and Drug Administration approved SKBHT as a therapeutics for relieving the symptoms, experimental evidence for SKBHT suppressing inflammation is scarce. Here, we presented evidence that SKBHT can suppress inflammation in an acute lung injury (ALI) mouse model and explored the possible underlying mechanisms of SKBHT's anti-inflammatory activity. Single intratracheal (i.t.) injection of SKBHT (1 mg/kg or 10 mg/kg body weight) into mouse lungs decreased prototypic features of lung inflammation found in ALI, such as a high level of proinflammatory cytokines, neutrophil infiltration, and the formation of hyaline membrane, which were induced by a single i.t. LPS (2 mg/kg body weight). When added to a murine macrophage RAW 264.7 cells, SKBHT activated an anti-inflammatory factor Nrf2, increasing the expression of genes regulated by Nrf2. SKBHT suppressed the ubiquitination of Nrf2, suggesting that SKBHT increases the level of and thus activates Nrf2 by blunting the ubiquitin-dependent degradation of Nrf2. SKBHT induced the expression of tumor necrosis factor α-induced protein 3 (TNFAIP3), an ubiquitin-modulating protein that suppresses various cellular signals to NF-κB. Concordantly, SKBHT suppressed NF-κB activity and the expression of inflammatory cytokine genes regulated by NF-κB. Given that Nrf2 and TNFAIP3 are involved in regulating inflammation, our results suggest that SKBHT suppresses inflammation in the lung, the effect of which is related to SKBHT activating Nrf2 and TNFAIP3.

The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice.

BACKGROUND: Guettarda speciosa is mainly found in tropical areas in Asia. Although G. speciosa is traditionally used to treat some of the inflammatory disorders, the experimental evidence supporting the anti-inflammatory effect of G. speciosa is limited. Here, we sought to obtain evidence that G. speciosa has anti-inflammatory activity using an acute lung injury (ALI) mouse model and to explore possible underlying mechanisms for the activity. METHODS: The methanol extract of G. speciosa Linn. (MGS) was fingerprinted by HPLC. Cytotoxicity was determined by MTT and flow cytometer. As for an ALI mouse model, C57BL/6 mice received an intratracheal (i.t.) injection of lipopolysaccharide (LPS). The effects of MGS on lung inflammation in the ALI mice were assessed by differential cell counting and FACS of inflammatory cells and hematoxylin and eosin staining of lung tissue. Proteins were analyzed by immunoprecipitation and immunoblotting, and gene expression was by real-time qPCR. Neutrophil elastase activity was measured by ELISA. RESULTS: MGS did not cause metabolic disarray or produce reactive oxygen species that could induce cytotoxicity. Similar to ALI patients, C57BL/6 mice that received an i.t. LPS developed a high level of neutrophils, increased pro-inflammatory cytokines, and inflicted tissue damage in the lung, which was suppressed by i.t. MGS administered at 2 h after LPS. Mechanistically, MGS activated Nrf2, which was related to MGS interrupting the ubiquitin-dependent degradation of Nrf2. MGS suppressed the nuclear localization of NF-κB induced by LPS, suggesting the inhibition of NF-κB activity. Furthermore, MGS inhibited the enzymatic activity of neutrophil elastase. CONCLUSION: MGS could suppress lung inflammation in an ALI mouse model, the effect of which could be attributed to multiple mechanisms, including the activation of Nrf2 and the suppression of NF-κB and neutrophil elastase enzymatic activity by MGS.

Suppression of lung inflammation by the ethanol extract of Chung-Sang and the possible role of Nrf2.

