Anti-inflammatory Property of Imperatorin on Alveolar Macrophages

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Anti-inflammatory Property of Imperatorin on Alveolar Macrophages and Inflammatory Lung Injury Ya-Zhen Li,†,# Jia-Hong Chen,‡,# Cheng-Fang Tsai,§ and Wei-Lan Yeh*,⊥,∥ †

Department of Biological Science and Technology, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402, Taiwan Department of General Surgery, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung, 42743, Taiwan § Department of Biotechnology, Asia University, No. 500 Lioufeng Road, Taichung, 41354, Taiwan ⊥ Institute of New Drug Development, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402, Taiwan ∥ Research Center for Tumor Medical Science, China Medical University, No. 91 Hsueh-Shih Road, Taichung, 40402, Taiwan J. Nat. Prod. Downloaded from pubs.acs.org by WESTERN UNIV on 03/20/19. For personal use only.

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ABSTRACT: Imperatorin is one of the furanocoumarin derivatives and exists in many medicinal herbs with anticancer, antiviral, antibacterial, and antihypertensive activities. In this study, we examined the anti-inflammatory effects of imperatorin on inflammation-associated lung diseases. Imperatorin reduced iNOS and COX-2 expression and also IL-6 and TNFα production enhanced by zymosan. Imperatorin also inhibited the signaling pathways of JAK/STAT and NF-κB. Moreover, in vivo study also revealed that zymosan-induced immune cell infiltration, pulmonary fibrosis, and edema were relieved by imperatorin in mice. We found that imperatorin exerts anti-inflammatory effects that are associated with amelioration of lung inflammation, edema, and rapid fibrosis. Studies on alveolar macrophages also reveal that imperatorin reduced the production of pro-inflammatory mediators and cytokines and inhibited pro-inflammatory JAK1/STAT3 and NF-κB signaling pathways. These results indicate that imperatorin may be a potential anti-inflammatory agent for inflammatory-associated lung diseases.

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cerevisiae.7,8 When administered into animals, zymosan provokes inflammation by triggering nuclear factor-κB (NFκB) activation, cyclooxygenase 2 (COX-2), and nitric oxide synthase (NOS) expressions and pro-inflammatory cytokine and chemokine production.9,10 Imperatorin is one of the furanocoumarin derivatives and exists in many medicinal herbs.11 Imperatorin has been reported to have many pharmacological properties including anticancer, antiviral, antibacterial, and antihypertensive activities.12−15 Studies have revealed that imperatorin also possesses anti-inflammatory effects against lipopolysaccharide (LPS), rheumatoid arthritis, colitis, and allergic rhinitis.16−19 However, little information is available on the effect of imperatorin against pulmonary inflammation. Therefore, in this study, we examined the anti-inflammatory effects of imperatorin on pulmonary inflammation provoked by zymosan in vitro and in vivo. The manifested therapeutic effect of imperatorin on pulmonary inflammation reveals it as a potential candidate for drug development.

cute lung injury (ALI) and acute respiratory distress syndrome (ARDS) frequently occur in critically ill patients, with high mortality and extensive effects on public health.1 They are the manifestations of inflammatory responses of the lung, which may result from lung infection, sepsis, trauma, or other insults.2 Upregulation of pro-inflammatory cytokines occurs as a direct response and as a marker of ongoing injury.3 ALI and ARDS are characterized by damaged alveolar capillary barrier, edema, pulmonary infiltrates, and hypoxemia, and persistence and progression of the injury can lead to pulmonary vascular destruction, pulmonary fibrosis, and multiple organ failure.4 Hence, exploring innovative therapies and potential medications for ALI is an urgent need. The pivotal event of pulmonary inflammatory response is the activation of alveolar macrophages and recruitment of immune cells into the bronchoalveolar spaces.5 Alveolar macrophages form the first line of defense against external insults and play a key role in lung inflammation initiation, resolution, and tissue repair.6 Among experimental models demonstrated for investigating mechanisms involved in ALI, zymosan is one of the most commonly used substances, which is derived from the cell wall of the yeast Saccharomyces © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 15, 2019

