Loranthus tanakae Franch. & Sav. Suppresses Inflammatory Response in Cigarette Smoke Condensate Exposed Bronchial Epithelial Cells and Mice

Loranthus tanakae Franch. & Sav. found in China, Japan, and Korea is traditionally used for managing arthritis and respiratory diseases. In this study, we analyzed the components of L. tanakae 70% ethanol extract (LTE) and investigated the therapeutic effects of LTE on pulmonary inflammation using cells exposed to cigarette smoke condensate (CSC) and lipopolysaccharide (LPS) in vitro and in vivo in mice and performed a network analysis between components and genes based on a public database. We detected quercitrin, afzelin, rhamnetin 3-rhamnoside, and rhamnocitrin 3-rhamnoside in LTE, which induced a significant reduction in inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and inflammatory cells in CSC exposed H292 cells and in mice, accompanied by a reduction in inflammatory cell infiltration into lung tissue. In addition, LTE increased translocation into the nuclei of nuclear factor erythroid-2-related factor 2 (Nrf2). By contrast, the activation of nuclear factor (NF)-κB, induced by CSC exposure, decreased after LTE application. These results were consistent with the network pharmacological analysis. In conclusion, LTE effectively attenuated pulmonary inflammation caused by CSC+LPS exposure, which was closely involved in the enhancement of Nrf2 expression and suppression of NF-κB activation. Therefore, LTE may be a potential treatment option for pulmonary inflammatory diseases including chronic obstructive pulmonary disease (COPD).


Introduction
Cigarette smoke (CS) is associated with the progression of numerous diseases and is regarded as a primary cause in the pathogenesis of chronic obstructive pulmonary disease (CODP) [1]. CS contains several harmful substances, including chemicals, heavy metals, oxidant radicals, and carcinogens, which increase the risk of developing various diseases [2]. Exposure to CS induces the generation of cytokines, reactive oxygen species (ROS), and The aerial parts of L. tanakae were purchased from a herbalist in Jeongseon, Gangwondo in Republic of Korea. The plant (voucher specimen No. 2-16-0335) was authenticated by Drs. Sungyu Yang and Byeong Cheol Moon at the Korea Institute of Oriental Medicine (KIOM). After air-drying, the plant was pulverized using a blender (Hanil, Seoul, Republic of Korea) and passed through a 600 µm sieve.

Extraction and Isolation
L. tanakae (1.032 kg) was refluxed with 6 L of 70% ethanol for 2 h twice. The extraction filtered through chromatography paper (46 × 57 cm) and removed the solvent in vacuo. The yield of the L. tanakae 70% ethanol extract (LTE) was 126.42 g (12.25%, w/w) and it was stored at −20 • C. Four of the major compounds were isolated using preparative high performance liquid chromatography (HPLC, Prep LC) from the 70% ethanol extract. A 100 mg aliquot of LTE was dissolved in 70% ethanol (1 mL) and filtered through a syringe filter (0.45 µm) before injecting it into the HPLC system. The Prep LC system (Waters, Milford, MA, USA) consisted of a Waters 2545 quaternary gradient module, Waters 2998 photodiode array detector, Waters flexinject, and Waters fraction collector III. Four components were isolated using a Phenomenex Synergi 4µ Fusion-RP 80A (250 × 21.20 mm, Phenomenex Inc., Torrance, CA, USA). The mobile phase was prepared by mixing 0.05% aqueous formic Antioxidants 2022, 11, 1885 3 of 15 acid (A) and acetonitrile (B), and set in a linear gradient program; 5% A → 30% A for 60 min. The flow rate was 5 mL/min and injected volume was 500 µL at room temperature and detection was conducted at a wavelength of 254 nm. Chemical structures were determined with spectroscopic methods using mass spectroscopy and NMR.

Small Molecules and Potential Target Genes
Genes related to the four active components were collected from the SwissTarget-Prediction database (http://www.swisstargetprediction.ch/, accessed on 22 May 2022) with 'Homo sapiens' species and probability > 0. In this database, we used the probability originated from our cross-validation analysis to rank the targets and assess the accuracy of the predictions [17]. Potential target genes were searched in GeneCards: Human Gene Database (https://www.genecards.org/. version 5.11, accessed on 18 May 2022), and selected according to the intersection of the genes related to the active small molecules with the COPD-related genes.

