Antinociceptive and Anti-Inflammatory Effects of Zerumbone against Mono-Iodoacetate-Induced Arthritis

The fresh rhizome of Zingiber zerumbet Smith (Zingiberaceae) is used as a food flavoring and also serves as a folk medicine as an antipyretic and for analgesics in Taiwan. Zerumbone, a monocyclic sesquiterpene was isolated from the rhizome of Z. zerumbet and is the major active compound. In this study, the anti-inflammatory and antinociceptive effects of zerumbone on arthritis were explored using in vitro and in vivo models. Results showed that zerumbone inhibited inducible nitric oxide (NO) synthase (iNOS), cyclooxygenase (COX)-2 expressions, and NO and prostaglandin E₂ (PGE₂) production, but induced heme oxygenase (HO)-1 expression in a dose-dependent manner in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. When zerumbone was co-treated with an HO-1 inhibitor (tin protoporphyrin (SnPP)), the NO inhibitory effects of zerumbone were recovered. The above results suggest that zerumbone inhibited iNOS and COX-2 through induction of the HO-1 pathway. Moreover, matrix metalloproteinase (MMP)-13 and COX-2 expressions of interleukin (IL)-1β-stimulated primary rat chondrocytes were inhibited by zerumbone. In an in vivo assay, an acetic acid-induced writhing response in mice was significantly reduced by treatment with zerumbone. Furthermore, zerumbone reduced paw edema and the pain response in a mono-iodoacetate (MIA)-induced rat osteoarthritis model. Therefore, we suggest that zerumbone possesses anti-inflammatory and antinociceptive effects which indicate zerumbone could be a potential candidate for osteoarthritis treatment.     

Anti-Inflammatory Components from the Root of Solanum erianthum

Two new norsesquiterpenoids, solanerianones A and B (1–2), together with nine known compounds, including four sesquiterpenoids, (−)-solavetivone (3), (+)-anhydro-β-rotunol (4), solafuranone (5), lycifuranone A (6); one alkaloid, N-trans-feruloyltyramine (7); one fatty acid, palmitic acid (8); one phenylalkanoid, acetovanillone (9), and two steroids, β-sitosterol (10) and stigmasterol (11) were isolated from the n-hexane-soluble part of the roots of Solanum erianthum. Their structures were elucidated on the basis of physical and spectroscopic data analyses. The anti-inflammatory activity of these isolates was monitored by nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells. The cytotoxicity towards human lung squamous carcinoma (CH27), human hepatocellular carcinoma (Hep 3B), human oral squamous carcinoma (HSC-3) and human melanoma (M21) cell lines was also screened by using an MTT assay. Of the compounds tested, 3 exhibited the strongest NO inhibition with the average maximum inhibition (E_max) at 100 μM and median inhibitory concentration (IC₅₀) values of 98.23% ± 0.08% and 65.54 ± 0.18 μM, respectively. None of compounds (1–9) was found to possess cytotoxic activity against human cancer cell lines at concentrations up to 30 μM.     

Activation of Nrf2/HO-1 by Peptide YD1 Attenuates Inflammatory Symptoms through Suppression of TLR4/MYyD88/NF-κB Signaling Cascade

In this study, we investigate the immunomodulatory effects of a novel antimicrobial peptide, YD1, isolated from Kimchi, in both in vitro and in vivo models. We establish that YD1 exerts its anti-inflammatory effects via up-regulation of the Nrf2 pathway, resulting in the production of HO-1, which suppresses activation of the NF-κB pathway, including the subsequent proinflammatory cytokines IL-1β, IL-6, and TNF-α. We also found that YD1 robustly suppresses nitric oxide (NO) and prostaglandin E2 (PGE₂) production by down-regulating the expression of the upstream genes, iNOS and COX-2, acting as a strong antioxidant. Collectively, YD1 exhibits vigorous anti-inflammatory and antioxidant activity, presenting it as an interesting potential therapeutic agent.     

Anti-Inflammatory and Analgesic Effects of Pyeongwisan on LPS-Stimulated Murine Macrophages and Mouse Models of Acetic Acid-Induced Writhing Response and Xylene-Induced Ear Edema

Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance.     