BACKGROUND: Asian traditional herbal remedies are typically a concoction of a major and several complementary herbs. While balancing out any adverse effect of the major herb, the complementary herbs could dilute the efficacy of the major herb, resulting in a suboptimal therapeutic effect of an herbal remedy. Here, we formulated Chung-Sang (CS) by collating five major herbs, which are used against inflammatory diseases, and tested whether an experimental formula composed of only major herbs is effective in suppressing inflammation without significant side effects. METHODS: The 50% ethanol extract of CS (eCS) was fingerprinted by HPLC. Cytotoxicity to RAW 264.7 cells was determined by an MTT assay and a flow cytometer. Nuclear NF-κB and Nrf2 were analyzed by western blot. Ubiquitinated Nrf2 was similarly analyzed following immunoprecipitation of Nrf2. Acute lung inflammation and sepsis were induced in C57BL/6 mice. The effects of eCS on lung disease were measured by HE staining of lung sections, a differential cell counting of bronchoalveolar lavage fluid, a myeloperoxidase (MPO) assay, a real-time qPCR, and Kaplan-Meier survival of mice. RESULTS: eCS neither elicited cytotoxicity nor reactive oxygen species. While not suppressing NF-κB, eCS activated Nrf2, reduced the ubiquitination of Nrf2, and consequently induced the expression of Nrf2-dependent genes. In an acute lung inflammation mouse model, an intratracheal (i.t.) eCS suppressed neutrophil infiltration, the expression of inflammatory cytokine genes, and MPO activity. In a sepsis mouse model, a single i.t. eCS was sufficient to significantly decrease mouse mortality. CONCLUSIONS: eCS could suppress severe lung inflammation in mice. This effect seemed to associate with eCS activating Nrf2. Our findings suggest that herbal remedies consisting of only major herbs are worth considering.

Anti-inflammatory activity of the decoction of Forsythia suspensa (Thunb.) Vahl is related to Nrf2 and A20.

ETHNOPHARMACOLOGICAL RELEVANCE: The water extract of Forsythiae Fructus (WFF) is an herbal remedy that is prescribed to treat various inflammatory diseases in traditional Chinese medicine. Although the anti-inflammatory activity of WFF has been reported, the underlying mechanisms for the activity remain unclear. Here, we examined whether the anti-inflammatory activity of WFF is associated with Nrf2, an anti-inflammatory factor, and A20, an ubiquitin-regulator protein that inhibits signaling cascades of endotoxin or cytokines. MATERIALS AND METHODS: The water extract of Forsythia suspensa (Thunb.) Vahl was prepared and fingerprinted by HPLC. Cytotoxicity and intracellular ROS induced by WFF were determined by MTT and FACS analyses, respectively. Nuclear and cytoplasmic proteins were analyzed by immunoblot. Expression of mRNA was analyzed by a semi-quantitative RT-PCR. Expression of proteins or genes was quantitated by Image J. RESULTS: WFF activated Nrf2, inducing the expression of Nrf2-dependent genes, such as HO-1, NQO1, and GCLC in RAW 264.7 cells. On the other hand, WFF suppressed NF-κB induced by LPS or TNF-α, which was coincided with the expression of A20. Conversely, WFF failed to suppress NF-κB when A20 expression was silenced by siRNA. CONCLUSION: WFF activated Nrf2 and expressed A20. Given that Nrf2 suppresses inflammation and A20 broadly disrupts inflammatory signaling cascades, our results suggest that the anti-inflammatory activity of WFF is attributable to Nrf2 and A20.

Hominis placenta Suppresses Acute Lung Inflammation by Activating Nrf2.

Hominis placenta (HP), a dried human placenta, has been known to target liver, lung, or kidney meridians, improving the functions associated with these meridians in traditional Chinese or Asian medicine (TCM). Since recent studies implicate an HP extract in suppressing inflammation, we investigated whether an aqueous HP extract can ameliorate inflammation that occurred in the lungs. When administered with a single intratracheal lipopolysaccharide (LPS), C57BL/6 mice developed an acute neutrophilic lung inflammation along with an increased expression of pro-inflammatory cytokine genes. However, this was diminished by the administration HP extract via an intraperitoneal route 2 h after LPS treatment. Western blot and semi-quantitative RT-PCR analyses revealed that while suppressing the activity of a proinflammatory factor NF-[Formula: see text]B marginally, the HP extract strongly activated an anti-inflammatory factor Nrf2, with concomitant expression of Nrf2-dependent genes. Mechanistically, the HP extract suppressed the ubiquitin-mediated degradation of Nrf2, functioning similarly to a 26S proteasome inhibitor, MG132. Collectively, these results suggest that the HP extract suppresses inflammation in mouse lungs, which is in part related to the HP extract perturbing the ubiquitin-dependent degradation of Nrf2 and thus increasing the function of Nrf2.

Differential Regulation of NF-κB and Nrf2 by Bojungikki-Tang Is Associated with Suppressing Lung Inflammation.