Protein-Protein Interaction (PPI)
PPI is the process by which two or more proteins form a complex through noncovalent bonds [18]. Sophisticated network-based tools have been designed to anticipate potential disease genes [19]. PPI analyses were performed with the STITCH database (http://stitch.embl.de/, version 5, accessed on 18 May 2022) with a medium confidence score (≥0.400), and topology of PPI was conducted by Cytoscape version 3.7.2 (https: //cytoscape.org/, accessed on 18 May 2022) [ A human airway epithelial cells (NCI-H292, American Type Culture Collection (ATCC), Manassas, VA, USA) were maintained in RPMI 1640 added with 10% heat-inactivated fetal bovine serum and antibiotics at 37 • C in a 5% CO 2 incubator. Cell viability was determined using an EZ-Cytox kit (DAELIL, Seoul, Korea).

Evaluation of Inflammatory Cytokines in H292 Cells
The cells were seeded on 6 well plates (4 × 10 5 cells/well) for 24 h, then treated with LTE at 12.5, 25, 50 and 100 µg/mL. After incubation for 1 h, the cells were treated with CSC (100 µg/mL) for 24 h. The supernatant of cells was collected and determined the generation of interleukin (IL)-1β and IL-6 using commercial ELISA kit (BD Science, San Diego, CA, USA). The CSC was prepared as previously described [20]. Briefly, the CSC sample was prepared by smoking the cigarettes (research reference cigarette 3R4F, University of Kentucky, Lexington, KY, USA) on the 30-port smoking machine in according to International Organization for Standardization (ISO) 3308 (puff duration 2 s, puff volume 35 mL, puff interval 60 s and no vent blocking). The CSC was collected from generated cigarette smoke at 1 L/min for 5 min using a Whatman Cambridge filter pad (44 mm diameter glass fiber filter, GE Healthcare, Buckinghamshire, UK) and a mini vacuum pump (XR5000, SKC Inc., Covington, GA, USA). The total particulate matter (TPM) on the filter pad was extracted with methanol for 30 min with twist shaker to yield a concentration of 5 mg/mL (TPM mass per methanol volume). The 50 mL tubes containing the aliquots of this TPM (40 mL/tube) were subsequently vaporized in a vacuum dry oven overnight.

DPPH Radical Scavenging Activity
1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -zaino-bis-3-ethylbenzthiazoline-6sulphonic acid (ABTS) free radical scavenging activity for measurement of antioxidant capacities were determined according to reported [22,23]. DPPH solution dissolved in methanol and 100 µL LTE (62.5, 125, 250, and 500 µg/mL) reacted at 37 • C in incubator for 30 min. The absorbance of 100 µL DPPH radical was measured at 517 nm. For the ABTS assay, radical solution reacted with phosphate was adjusted to a final absorbance of 0.7 ± 0.02 at 734 nm. Gallic acid was used as a positive control in this study. The scavenging rate of DPPH and ABTS of the LTE was calculated according to the following formula: % scavenging rate = [(Abs free radical − Abs control )/Abs free radical ] × 100

Procedure of Animal Experiments
To establish COPD model, we used CSC and LPS exposed mouse model. In the development of COPD, besides exposure to CS, bacterial, fungi, virus and air-pollutants are also important risk factors potentially aggravating clinical sign of COPD [24]. In previous report, additional exposure to LPS in cigarette smoke induced experimental model for COPD induces an excessive inflammatory response in the respiratory tract [20,25]. The animals were assigned into 5 groups (n = 5) as follows: normal control (PBS intranasal instillation and oral gavage), CSC+LPS (CSC+LPS intranasal instillation and PBS oral gavage), ROF (CSC+LPS intranasal instillation and roflumilast (10 mg/kg of body weight) oral gavage), LTE 50 and 100 (CSC+LPS intranasal instillation and LTE oral gavage (50 and 100 mg/kg of body weight, respectively)). CSC (12.5 mg/kg of body weight) and LPS (0.5 mg/kg of body weight) were intranasally administered to animals under slight anesthesia on days 1, 6 and 13; the animals were sacrificed on day 15. To obtain bronchoalveolar lavage fluids (BALF), the mice were tracheostomized under anesthesia and endotracheal tubes were then inserted. PBS (0.7 mL) was infused into the lungs and withdrawn, and this procedure was repeated once (total volume: 1.4 mL). The inflammatory cell counts of BALF were assessed as previously described [21]. The supernatant of BALF was used for the evaluation of cytokines.