An Amphiprotic Novel Chitosanase from Bacillus mycoides and Its Application in the Production of Chitooligomers with Their Antioxidant and Anti-Inflammatory Evaluation

The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The transformation of chitinous biowaste into valuable bioactive chitooligomers (COS) is one of the most exciting applications of chitosanase. An amphiprotic novel chitosanase from Bacillus mycoides TKU038 using squid pen powder (SPP)-containing medium was retrieved from a Taiwan soil sample, which was purified by column chromatography, and characterized by biochemical protocol. Extracellular chitosanase (CS038) was purified to 130-fold with a 35% yield, and its molecular mass was roughly 48 kDa. CS038 was stable over a wide range of pH values (4–10) at 50 °C and exhibited an optimal temperature of 50 °C. Interestingly, the optimum pH values were estimated as 6 and 10, whereas CS038 exhibited chitosan-degrading activity (100% and 94%, respectively). CS038 had K_m and V_max values of 0.098 mg/mL and 1.336 U/min, separately, using different concentrations of water-soluble chitosan. A combination of the high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer data revealed that the chitosan oligosaccharides obtained from the hydrolysis of chitosan by CS038 comprise oligomers with multiple degrees of polymerization (DP), varying from 3–9, as well as CS038 in an endolytic fashion. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities. The COS activities were dose dependent and correlated to their DP. The COS with high DP exhibited enhanced DPPH radical scavenging capability compared with COS with low DP. Furthermore, the COS exhibited inhibitory behavior on nitric oxide (NO) production in murine RAW 264.7 macrophage cells, which was induced by Escherichia coli O111 lipopolysaccharide (LPS). The COS with low DP possesses a more potent anti-inflammatory capability to decrease NO production (IC₅₀, 76.27 ± 1.49 µg/mL) than that of COS with high DP (IC₅₀, 82.65 ± 1.18 µg/mL). Given its effectiveness in production and purification, acidophilic and alkalophilic properties, stability over ranges of pH values, ability to generate COS, antioxidant activity, and anti-inflammatory, CS038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic.     

Secondary Metabolites of the Endophytic Fungus Lachnum abnorme from Ardisia cornudentata

Fractionation of an EtOAc-soluble fraction of the solid fermentate of an endophytic fungus, Lachnum abnorme Mont. BCRC 09F0006, derived from the endemic plant, Ardisia cornudentata Mez. (Myrsinaceae), resulted in the isolation of three new chromones, lachnochromonins D–F (1–3), one novel compound, lachabnormic acid (4), along with nine known compounds (5–13). Their structures were elucidated by spectroscopic analyses. Alternariol-3,9-dimethyl ether (6) was given the correct data as well as 2D spectral analyses for the first time. This is the first report of the isolation of one unprecedented compound 4 from Lachnum genus, while all known compounds were also found for the first time from Lachnum. The effects of some isolates (3, 4, 7–9, 10, and 13) on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages were also evaluated. Several compounds exhibited weak inhibitory activity on lipopolysaccharide (LPS)-stimulated NO production in RAW 264.7 macrophages.     

Moracin C,A Phenolic Compound Isolated from Artocarpus heterophyllus,Suppresses Lipopolysaccharide-Activated Inflammatory Responses in Murine Raw264.7 Macrophages

Artocarpus heterophyllus, a popular tropical fruit commonly known as the jackfruit tree, is normally planted in subtropical or tropical areas. Since a variety of phytochemicals isolated from A. heterophyllus have been found to possess potently anti-inflammatory, antiviral and antimalarial activities, researchers have devoted much interest to its potential pharmaceutical value. However, the exact mechanism underlying its anti-inflammatory activity is not well characterized. In this study, seven natural products isolated from A. heterophyllus, including 25-Hydroxycycloart-23-en-3-one (HY), Artocarpin (AR), Dadahol A (DA), Morachalcone A (MA), Artoheterophyllin B (AB), Cycloheterophyllin (CY) and Moracin C (MC) were collected. Lipopolysaccharide (LPS)-stimulated inflammatory response in RAW264.7 macrophages were used in this study. Among these compounds, MC significantly inhibited LPS-activated reactive oxygen species (ROS) and nitric oxide (NO) release without marked cytotoxicity. Furthermore, MC effectively reduced LPS stimulated up-regulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and serval pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α)). Mechanistic studies revealed that the anti-inflammatory effect of MC was associated with the activation of the mitogen activated protein kinases (MAPKs) (including p38, ERK and JNK) and nuclear factor-κB (NF-κB) pathways, especially reducing the nuclear translocation of NF-κB p65 subunit as revealed by nuclear separation experiment and confocal microscopy.     