Bojungikki-tang (BT), an Asian herbal remedy, has been prescribed to increase the vitality of debilitated patients. Since a compromised, weakened vitality often leads to illness, BT has been widely used to treat various diseases. However, little is known about the mechanism by which BT exerts its effect. Given that BT ameliorates inflammatory pulmonary diseases including acute lung injury (ALI), we investigated whether BT regulates the function of key inflammatory factors such as NF-κB and Nrf2, contributing to suppressing inflammation. Results show that BT interrupted the nuclear localization of NF-κB and suppressed the expression of the NF-κB-dependent genes in RAW 264.7 cells. In similar experiments, BT induced the nuclear localization of Nrf2 and the expression of the Nrf2-dependent genes. In a lipopolysaccharide-induced ALI mouse model, a single intratracheal administration of BT to mouse lungs ameliorated alveolar structure and suppressed the expression of proinflammatory cytokine genes and neutrophil infiltration to mouse lungs. Therefore, our findings suggest that suppression of NF-κB and activation of Nrf2, by which BT suppresses inflammation, are ways for BT to exert its effect.

Suppression of lung inflammation by the methanol extract of Spilanthes acmella Murray is related to differential regulation of NF-κB and Nrf2.

ETHNOPHARMACOLOGICAL RELEVANCE: Although Spilanthes acmella has been used to relieve inflammation, fever, pain, or infection in traditional Asian medicine, experimental evidence supporting these functions is scarce. Here, we examined an anti-inflammatory function and a possible underlying mechanism of S. acmella Murray (SAM). MATERIALS AND METHOD: The methanol extract of SAM was fingerprinted by HPLC. C57BL/6 mice were administered with a single intratracheal (i.t.) LPS and 2 h later with a single i.t. SAM. The effect of SAM on lung inflammation was assessed by histology, semi-quantitative RT-PCR, and MPO assay of lung tissue. The effects of SAM on a pro-inflammatory factor NF-κB and an anti-inflammatory factor Nrf2 were analyzed by immunoblotting of nuclear proteins and by semi-quantitative RT-PCR analysis of mRNA of the genes governed by these transcription factors. V5-Nrf2 was precipitated by an anti-V5 antibody and the ubiquitinated V5-Nrf2 was revealed by immunoblotting of HA-tagged ubiquitin. RESULTS: The i.t. SAM robustly diminished a neutrophilic lung inflammation induced by i.t. LPS treatment of mice. In RAW 264.7 cells, SAM suppressed the nuclear localization of NF-κB and the expression of NF-κB-dependent cytokine genes. SAM increased the level of Nrf2 in the nucleus and the expression of Nrf2-dependent genes while suppressing ubiquitination of Nrf2. CONCLUSION: Our results suggest that SAM can suppress a neutrophilic inflammation in mouse lungs, which is associated with suppressed NF-κB and activated Nrf2. Our results provide experimental evidence supporting the anti-inflammatory function of S. acmella.

Kaurenoic acid activates TGF-ß signaling.

BACKGROUND: Kaurenoic acid (ent-kaur-16-en-19-oic acid: KA) is a key constituent found in the roots of Aralia continentalis Kitagawa (Araliaceae) that has been used for treating rheumatism in traditional Asian medicine. HYPOTHESIS: Although KA was reported to suppress inflammation by activating Nrf2, the anti-inflammatory function of KA is less characterized. Given the complex nature of the inflammatory response and the critical role of TGF-ß in resolving inflammation, we hypothesized that KA suppresses inflammatory response by activating TGF-ß signaling. METHODS: Murine macrophage RAW 264.7, human lung epithelial cell MRC-5, and a TGFßRII defective cell HCT116 were treated with various amounts of KA. KA was also administered to mouse lung via intratracheal (i.t.) route. Phosphorylated Smad2 and Smad3 were analyzed by western blot. TGFß-dependent gene expression was determined by immunoblotting of α-SMA and luciferase assay. RESULTS: KA induced the phosphorylation of Smad2 and Smad3, key activator molecules in TGF-ß signaling. EW7197, an inhibitor for activin receptor-like kinase 5/TGF-ß receptor I (TGFßR1) suppressed KA-mediated phosphorylation of Smad2. Similarly, KA failed to phosphorylate Smad2 in HCT116, suggesting that KA acts through the prototypic TGFßR. KA treatment increased the transcriptional activity driven by a Smad-binding element in a luciferase reporter assay and induced the α-smooth muscle actin (α-SMA). Similarly, i.t. KA induced the phosphorylation of Smad2 and increased the expression ofα-SMA in mouse lungs. CONCLUSION: KA activated TGF-ß signaling, suggesting that TGFß signaling is associated with KA suppressing inflammation.