Measurement of Inflammatory Mediators in BALF
The generation of IL-6 and TNF-α in BALF were assessed using commercial ELISA kits (BD Science) according to the manufacturer's protocols.

Histopathology of Lung Tissue
The left lung tissue was fixed in neutralized formalin and stained with hematoxylin and eosin per standard procedures (thickness of sections: 4 µm) to evaluate pulmonary inflammation. To evaluate the expression NRF-2 and NF-κB on lung tissue, we conducted immunohistochemistry as previously described [19]. The following primary antibodies were used: NF-κB (1:100 dilution, Abcam) and NRF-2 (1:100 dilution, Abcam). Quantitative analysis of pulmonary inflammation, mucus production and protein expression was performed using an image analyzer (IMT i-Solution Inc., Vancouver, BC, Canada).

Statistical Analysis
The data were expressed as the mean ± standard deviation (SD). Statistical significance was determined using an analysis of variance (ANOVA) followed by a multiple comparison test with Dunnet's adjustment. p values < 0.05 were considered significant.

Network of Active Molecules and Potential Target Genes
A total of 173 genes was searched for linking to the four molecules according to Swis-sTargetPrediction database with 'Homo sapiens' species (Supplementary Materials Table  S1). After searching for genes associated with COPD within GeneCards DB (Supplementary Materials Table S2)

Network of Active Molecules and Potential Target Genes
A total of 173 genes was searched for linking to the four molecules according to Swis-sTargetPrediction database with 'Homo sapiens' species (Supplementary Materials Table S1). After searching for genes associated with COPD within GeneCards DB (Supplementary  Materials Table S2)

Signal Pathway Analysis
To explore the mechanism of the effect of L. tanakae on COPD, we analyzed KEGG pathways (p < 0.05). The top 10 COPD-related pathways were associated with cancer, PI3K-Akt signaling, fluid shear stress and atherosclerosis, MAPL signaling, Fc epsilon RI signaling, longevity regulation, chemokine signaling, NF-κB signaling, TNF signaling, and T cell receptor signaling (Figure 4a). The COPD-related pathways were classified to be associated with cancer overview, signal transduction, cardiovascular disease, immune system, aging, and sensory systems. Signal transduction and immune system were also potential pathways (Figure 4b). The genes associated with more than 10 pathways were IKBKB, MAPK8, PIK3CA, PRKACA, PRKCA, and TNF ( Figure 4c).

Signal Pathway Analysis
To explore the mechanism of the effect of L. tanakae on COPD, we analyzed KEGG pathways (p < 0.05). The top 10 COPD-related pathways were associated with cancer, PI3K-Akt signaling, fluid shear stress and atherosclerosis, MAPL signaling, Fc epsilon RI signaling, longevity regulation, chemokine signaling, NF-κB signaling, TNF signaling, and T cell receptor signaling (Figure 4a). The COPD-related pathways were classified to be associated with cancer overview, signal transduction, cardiovascular disease, immune system, aging, and sensory systems. Signal transduction and immune system were also potential pathways (Figure 4b). The genes associated with more than 10 pathways were IKBKB, MAPK8, PIK3CA, PRKACA, PRKCA, and TNF (Figure 4c).

Signal Pathway Analysis
To explore the mechanism of the effect of L. tanakae on COPD, we analyzed KEGG pathways (p < 0.05). The top 10 COPD-related pathways were associated with cancer, PI3K-Akt signaling, fluid shear stress and atherosclerosis, MAPL signaling, Fc epsilon RI signaling, longevity regulation, chemokine signaling, NF-κB signaling, TNF signaling, and T cell receptor signaling (Figure 4a). The COPD-related pathways were classified to be associated with cancer overview, signal transduction, cardiovascular disease, immune system, aging, and sensory systems. Signal transduction and immune system were also potential pathways (Figure 4b). The genes associated with more than 10 pathways were IKBKB, MAPK8, PIK3CA, PRKACA, PRKCA, and TNF (Figure 4c).