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DOI: 10.1021/acs.jnatprod.9b00145 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Imperatorin attenuated acute lung injury and rapid fibrosis induced by intratracheal administration of zymosan in mice. Lung sections were analyzed by H&E staining (A) and Masson’s trichrome staining (B). H&E staining colors the cytoplasm pink and nuclei blue. Masson’s trichrome staining colors collagen blue, cytoplasm pink, and nuclei dark brown to black. Collagen deposition was also evaluated by the hydroxyproline assay (C). Note that imperatorin treatment ameliorated zymosan-induced hypertrophic smooth muscle cells, thickened basement membrane, and subepithelial collagen deposition/fibrosis. Lung edema induced by zymosan was also ameliorated by imperatorin by detecting the wet/dry ratio (D). Total protein (E) and PGE2 expression (F) in BALF were also analyzed. Immune cells infiltrated in BALF were counted after Diff Quick Staining (G). Graphs showed mean ± SD of at least four independent experiments, and representative pictures are shown in A and B. In both A and B, scale bars in low power fields (upper panels) are 200 μm, and scale bars in high power field (lower panels) are 100 μm. *p < 0.05; **p < 0.01; ***p < 0.001 compared to sham group; ##p < 0.01 compared to zymosan-treated group. Abbreviations: Con, control; Imp, imperatorin; Zym, zymosan. B



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RESULTS AND DISCUSSION Imperatorin Relieves Zymosan-Induced Lung Injury. Among the animal models established for the investigation of the mechanism involved in acute lung injury and fibrosis, intraperitoneal (i.p.) or intratracheal (i.t.) injection of zymosan is one of the most commonly used models.10 It has been reported that acute lung injury causes not only inflammatory damages but also rapid fibrosis, with the first rapid fibrosis stage arising at 7 days and second stage occurring on days 14− 21.20 When injected into animals, zymosan induces inflammation by a series of mechanisms leading to severe organ dysfunction syndrome.21 Lung injury was characterized by alveolar thickening, infiltration of immune cells, and increased permeability of the alveolar capillary membrane.22,23 As shown in Figure 1A, the mice in the sham and imperatorin-treated alone groups showed no morphologic changes after H&E staining, indicating that intraperitoneal administration of saline or imperatorin alone did not induce additional response in this experimental procedure. However, zymosan-stimulated mice had significant immune cell infiltration, hypertrophic smooth muscle cells, and thickened basement membrane, and these phenomena were deteriorated by imperatorin treatment. In addition, collagen deposition resulting from acute fibrosis was also evaluated by Masson’s trichrome staining and a hydroxyproline assay kit.24,25 We found that subepithelial collagen deposition/fibrosis induced by zymosan was mitigated by imperatorin (Figure 1B). Analyzing lung tissue lysates by hydroxyproline assay also revealed that zymosan-induced collagen production was also reduced by imperatorin (Figure 1C). The pathogenesis of lung injury also involves accumulation of protein-rich fluid in the airspaces and pulmonary edema.26 Hence, the lung tissues were obtained and subjected to calculate wet/dry (W/D) ratio, and the collected bronchoalveolar lavage fluid (BALF) was also analyzed. As shown in Figure 1D, the W/D ratio was markedly increased in the zymosan-stimulated group, and the ratio was significantly decreased in the zymosan + imperatorin group. Furthermore, the protein concentration in BALF was elevated in the zymosan group, whereas the level in the zymosan + imperatorin group was significantly lower (Figure 1E). The production of PGE2 was also enhanced by zymosan and decreased by imperatorin (Figure 1F). BALF smear was also analyzed by Diff-Quik staining. As shown in Figure 1G, zymosan enhanced immune cell infiltration in BALF, whereas imperatorin ameliorated immune cell infiltration. In summary, imperatorin may deteriorate zymosan-induced lung injury by relieving inflammatory responses and rapid fibrosis. Imperatorin Ameliorates Zymosan-Induced iNOS, COX-2, and Pro-inflammatory Cytokine Upregulation on Alveolar Macrophages. The treatment of diseases associated with or initiated by inflammation has been established by discovering their inflammatory mechanisms involving a variety of signaling molecules, effector proteins, and transcription factors. Zymosan is reported to trigger inflammatory signal cascades and activate downstream effectors such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory factors such as interleukin (IL)-6 and tumor necrosis factor (TNF) α.10,27,28 As shown in Figure 2A,B, we observed that zymosan-induced protein and mRNA expression of iNOS and COX-2 were both significantly reduced by the treatment of imperatorin in a concentrationdependent manner in alveolar macrophages. In Figure 2B,