The function of cancer-shed gangliosides in macrophage phenotype: involvement with angiogenesis.

Tumor-derived gangliosides in the tumor microenvironment are involved in the malignant progression of cancer. However, the molecular mechanisms underlying the effects of gangliosides shed from tumors on macrophage phenotype remain unknown. Here, we showed that ganglioside GM1 highly induced the activity and expression of arginase-1 (Arg-1), a major M2 macrophage marker, compared to various gangliosides in bone marrow-derived macrophages (BMDM), peritoneal macrophages and Raw264.7 macrophage cells. We found that GM1 bound to macrophage mannose receptor (MMR/CD206) and common gamma chain (γc). In addition, GM1 increased Arg-1 expression through CD206 and γc-mediated activation of Janus kinase 3 (JAK3) and signal transducer and activator of transcription- 6 (STAT-6). Interestingly, GM1-stimulated macrophages secreted monocyte chemoattractant protein-1 (MCP-1/CCL2) through a CD206/γc/STAT6-mediated signaling pathway and induced angiogenesis. Moreover, the angiogenic effect of GM1-treated macrophages was diminished by RS102895, an MCP-1 receptor (CCR2) antagonist. From these results we suggest that tumor-shed ganglioside is a secretory factor regulating the phenotype of macrophages and consequently enhancing angiogenesis.

The Fruit Hull of Gleditsia sinensis Enhances the Anti-Tumor Effect of cis-Diammine Dichloridoplatinum II (Cisplatin).

Lung cancer has substantial mortality worldwide, and chemotherapy is a routine regimen for the treatment of patients with lung cancer, despite undesirable effects such as drug resistance and chemotoxicity. Here, given a possible antitumor effect of the fruit hull of Gleditsia sinensis (FGS), we tested whether FGS enhances the effectiveness of cis-diammine dichloridoplatinum (II) (CDDP), a chemotherapeutic drug. We found that CDDP, when administered with FGS, significantly decreased the viability and increased the apoptosis and cell cycle arrest of Lewis lung carcinoma (LLC) cells, which were associated with the increase of p21 and decreases of cyclin D1 and CDK4. Concordantly, when combined with FGS, CDDP significantly reduced the volume and weight of tumors derived from LLC subcutaneously injected into C57BL/6 mice, with concomitant increases of phosphor-p53 and p21 in tumor tissue. Together, these results show that FGS could enhance the antitumor activity of CDDP, suggesting that FGS can be used as a complementary measure to enhance the efficacy of a chemotherapeutic agent such as CDDP.

Therapeutic effect of Mahaenggamseok-tang on neutrophilic lung inflammation is associated with NF-κB suppression and Nrf2 activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Mahaenggamseok-tang (MHGST), an herbal formula in traditional Asian medicine, has been used to treat patients with various pulmonary diseases including common cold and influenza. However, the potential therapeutic effect of MHGST on acute lung injury (ALI), a leading cause of death worldwide, and the anti-inflammatory mechanisms of MHGST remained less understood. MATERIALS AND METHODS: The methanol extract of MHGST was prepared and fingerprinted by HPLC. For the induction of ALI, C57BL/6 mice (n=5/group) received a single intraperitoneal (i.p.) injection of LPS. Referring to the dose for patients, two different amounts of MHGST were delivered in an aerosol to mouse lungs via trachea 2h after the i.p. LPS administration. Lung histology, bronchoalveolar lavage fluid, myeloperoxidase (MPO) activity, and the expression of inflammatory and Nrf2-dependent genes were analyzed to determine the effect of MHGST on lung inflammation. For mechanistic studies, western blotting and semi-quantitative RT-PCR were conducted using RAW 264.7 cells. RESULTS: When administered 2h after the onset of ALI, MHGST relieved lung pathology characteristic to ALI, with decreases of neutrophil infiltration and MPO activity. While suppressing the expression of inflammatory genes, MHGST increased the expression of Nrf2-dependent genes in ALI mouse lungs. Concordantly, MHGST activated Nrf2 activity while suppressing NF-κB in RAW 264.7 cells. CONCLUSION: MHGST suppressed neutrophilic lung inflammation, a hallmark of ALI, which was associated with the activation of anti-inflammatory Nrf2 and the suppression of pro-inflammatory NF-κB. Our results suggest that MHGST has a therapeutic potential against ALI.

Glycosylation enables aesculin to activate Nrf2.