Effects of LTE on the Generation of Inflammatory Cytokines in CSC Exposed H292 Cells
Based on the results of cell viability, we determined 100 μg/mL as a high concentration of LTE that resulted in a non-toxic effect (Figure 5a). CSC exposed H292 cells showed the marked elevation of IL-1β ( Figure 5b) and IL-6 ( Figure 5c) compared with non-treated cells. However, LTE treatment significantly declined the production of IL-1β and IL-6 in CSC exposed H292 cells, in a concentration dependent manner.

Effects of LTE on the Expression of Nrf2 and NF-κB in CSC Exposed H292 Cells
A greater translocation of Nrf2 into nuclei was observed in CSC exposed H292 cells in comparison to that in non-treated cells (Figure 6a,c). LTE treatment further increased Nrf2 translocation into the nucleus in CSC exposed H292 cells. The translocation of NF-κB also increased into the nuclei of CSC exposed H292 cells compared with that in non-

Effects of LTE on the Generation of Inflammatory Cytokines in CSC Exposed H292 Cells
Based on the results of cell viability, we determined 100 µg/mL as a high concentration of LTE that resulted in a non-toxic effect (Figure 5a). CSC exposed H292 cells showed the marked elevation of IL-1β ( Figure 5b) and IL-6 ( Figure 5c) compared with non-treated cells. However, LTE treatment significantly declined the production of IL-1β and IL-6 in CSC exposed H292 cells, in a concentration dependent manner.

Effects of LTE on the Generation of Inflammatory Cytokines in CSC Exposed H292 Cells
Based on the results of cell viability, we determined 100 μg/mL as a high concentration of LTE that resulted in a non-toxic effect (Figure 5a). CSC exposed H292 cells showed the marked elevation of IL-1β ( Figure 5b) and IL-6 ( Figure 5c) compared with non-treated cells. However, LTE treatment significantly declined the production of IL-1β and IL-6 in CSC exposed H292 cells, in a concentration dependent manner.

Effects of LTE on the Expression of Nrf2 and NF-κB in CSC Exposed H292 Cells
A greater translocation of Nrf2 into nuclei was observed in CSC exposed H292 cells in comparison to that in non-treated cells (Figure 6a,c). LTE treatment further increased Nrf2 translocation into the nucleus in CSC exposed H292 cells. The translocation of NF-κB also increased into the nuclei of CSC exposed H292 cells compared with that in non-

Effects of LTE on the Expression of Nrf2 and NF-κB in CSC Exposed H292 Cells
A greater translocation of Nrf2 into nuclei was observed in CSC exposed H292 cells in comparison to that in non-treated cells (Figure 6a,c). LTE treatment further increased Nrf2 translocation into the nucleus in CSC exposed H292 cells. The translocation of NF-κB also increased into the nuclei of CSC exposed H292 cells compared with that in non-treated cells

Effects of LTE and Its Components on ROS Production
The scavenging DPPH and ABTS activities of the extracts and four ingredients were presented in Table 1. Antioxidant effect for DPPH and ABTS of LTE were 124.37 and 226.1 μg/mL, respectively. Additionally, rhamnetin 3-ramnoside and quercitrin showed antioxidant effects, but rhamnocitrin 3-rhamnoside and afzelin showed no antioxidant effect in DPPH and ABTS assay. Additionally, we evaluated the effects of rhamnetin 3-ramnoside and quercitrin on H2O2-treated HeLa cells. H2O2-treated cells showed excessive ROS production, but rhamnetin 3-ramnoside and quercitrin-treated cells exhibited the significant reduction in ROS production compared with H2O2-treated cells (Figure 7a-c).

Effects of LTE and Its Components on ROS Production
The scavenging DPPH and ABTS activities of the extracts and four ingredients were presented in Table 1. Antioxidant effect for DPPH and ABTS of LTE were 124.37 and 226.1 µg/mL, respectively. Additionally, rhamnetin 3-ramnoside and quercitrin showed antioxidant effects, but rhamnocitrin 3-rhamnoside and afzelin showed no antioxidant effect in DPPH and ABTS assay. Additionally, we evaluated the effects of rhamnetin 3-ramnoside and quercitrin on H 2 O 2 -treated HeLa cells. H 2 O 2 -treated cells showed excessive ROS production, but rhamnetin 3-ramnoside and quercitrin-treated cells exhibited the significant reduction in ROS production compared with H 2 O 2 -treated cells (Figure 7a-c).