Figure 2. Imperatorin ameliorates zymosan-induced iNOS and COX2 expression on alveolar macrophages. Cells were treated with imperatorin for 30 min and zymosan (10 μg/mL) for another 6 or 24 h, and the expressions of mRNA and protein was analyzed, respectively. iNOS and COX-2 mRNA (A) and protein expression (B) induced by zymosan was significantly decreased by imperatorin. (C) Zymosan-induced nitrite production and PGE2 secretion were also reduced by imperatorin by analyzing cultural supernatant. (D) The dosages of zymosan and imperatorin used on alveolar macrophages were confirmed to be harmless and did not affect cell viability. Statistics showed mean ± SD of at least three independent experiments, and representative blotting is shown in B. Control values were used as baseline to normalize the treatment group values. **p < 0.01; ***p < 0.001 compared to control group; #p < 0.05; ##p < 0.01; ### p < 0.001 compared to zymosan-treated group. Abbreviations: Con, control; Imp, imperatorin.

zymosan induced iNOS protein expression to 2.93 ± 0.84-fold of control (p < 0.05), and imperatorin (15 μg/mL) reduced iNOS protein expression to 1.47 ± 0.29-fold of control (p < 0.05, compared to the zymosan group). On the other hand, zymosan induced COX-2 protein expression to 2.74 ± 0.49fold of control (p < 0.01), and imperatorin (15 μg/mL) reduced COX-2 protein expression to 1.80 ± 0.31-fold of control (p < 0.05, compared to the zymosan group). Moreover, the downstream products nitrite and prostaglandin E2 (PGE2) induced by zymosan were also markedly decreased by imperatorin, while imperatorin alone did not affect any of the effects (Figure 2C). In addition, cell viability was also evaluated and exhibited that the concentrations of zymosan and imperatorin used did not have any effect (Figure 2D). Furthermore, the effect of imperatorin on zymosan-induced pro-inflammatory cytokine production was also examined. As shown in Figure 3, zymosan markedly provoked IL-6 and TNFα mRNA and protein expression, analyzed by real-time PCR and cytokine array, respectively. Treatment with imperatorin significantly decreased zymosan-induced IL-6 and TNFα mRNA and protein expression. Taken together, these results suggest that imperatorin may exert antiinflammatory effects against zymosan stimulation. The JAK1/STAT3 and NF-κB Signaling Pathway May Be Involved in the Anti-inflammatory Effects of Imperatorin on Alveolar Macrophages. It is well known that inflammatory signaling cascades have been identified in a series of promising targets, especially in pathways involving Janus kinase (JAK)/signal transducer and activators of C