Since aesculin, 6,7-dihydroxycoumarin-6-O-ß-glucopyranoside, suppresses inflammation, we asked whether its anti-inflammatory activity is associated with the activation of nuclear factor-E2-related factor 2 (Nrf2), a key anti-inflammatory factor. Our results, however, show that aesculin marginally activated Nrf2. Since glycosylation can enhance the function of a compound, we then asked whether adding a glucose makes aesculin activate Nrf2. Our results show that the glycosylated aesculin, 3-O-ß-d-glycosyl aesculin, robustly activated Nrf2, inducing the expression of Nrf2-dependent genes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinone oxidoreductase 1 in macrophages. Mechanistically, 3-O-ß-d-glycosyl aesculin suppressed ubiquitination of Nrf2, retarding degradation of Nrf2. Unlike aesculin, 3-O-ß-d-glycosyl aesculin significantly suppressed neutrophilic lung inflammation, a hallmark of acute lung injury (ALI), in mice, which was not recapitulated in Nrf2 knockout mice, suggesting that the anti-inflammatory function of the compound largely acts through Nrf2. In a mouse model of sepsis, a major cause of ALI, 3-O-ß-d-glycosyl aesculin significantly enhanced the survival of mice, compared with aesculin. Together, these results show that glycosylation could confer the ability to activate Nrf2 on aesculin, enhancing the anti-inflammatory function of aesculin. These results suggest that glycosylation can be a way to improve or alter the function of aesculin.

Therapeutic effect of ent-kaur-16-en-19-oic acid on neutrophilic lung inflammation and sepsis is mediated by Nrf2.

Kaurenoic acid (ent-kaur-16-en-19-oic acid: KA) is a key constituent found in the roots of Aralia continentalis Kitagawa (Araliaceae), a remedy to treat patients with inflammatory diseases in traditional Asian medicine. Since KA activates Nrf2, a key anti-inflammatory factor, at the cellular level, we explored a possible therapeutic usage of KA against neutrophilic inflammatory lung disease such as acute lung injury (ALI). Intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) to C57BL/6 mice induced lung inflammation as in ALI. 2 h after i.p. LPS, intratracheal (i.t.) delivery of KA (0.3, 3, or 30 µg/kg body weight) improved lung structure and significantly suppressed neutrophil infiltrations to mouse lungs, with concomitant reduction of myeloperoxidase activity and of the expression of pro-inflammatory cytokine genes. While activating Nrf2 and expressing Nrf2-dependent genes in mouse lungs, KA did not significantly suppress neutrophil lung inflammation in Nrf2 KO mice. In a mouse model of sepsis, a major cause of ALI, single i.t. KA (3 µg/kg) 2 h after the onset of sepsis significantly decreased the mortality of mice. Together, these results suggest that KA has a therapeutic potential against inflammatory lung disease, the effect of which is associated with Nrf2 activation.

Ethanol extract of the tuber of Alisma orientale reduces the pathologic features in a chronic obstructive pulmonary disease mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The tuber of Alismataceae Alisma orientale Juzepzuk has been prescribed as a remedy for treating the diseases associated with body fluid dysfunction such as edema and inflammatory lung diseases. Chronic obstructive pulmonary disease (COPD) is a debilitating, inflammatory lung disease without effective treatment. Along with persistent inflammation, autophagy has been recently reported to contribute to COPD. Here, by employing a murine model, we examined whether the tuber of the plant is effective against COPD MATERIALS AND METHODS: The ethanol extract of the tuber of A. orientale Juzepzuk (EEAO) was fingerprinted by HPLC. For the establishment of COPD lung, mice received single intratracheal (i.t.) spraying of elastase and LPS per week for 2 weeks. After approximated to the dose prescribed typically to patients, EEAO was administered to the lung 2h after each LPS treatment. Morphometric analyses, semi-quantitative RT-PCR, and western blot were performed to evaluate the effects of EEAO on emphysema, inflammation, and autophagy in mouse lungs. The effect of EEAO on autophagy was also assessed by western blot at the cellular level with murine macrophages and human lung epithelial cells. RESULTS: When receiving i.t. elastase and LPS for 2 weeks, mice developed emphysema and inflammation in the lung. EEAO treatment, however, significantly reduced emphysema and inflammatory cell infiltration to the lung with concomitant decrease of the production of pro-inflammatory cytokines including TNF-α, IL-6, and TGF-ß, signature cytokines of COPD. Unlike control mice, the lungs of the COPD mice expressed LC3-II, a biomarker for autophagy formation, which was decreased by EEAO treatment. EEAO also lowered the expression of LC3-II in murine macrophage, RAW 264.7, and human lung epithelial cell, BEAS-2B, which was associated with EEAO activating mTOR. CONCLUSION: EEAO relieved COPD pathologic features in a mouse model, which was associated with suppression of lung inflammation, emphysema, and autophagy. Our results suggest an effectiveness of the tuber of A. orientale in chronic inflammatory lung diseases such as COPD.