The Constituents of Michelia compressa var. formosana and Their Bioactivities

Phytochemical investigation of the heartwood of Michelia compressa afforded forty-four compounds, which were identified by comparison of experimental and literature analytical and spectroscopic data. Some compounds were evaluated for their anti-inflammatory and anticancer bioactivities. The result showed that soemerine (1) and cyathisterol (2) exhibited significant nitric oxide (NO) inhibition, with IC₅₀ values of 8.5 ± 0.3 and 9.6 ± 0.5 µg/mL, respectively. In addition, liriodenine (3) and oliveroline (4) exhibited cytotoxicity to human nasopharyngeal carcinoma (NPC-TW01), non-small cell lung carcinoma (NCI-H226), T cell leukemia (Jurkat), renal carcinoma (A498), lung carcinoma (A549) and fibrosarcoma (HT1080) cell lines with IC₅₀ values in the range of 15.7–3.68 μM.     

Achillea millefolium L. Essential Oil Inhibits LPS-Induced Oxidative Stress and Nitric Oxide Production in RAW 264.7 Macrophages

Achillea millefolium L. is a member of the Asteraceae family and has been used in folk medicine in many countries. In this study, 19 compounds in A. millefolium essential oil (AM-EO) have been identified; the major components are artemisia ketone (14.92%), camphor (11.64%), linalyl acetate (11.51%) and 1,8-cineole (10.15%). AM-EO can suppress the inflammatory responses of lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages, including decreased levels of cellular nitric oxide (NO) and superoxide anion production, lipid peroxidation and glutathione (GSH) concentration. This antioxidant activity is not a result of increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, but rather occurs as a result of the down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and heme oxygenase-1 (HO-1) expression, thus reducing the inflammatory response. Therefore, AM-EO can be utilized in many applications, including the treatment of inflammatory diseases in the future.     

The Effect of the Aerial Part of Lindera akoensis on Lipopolysaccharides (LPS)-Induced Nitric Oxide Production in RAW264.7 Cells

Four new secondary metabolites, 3α-((E)-Dodec-1-enyl)-4β-hydroxy-5β-methyldihydrofuran-2-one (1), linderinol (6), 4′-O-methylkaempferol 3-O-α-l-(4″-E-p-coumaroyl)rhamnoside (11) and kaempferol 3-O-α-l-(4″-Z-p-coumaroyl) rhamnoside (12) with eleven known compounds—3-epilistenolide D₁ (2), 3-epilistenolide D₂ (3), (3Z,4α,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (4), (3E,4β,5β)-3-(dodec-11-ynylidene)-4-hydroxy-5-methylbutanolide (5), matairesinol (7), syringaresinol (8), (+)-pinoresinol (9), salicifoliol (10), 4″-p-coumaroylafzelin (13), catechin (14) and epicatechin (15)—were first isolated from the aerial part of Lindera akoensis. Their structures were determined by detailed analysis of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Lindera akoensis showed that in vitro anti-inflammatory activity decreases the LPS-stimulated production of nitric oxide (NO) in RAW 264.7 cell, with IC₅₀ values of 4.1–413.8 μM.     