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Figure 3. Imperatorin downregulates zymosan-induced IL-6 and TNFα expression on alveolar macrophages. Cells were treated with imperatorin for 30 min and zymosan (10 μg/mL) for another 6 or 24 h, and mRNA and cultural supernatants were analyzed, respectively. The mRNA expression of IL-6 (A) and TNFα (B) induced by zymosan was dose-dependently reduced by imperatorin. (C) By cytokine array analysis, zymosan-induced IL-6 (indicated in red) and TNFα (indicated in blue) protein expression in cultural supernatant was also decreased by imperatorin (15 μg/mL). Graphs show mean ± SD of at least three independent experiments. Control values were used as baseline to normalize the treatment group values. **p < 0.01; ***p < 0.001 compared to control group; ##p < 0.01; ###p < 0.001 compared to zymosan-treated group. Abbreviations: Con, control; Imp, imperatorin; Zym; zymosan.

abrogated dose-dependently. As shown in Figure 4B, zymosaninduced phosphorylation of JAK1 was reduced to 1.29 ± 0.04fold of control (p < 0.05, compared to the zymosan group) by 15 μg/mL imperatorin. Zymosan-induced phosphorylation of STAT3 was also reduced to 1.32 ± 0.14-fold of control (p < 0.05, compared to the zymosan group) by 15 μg/mL imperatorin. In addition, zymosan also elevated IKK phosphorylation in a time-dependent manner. In Figure 5A, zymosan induced IKK

transcription (STAT) and NF-κB transcription factor. Hence, we have analyzed JAK1, JAK2, STAT1, and STAT3 phosphorylation in a time-dependent manner after zymosan treatment. Among them, phosphorylation of JAK1 and STAT3 was significantly increased, while JAK2 and STAT1 were not affected by zymosan treatment. As shown in Figure 4A,

Figure 5. NF-κB signaling may be involved in the anti-inflammatory effect of imperatorin on alveolar macrophages. (A) Cells were treated with zymosan (10 μg/mL) for 1 to 60 min, and phosphorylation of IKK was elevated by zymosan in a time-dependent manner. (B) Zymosan-induced IKK phosphorylation was reduced by imperatorin dose-dependently. (C) Translocation of p65 from the cytosol to the nucleus induced by zymosan (10 μg/mL, 3 h) was also abrogated by imperatorin (15 μg/mL). Statistics show mean ± SD of at least three independent experiments, and representative blotting is shown. Each experiment was conducted at least three times, and representative data are shown here. Green, p65; blue, DAPI. Abbreviations: Con, control; Imp, imperatorin; Zym, zymosan.

Figure 4. JAK1/STAT3 signaling may be involved in the antiinflammatory effect of imperatorin on alveolar macrophages. Cells were treated with zymosan (10 μg/mL) for 1 to 30 min, and phosphorylation of JAK1, JAK2, STAT1, and STAT3 was analyzed. As shown in A, p-JAK1 and p-STAT3 were elevated by zymosan in a time-dependent manner, while p-JAK2 and p-STAT1 were not affected. (B) Zymosan-induced JAK1 and STAT3 phosphorylation was reversed by imperatorin dose-dependently. Statistics showed mean ± SD of at least three independent experiments, and representative blotting is shown. Each experiment was conducted at least three times, and representative blotting is shown here. Abbreviations: Con, control; Imp, imperatorin.

phosphorylation until 30 min of treatment (2.73 ± 0.16-fold of control; p < 0.01, compared to control). In addition, imperatorin reversed zymosan-induced phosphorylation of IKK dose-dependently. As shown in Figure 5B, zymosaninduced phosphorylation of IKK was reduced to 1.02 ± 0.08fold of control (p < 0.01, compared to the zymosan group) by 15 μg/mL imperatorin. Moreover, analyzed by immunofluorescence, zymosan stimulation for 3 h enhanced p65 trans-

zymosan induced JAK1 phosphorylation to the peak at 5 min treatment (2.44 ± 0.22-fold of control; p < 0.001, compared to control). Zymosan also enhanced STAT3 phosphorylation to the peak at 5 min treatment (2.66 ± 0.78-fold of control; p < 0.05, compared to control). By treating with imperatorin, zymosan-induced phosphorylation of JAK1 and STAT3 was D