Effects of LTE on Inflammatory Indexes in CSC+LPS Exposed Mice
CSC+LPS exposed mice showed markedly elevated inflammatory cell counts for neutrophils, macrophages and total cells in BALF compared with those in the control group (Figure 8a-c, respectively). Roflumilast-treated mice exhibited the significant reduction in the inflammatory cell counts in BALF compared with CSC+LPS exposed mice. The administration of LTE significantly reduced inflammatory cell counts in BALF compared with those in CSC+LPS exposed mice, which was observed in dose-dependently manner. The levels of IL-1β, IL-6, and TNF-α in BALF were significantly elevated in CSC+LPS exposed mice than those in the control group (Figure 8d-f, respectively). Roflumilast-treated mice exhibited the significant reduction in IL-1β, IL-6, and TNF-α in BALF compared with CSC+LPS exposed mice. The administration of LTE reduced the levels of IL-1β, IL-6, and TNF-α in BALF in comparison with those in CSC+LPS exposed mice, which were noticeably observed in the group with a high dose of LTE. Although there is no significant difference between ROF group and LTE groups, ROF group slightly more decreased inflammatory cell count and cytokines than LTE groups.

Effects of LTE on Inflammatory Indexes in CSC+LPS Exposed Mice
CSC+LPS exposed mice showed markedly elevated inflammatory cell counts for neutrophils, macrophages and total cells in BALF compared with those in the control group (Figure 8a-c, respectively). Roflumilast-treated mice exhibited the significant reduction in the inflammatory cell counts in BALF compared with CSC+LPS exposed mice. The administration of LTE significantly reduced inflammatory cell counts in BALF compared with those in CSC+LPS exposed mice, which was observed in dose-dependently manner. The levels of IL-1β, IL-6, and TNF-α in BALF were significantly elevated in CSC+LPS exposed mice than those in the control group (Figure 8d-f, respectively). Roflumilast-treated mice exhibited the significant reduction in IL-1β, IL-6, and TNF-α in BALF compared with CSC+LPS exposed mice. The administration of LTE reduced the levels of IL-1β, IL-6, and TNF-α in BALF in comparison with those in CSC+LPS exposed mice, which were noticeably observed in the group with a high dose of LTE. Although there is no significant difference between ROF group and LTE groups, ROF group slightly more decreased inflammatory cell count and cytokines than LTE groups.

Effects of LTE on Pathophysiological Alteration of Lung Tissue in CSC+LPS Exposed Mice
CSC+LPS exposure to mice induced the recruitment of inflammatory cells and mucus production into lung tissue. Roflumilast-treated mice showed the marked reduction in inflammatory response and mucus production compared with the CSC+LPS exposed mice. The LTE treatment reduced the inflammatory response and mucus production caused by CSC+LPS exposure (Figure 9a-c). Although there is no significant difference between ROF group and LTE groups, ROF group slightly more decreased mucus production than LTE groups. The expression of Nrf2 on lung tissue increased to a greater extent in Roflumilast and LTE-treated mice with CSC+LPS exposure than that in CSC+LPS exposed mice (Figure 9a,d). In contrast, the administration of Roflumilast and LTE clearly reduced the expression of NF-κB on lung tissue owing to CSC+LPS exposure (Figure 9a,e). mice exhibited the significant reduction in IL-1β, IL-6, and TNF-α in BALF compared with CSC+LPS exposed mice. The administration of LTE reduced the levels of IL-1β, IL-6, and TNF-α in BALF in comparison with those in CSC+LPS exposed mice, which were noticeably observed in the group with a high dose of LTE. Although there is no significant difference between ROF group and LTE groups, ROF group slightly more decreased inflammatory cell count and cytokines than LTE groups.

Effects of LTE on Pathophysiological Alteration of Lung Tissue in CSC+LPS Exposed Mice
CSC+LPS exposure to mice induced the recruitment of inflammatory cells and mucus production into lung tissue. Roflumilast-treated mice showed the marked reduction in inflammatory response and mucus production compared with the CSC+LPS exposed mice. The LTE treatment reduced the inflammatory response and mucus production caused by CSC+LPS exposure (Figure 9a-c). Although there is no significant difference between ROF group and LTE groups, ROF group slightly more decreased mucus production than LTE groups. The expression of Nrf2 on lung tissue increased to a greater extent in Roflumilast and LTE-treated mice with CSC+LPS exposure than that in CSC+LPS exposed mice (Figure 9a,d). In contrast, the administration of Roflumilast and LTE clearly reduced the expression of NF-κB on lung tissue owing to CSC+LPS exposure (Figure 9a,e)