Discovery of Phenolic Glycoside from Hyssopus cuspidatus Attenuates LPS-Induced Inflammatory Responses by Inhibition of iNOS and COX-2 Expression through Suppression of NF-κB Activation

It seems quite necessary to obtain effective substances from natural products against inflammatory response (IR) as there are presently clinical problems regarding accompanying side effects and lowered quality of life. This work aimed to investigate the abilities of hyssopuside (HY), a novel phenolic glycoside isolated from Hyssopus cuspidatus (H. cuspidatus), against IR in lipopolysaccharide (LPS)-induced RAW 264.7 cells and mouse peritoneal macrophages. The results indicated that HY could reduce nitric oxide (NO) production and inhibit the production and secretion of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated macrophages. Moreover, data from the immunofluorescence study showed that HY suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. The Western blot results suggested that HY reversed the LPS-induced degradation of IκB (inhibitor of NF-κB), which is normally required for the activation of NF-κB. Meanwhile, the overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) diminished significantly with the presence of HY in response to LPS stimulation. On the other hand, HY had a negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Moreover, an in silico study of HY against four essential proteins/enzymes revealed that COX-2 was the most efficient enzyme for the interaction, and binding of residues Phe179, Asn351, and Ser424 with HY played crucial roles in the observed activity. The structure analysis indicated the typical characterizations with phenylethanoid glycoside contributed to the anti-inflammatory effects of HY. These results indicated that HY manipulated its anti-inflammatory effects mainly through blocking the NF-κB signal transduction pathways. Collectively, we believe that HY could be a potential alternative phenolic agent for alleviating excessive inflammation in many inflammation-associated diseases.     

Antioxidant,Analgesic, Anti-Inflammatory,and Hepatoprotective Effects of the Ethanol Extract of Mahonia oiwakensis Stem

The aim of this study was to evaluate pharmacological properties of ethanol extracted from Mahonia oiwakensis Hayata stems (MOS_EtOH). The pharmacological properties included antioxidant, analgesic, anti-inflammatory and hepatoprotective effects. The protoberberine alkaloid content of the MOS_EtOH was analyzed by high-performance liquid chromatography (HPLC). The results revealed that three alkaloids, berberine, palmatine and jatrorrhizine, could be identified. Moreover, the MOS_EtOH exhibited antioxidative activity using the DPPH assay (IC₅₀, 0.743 mg/mL). The DPPH radical scavenging activity of MOS_EtOH was five times higher that that of vitamin C. MOS_EtOH was also found to inhibit pain induced by acetic acid, formalin, and carrageenan inflammation. Treatment with MOS_EtOH (100 and 500 mg/kg) or silymarin (200 mg/kg) decreased the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the CCl₄-treated group. Histological evaluation showed that MOS_EtOH reduced the degree of liver injury, including vacuolization, inflammation and necrosis of hepatocytes. The anti-inflammatory and hepatoprotective effect of MOS_EtOH were found to be related to the modulation of antioxidant enzyme activity in the liver and decreases in malondialdehyde (MDA) level and nitric oxide (NO) contents. Our findings suggest that MOS_EtOH has analgesic, anti-inflammatory and hepatoprotective effects. These effects support the use of MOS_EtOH for relieving pain and inflammation in folk medicine.     

NF-κB-Targeted Anti-Inflammatory Activity of Prunella vulgaris var. lilacina in Macrophages RAW 264.7

Prunella vulgaris var. lilacina, a herbal medicine, has long been used in Korea for the treatment of sore throat, and to alleviate fever and accelerate wound healing. Although the therapeutic effect of P. vulgaris var. lilacina is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here, we sought to elucidate the possible mechanisms of the anti-inflammatory activity. We have investigated the anti-inflammatory activity of the various solvent fractions (hexane, butanol, chloroform and water) from the ethanol extract of P. vulgaris var. lilacina in activated macrophages. The hexane fraction exhibited higher anti-inflammatory activities, inducing inhibition of nitric oxide and prostaglandin E2 production as well as inducible nitric oxide synthase, cyclooxygenase-2, and tumor necrosis factor-α mRNA expression in response to lipopolysaccharide stimulation. Moreover, the hexane fraction from P. vulgaris var. lilacina significantly inhibited the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and the nuclear translocation of the NF-κB p50 and p65 subunits. These results indicate that P. vulgaris var. lilacina has an anti-inflammatory capacity in vitro, suggesting that it could be a potential source of natural anti-inflammatory agents.     

Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile,Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and βO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18βH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and β-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC₅₀^(24h) = 0.7–4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and βO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and βO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.     