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location from cytosol to the nucleus, and 15 μg/mL imperatorin abolished p65 translocation, as shown in Figure 5C. Taken together, these data suggest that imperatorin exerts an anti-inflammatory effect by inhibiting the JAK1/STAT3 and NF-κB signaling pathways. Alveolar macrophages form the front line of defense against pathogens or airborne particles. Inflammatory-associated lung diseases such as acute lung injury is characterized by excessive neutrophil infiltration, which is soon replaced by macrophages after neutrophil degranulation.29 Macrophages produce a variety of cytokines and chemokines that amplify the inflammatory responses and involve the alteration of basement membrane and the increase of membrane permeability.30,31 Besides, macrophages release cytokines, oxidants, and proteases, leading to the damage of the epithelial−alveolar barrier and extracellular matrix deposition.32,33 In the present study, zymosan-induced mice showed subepithelial collagen deposition, pulmonary edema, higher immune cell infiltration, and protein concentration in BALF, and the treatment with imperatorin showed significant protective effects against zymosan-induced lung injury. Inflammatory activity is largely mediated by nitric oxide (NO) and prostaglandin E2, which are robustly produced upon inflammatory stimulus.34,35 Excessive production of NO and PGE2 plays critical roles in the aggravation of inflammatory diseases; its rapid onset and continuous development often lead to high mortality rates.36,37 Suppression of NO and PGE2 production pathways, as shown by imperatorin, may satisfy the need for controlling the rapid progression of acute inflammatory injury. In addition, the release of inflammatory mediators is also essential in the inflammatory cascade.38 Among them, TNFα, IL-6, and IL-1β are considered to be important mediators in inflammatory responses. Evidence indicates that alveolar macrophages release these mediators in the early phase of acute lung injury,10 and enhanced TNFα expression is also associated with a poor prognosis of acute lung injury.39 In this study, the expression of iNOS and COX2, production of nitrite and PGE2, and secretion of TNFα and IL-6 were significantly elevated by zymosan and were all abrogated by the treatment with imperatorin, demonstrating the anti-inflammatory property of imperatorin. For developing therapies for inflammatory diseases, novel therapeutically relevant biological targets such as JAK/STAT and NF-κB signaling pathways have received growing attention, and regulators of these signaling cascades are considered to be promising anti-inflammatory therapeutics.40 In recent years, studies revealed that activation of the JAK/ STAT signaling pathway is pivotal in several inflammationassociated lung diseases, such as acute lung injury and COPD.41,42 It is well understood that JAKs interact with cytokine receptors and transduce the signals to amplify the release of inflammatory cytokines.43 Moreover, STATs are also known to mediate the transcription of cytokines. It has been observed not only that the JAK/STAT pathway is activated in COPD patients42 but that STATs are considered to be key players in initiating the inflammatory responses in acute lung injury.41 On the other hand, NF-κB activation is commonly observed in acute lung injury,44 and inhibition of NF-κB reduces the production of inflammatory mediators and exerts a protective effect against inflammatory stimulus.45−47 Taken together, inhibiting the JAK/STAT and/or NF-κB pathways may effectively ameliorate the progression of inflammatoryassociated lung diseases. In our study, imperatorin attenuated

zymosan-induced JAK1 and STAT3 phosphorylation and also IKK phosphorylation. Furthermore, imperatorin abolished zymosan-induced NF-κB p65 translocation from the cytosol to the nucleus. These findings suggest that imperatorin exerts protective effects against zymosan-initiated inflammatory response on alveolar macrophages. In summary, we have demonstrated the protective antiinflammatory effects of imperatorin on zymosan-induced pulmonary inflammation. This is evidenced by reducing iNOS and COX-2 expression, decreasing pro-inflammatory cytokines release, and inhibiting the JAK1/STAT3 and NF-κB pathways in alveolar macrophages. It is also observed that imperatorin alleviated lung inflammation and rapid fibrosis and decreased pulmonary edema in vivo. These results indicate that imperatorin may be considered to be an effective antiinflammatory agent for potential treatment of inflammatoryassociated lung diseases.