Cold stress aggravates inflammatory responses in an LPS-induced mouse model of acute lung injury.

Although the relationship between environmental cold temperature and susceptibility to respiratory infection is generally accepted, the effect of ambient cold temperature on host reactivity in lung inflammation has not been fully studied. To examine the function of ambient cold temperature on lung inflammation, mice were exposed to 4 °C for 8 h each day for 14 days. In the lungs of mice exposed to cold stress, inflammatory cells in bronchoalveolar lavage (BAL) fluid and lung tissues were slightly increased by about twofold. However, the structures of pulmonary epithelial cells were kept within normal limits. Next, we examined the effect of cold stress on the inflammatory responses in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The infiltration of neutrophils and inflammation of lung tissue determined by histology were significantly increased by exposure to ambient cold temperature. In addition, the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-17, and monokine induced by gamma interferon (MIG) was elevated by exposure to cold stress. Therefore, we suggest that cold stress is a factor that exacerbates lung inflammation including ALI. To our knowledge, this is the first report on the relationship between cold stress and severity of lung inflammation.

Neutrophilic Lung Inflammation Suppressed by Picroside II Is Associated with TGF-ß Signaling.

Although acute lung injury (ALI) is a leading cause of death in intensive care unit, effective pharmacologic means to treat ALI patients are lacking. The rhizome of Picrorhiza scrophulariiflora used in a traditional herbal medicine in Asian countries has been shown to have anti-inflammatory function, and picroside II (PIC II) is known as a major constituent in the plant. Here, we examined whether PIC II has an anti-inflammatory activity, which is applicable for treating ALI. We found that although it is not significantly effective in suppressing proinflammatory factor NF-κB or in activating anti-inflammatory factor Nrf2, PIC II induced the phosphorylation of Smad 2, with concomitant increase of luciferase activity from SBE luciferase reporter in RAW 264.7 cells. H&E staining of lung, differential counting of cells in bronchoalveolar lavage fluid, and semiquantitative RT-PCR analyses of lung tissues show that an intratracheal (i.t.) spraying of PIC II suppressed neutrophilic inflammation and the expression of proinflammatory cytokine genes in the lung, which were elicited by an i.t. LPS instillation to the lung. In addition, PIC II treatment increased the phosphorylation of Smad 2 in the lung tissue. Together, our results suggest that PIC II plays a role as an anti-inflammatory constituent in P. scrophulariiflora, whose activity is associated at least in part with TGF-ß signaling.

The inhibitory effects of Geranium thunbergii on interferon-γ- and LPS-induced inflammatory responses are mediated by Nrf2 activation.

Geranium thunbergii Sieb. et Zucc. (GT; which belongs to the Geraniaceae family) has been used as a traditional medicine in East Asia for the treatment of inflammatory diseases, including arthritis and diarrhea. However, the underlying mechanisms of the anti-inflammatory effects of GT remain poorly understood. In the present study, we examined the mechanisms responsible for the anti-inflammatory activity of GT in macrophages. The results revealed that GT significantly inhibited the lipopolysaccharide (LPS)- and interferon-γ (IFN-γ)-induced expression of pro-inflammatory genes, such as inducible nitric oxide synthase, tumor necrosis factor-α and interleukin-1ß, as shown by RT-PCR. However, the inhibitory effects of GT on LPS- and IFN-γ-induced inflammation were associated with an enhanced nuclear factor erythroid 2-related factor 2 (Nrf2) activity, but not with the suppression of nuclear factor (NF)-κB activity, as shown by western blot analysis. In addition, in bone marrow-derived macrophages (BMDM) isolated from Nrf2 knockout mice, GT did not exert any inhibitory effect on the LPS- and IFN-γ-induced inflammation. Taken together, our findings indicate that the anti-inflammatory effects of GT may be associated with the activation of Nrf2, an anti-inflammatory transcription factor.