Oleacein Attenuates Lipopolysaccharide-Induced Inflammation in THP-1-Derived Macrophages by the Inhibition of TLR4/MyD88/NF-κB Pathway †

It is known that plant phenolic compounds exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory factors. Recently, Olea europaea has been studied as a natural source of bioactive molecules; however, few studies have focused on the biological effect of oleacein (OLC), the most abundant secoiridoid. Therefore, the aim of this study was to investigate the potential anti-oxidant activity of OLC, as well as to study its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated THP-1-derived macrophages. LPS brought a dramatic increase of both release and gene expression of pro-inflammatory cytokines (IL-6, IL-1β and TNF-α), as well as a decrease of anti-inflammatory ones (IL-10), the effects of which are reverted by OLC. Moreover, it reduced the levels of COX-2, NO and PGE₂ elicited by LPS exposure in THP-1 macrophages. Interestingly, OLC modulated inflammatory signaling pathways through the inhibition of CD14/TLR4/CD14/MyD88 axis and the activation of NF-κB. Finally, OLC showed relevant anti-oxidant capability, assessed by abiotic assays, and reduced the intracellular amount of ROS generated by LPS exposure in THP-1 macrophages. Overall, these results suggest that the anti-oxidant activity and anti-inflammatory effect of OLC may cooperate in its protective effect against inflammatory stressors, thus being a possible alternative pharmacological strategy aimed at reducing the inflammatory process.     

Improved Chemotherapeutic Activity by Morus alba Fruits through Immune Response of Toll-Like Receptor 4

Morus alba L. fruits have long been used in traditional medicine by many cultures. Their medicinal attributes include cardiovascular, hepatoprotective, neuroprotective and immunomodulatory actions. However, their mechanism of macrophage activation and anti-cancer effects remain unclear. The present study investigated the molecular mechanisms of immune stimulation and improved chemotherapeutic effect of M. alba L. fruit extract (MFE). MFE stimulated the production of cytokines, nitric oxide (NO) and tumor necrosis factor-α (TNF-α) and tumoricidal properties of macrophages. MFE activated macrophages through the mitogen-activated protein kinase (MAPKinase) and nuclear factor-κB (NF-κB) signaling pathways downstream from toll-like receptor (TLR) 4. MFE was shown to exhibit cytotoxicity of CT26 cells via the activated macrophages, even though MFE did not directly affect CT26 cells. In a xenograft mouse model, MFE significantly enhanced anti-cancer activity combined with 5-fluorouracil and markedly promoted splenocyte proliferation, natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) activity and IFN-γ production. Immunoglobulin G (IgG) antibody levels were significantly increased. These results indicate the indirect anti-cancer activity of MFE through improved immune response mediated by TLR4 signaling. M. alba L. fruit extract might be a potential anti-tumor immunomodulatory candidate chemotherapy agent.     

GCR1 Positively Regulates UV-B- and Ethylene-Induced Stomatal Closure via Activating GPA1-Dependent ROS and NO Production

Heterotrimeric G proteins function as key players in guard cell signaling to many stimuli, including ultraviolet B (UV-B) and ethylene, but whether guard cell G protein signaling is activated by the only one potential G protein-coupled receptor, GCR1, is still unclear. Here, we found that gcr1 null mutants showed defects in UV-B- and ethylene-induced stomatal closure and production of reactive oxygen species (ROS) and nitric oxide (NO) in guard cells, but these defects could be rescued by the application of a Gα activator or overexpression of a constitutively active form of Gα subunit GPA1 (cGPA1). Moreover, the exogenous application of hydrogen peroxide (H₂O₂) or NO triggered stomatal closure in gcr1 mutants and cGPA1 transgenic plants in the absence or presence of UV-B or ethylene, but exogenous ethylene could not rescue the defect of gcr1 mutants in UV-B-induced stomatal closure, and gcr1 mutants did not affect UV-B-induced ethylene production in Arabidopsis leaves. These results indicate that GCR1 positively controls UV-B- and ethylene-induced stomatal closure by activating GPA1-dependent ROS and NO production in guard cells and that ethylene acts upstream of GCR1 to transduce UV-B guard cell signaling, which establishes the existence of a classic paradigm of G protein signaling in guard cell signaling to UV-B and ethylene.