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EXPERIMENTAL METHODS

Materials. Zymosan and anti-α-tubulin antibody were purchased from Sigma-Aldrich (St. Louise, MO, USA). Imperatorin was obtained from ChemFaces (Hubei, PRC). Anti-COX-2 antibody was purchased from Cayman Chemicals (Ann Arbor, MI, USA). AntiiNOS antibody was obtained from BD Transduction Laboratories (Franklin Lakes, NJ, USA). Antibodies against JAK1 and JAK2 were obtained from Elabscience (Houston, TX, USA). Antibodies against phospho-JAK1Y1022/1023, phospho-JAK2Y1007/1008, and HRP-conjugated secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against STAT1, STAT3, phospho-STAT1Y701, phospho-STAT3Y705, p65, IKKβ, and phospho-IKKα/β were obtained from Santa Cruz (Santa Cruz, CA, USA). FITC-conjugated secondary antibody was purchased from Leinco Technologies (St. Louis, MO, USA). Animals and Treatments. Male C57BL/6 mice weighing 25−30 g were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Animals were housed in standard laboratory cages under a 12 h light/dark cycle and provided with free access to food and water through the experiments. The studies were conducted under a protocol approved by the Institutional Animal Care Committee of China Medical University (No. 2018-084, Taichung, Taiwan). Animals were treated with 4 mg/kg imperatorin by i.p. injection for 3 consecutive days and then given 4 mg/kg (in 40 μL of saline) zymosan or saline by i.t. instillation. After 24 h, mice were sacrificed, and BALF was collected using five 0.2 mL aliquots of sterile phosphate-buffered saline (PBS). The BALF cell and supernatant fractions were separated by centrifugation. The cell-free supernatants were harvested, and the concentrations of PGE2 and total protein were measured. Total cell counts were performed on the BALF cell fraction. Samples of right lung were taken and prepared for immunohistochemistry. Samples of left lung were taken to examine water content by the W/D weight ratio. Diff-Quik Staining. The cell fraction of BALF was resuspended in PBS. The total cell numbers were counted using a hematocytometer. Smears were prepared and stained with Quick-Dip differential stain kit (ScyTek, Logan, UT, USA) to determine the differential cell counts.48 At least 300 cells per slide were evaluated to obtain the differential cell counts. Wet/Dry Weight Ratio. To quantify the magnitude of pulmonary edema, the harvested lungs were weighed and then placed in an oven at 48 °C for 48 h and weighed again when they were dried. The ratio of wet lungs to dry lungs was calculated.49 Immunohistochemistry (H&E, Trichrome). Lungs were harvested and subjected to 10% formalin fixation followed by paraffin embedment. Five-micrometer-thick slices were sectioned and stained with hematoxyline and eosin (H&E) or trichrome stain kit following the manufacturer’s instructions. Histologic changes were visualized and evaluated by a Leica microscope (Germany) and MetaMorph E



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mean ± SD of at least three independent experiments with significance defined as p < 0.05 analyzed by Student’s paired t-test.

Microscopy Automation and Image Analysis software (Molecular Devices, San Jose, CA, USA). Cell Culture. MH-S alveolar macrophages were obtained from Bioresource Collection and Research Center (Hsinchu, Taiwan) and maintained in RPMI 1640 medium adjusted to contain 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate and supplemented with 0.05 mM 2-mercaptoethanol, 10% fetal bovine serum, and 1% penicillin−streptomycin−amphotericin B solution (Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated in a 37 °C humidified incubator containing 5% CO2. Cell Viability Assay. Sulforhodamine B (SRB) colorimetric assay was conducted by fixing the cells in 10% trichloroacetic acid, followed by 0.4% (w/v) SRB staining for 30 min. After a brief wash, cells were dissolved by 10 mM Tris solution and subjected to spectrophotometric quantitation under OD 515 nm by a SpectraMax M5 plate reader.50 Western Blot Analysis. Cells were lysed by RIPA lysis buffer and allowed to sit on ice for 30 min. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA) followed by 1 h of blocking in 7.5% skim milk. The membrane was blotted by primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Proteins were visualized by Chemilucent Plus Western Blot enhancing kit (Millipore, Temecula, CA, USA) using Fuji medical X-ray films (Valhalla, NY, USA). Signal intensities were analyzed and quantitated by ImageJ.51 Quantitative PCR. Total RNA extracted by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) was reverse-transcribed into DNA by using cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA). SYBR Green Master Mix (Thermo Fisher Scientific) was used to perform PCR reaction by StepOne Plus RealTime PCR Systems (Applied Biosystem, Singapore) under the following conditions: 95 °C for 10 min, 42 cycles at 95 °C for 10 s, and then 60 °C for 60 s. The following primers were used. actin: 5′-AGATGCAGCAGATCCGCAT-3′ and 5′GTTCTTGCCCATCAGCACC-3′. COX-2: 5′-TGGGGTGATGAGCAACTATT-3′ and 5′AAGGAGCTCTGGGTCAAACT-3′. iNOS: 5′-CCCAGAGTTCCAGCTTCTGG-3′ and 5′CCAAGCCCCTCACCATTATCT-3′. IL-6: 5′-AGTTGCCTTCTTGGGACTGA-3′ and 5′-TCCACGATTTCCCAGAGAAC-3′. TNF-α: 5′-ACGGCATGGATCTCAAAGAC-3′ and 5′AGATAGCAAATCGGCTGACG-3′. PGE2 Assay. Production of PGE2 in the cultural supernatant was measured by a prostaglandin E2 ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s instructions. In brief, the cultural supernatant, PGE2 acetylcholinesterase tracer, and PGE2 monoclonal antibody were incubated in a 96-well plate at 4 °C for 18 h. After washing, Ellman’s reagent was added to develop the well, and the absorbance was determined at OD 405 nm using a SpectraMax M5 plate reader. Griess Assay. Production of NO was assayed by examining the presence of nitrite in the cultural supernatant. Briefly, the cultural supernatant was reacted with equal amounts of 0.1% NED solution and 1% sulfanilamide in 5% phosphoric acid for 10 min in the dark. The absorbance was determined at OD 520 nm using a SpectraMax M5 plate reader.52 Immunofluorescence Staining. Cells were seeded onto glass coverslips in a 12-well plate. After the indicated treatment, cells were fixed by 4% paraformaldehyde for 15 min and permeabilized by 1% Triton X-100 for 20 min. After blocking with 4% skim milk, cells were incubated with primary antibody at 4 °C overnight. After a brief wash, cells were incubated with FITC-conjugated secondary antibody for 1 h. Finally, cells were washed again, mounted, and visualized by a Leica fluorescence microscope (Germany) and MetaMorph Microscopy Automation and Image Analysis software (Molecular Devices). Statistics. Statistical analysis was conducted by using GraphPad Prism and SigmaPlot software. Control values were used as a baseline to normalize the treatment group values. Values are expressed as

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AUTHOR INFORMATION

Corresponding Author

*E-mail (W.-L. Yeh): [email protected]. Fax: 886-422071507. ORCID

Wei-Lan Yeh: 0000-0003-4398-7610 Author Contributions #

Y.-Z. Li and J.-H. Chen have contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Ministry of Science and Technology (MOST 106-2311-B-039-002), China Medical University (CMU106-N-09, CMU107-N-14, and CMU106-ASIA-07), and Taichung Tzu Chi Hospital (TTCRD107-03 and TTCRD108-07). We also thank Rapid Science Co., Ltd., for technically supporting immunohistochemistry.

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