Discussion
COPD is an important pulmonary disease with a high incidence and mortality [2]. Researchers have developed numerous therapeutic agents to treat COPD. However, candidates for controlling COPD have limited use owing to their low efficacy or toxicity. In this study, we analyzed the ingredients in LTE by HPLC and evaluated the therapeutic

Discussion
COPD is an important pulmonary disease with a high incidence and mortality [2]. Researchers have developed numerous therapeutic agents to treat COPD. However, candidates for controlling COPD have limited use owing to their low efficacy or toxicity. In this study, we analyzed the ingredients in LTE by HPLC and evaluated the therapeutic effects of LTE on CSC induced pulmonary inflammation using in vitro and in vivo experiments. In addition, the mechanisms of action of LTE on CSC induced airway inflammation were determined by network pharmacological analysis. HPLC revealed that the main components of LTE were quercitrin, afzelin, rhamnetin 3-rhamnoside, and rhamnocitrin 3-rhamnoside.
In the results of in vivo and in vitro experiments, LTE showed ROS scavenging activity in DPPH and ABTS assays and its ingredients including rhamnetin 3-rhamnoside and quercitrin markedly decreased the ROS production induced by H 2 O 2 treatment. Additionally, LTE significantly diminished inflammatory cytokines caused by the exposure to CSC+LPS or only CSC, accompanied by a reduction in inflammatory responses on lung tissue. LTE showed an elevation of Nrf2 and a decrease in NF-κB in both CSC exposed cells and CSC+LPS in the mice model. These results were consistent with those of network pharmacological analysis.
Result of network pharmacology analysis, total 17 effective COPD-related signaling pathways were analyzed as follows: pathways in cancer, PI3K-Akt, Fluid shear stress and atherosclerosis, MAPK, Fc epsilon RI, longevity regulating pathway-multi species, chemokine, T cell receptor, NF-κB, TNF, Inflammatory mediator regulation of TRP channels, Th 17 cell differentiation, chemical carcinogenesis-reactive oxygen species, NOD-like, mTOR, Toll-like and FoxO signaling pathways. A total 70 COPD-related genes were selected from four ingredients, of which 44 genes were involved in the COPD pathways. In particular, NF-κB, TNF, FoxO and PI3K-Akt are closely related with pathophysiological alteration induced by ROS. Exposure to irritant induces the activation of NF-κB and TNF signaling resulting the progression and aggravation of inflammatory responses on damaged lesions [29,30]. By contrast, FoxO and PI3K-Akt induce the enhancement of antioxidant system such as superoxide dismutase, catalase, Nrf2 and HO-1 in organism to reduce damage induced by ROS [31,32]. Therefore, the therapeutic effects of LTE on COPD is considered to be related with the reduction in damage caused by ROS.
CS is considered to be the most important factor in the progression of COPD owing to its many harmful constituents. CS induces the accumulation of inflammatory cells such as neutrophils and macrophages into lung tissue, which are associated with the alteration of the histology of lung tissue [4]. The neutrophils and macrophages involved in the generation of inflammatory mediators, including ROS, cytokines, and chemokines, result in the exacerbation of inflammatory responses [6]. In addition, neutrophils produce the protease that destroys normal lung tissue via the degradation of connective tissues [33]. In this study, LTE treatment reduced the number of inflammatory cells, including neutrophils and macrophages in the BALF of CSC exposed mice, which was accompanied by a decline of the inflammatory response of lung tissue. Furthermore, LTE decreased the production of TNF-α, IL-6, and IL-1β in CSC exposed cells and in CSC+LPS exposed mice. These results indicate that LTE effectively suppressed pulmonary inflammation induced by CSC exposure.
Inflammatory responses are involved in various signaling pathways. In particular, NF-κB is intimately linked with the progression of inflammatory response [2]. NF-κB can be activated by multiple stimuli, such as air pollutants, allergens, fine dust, bacteria, toxins, and chemicals in the airway [34]. Activated NF-κB translocates into the nucleus, which switches on various inflammatory genes, resulting in the amplification of inflammation on damaged lesions [10]. During the development of COPD, CS produces ROS, which stimulates the activation of NF-κB [6]. Therefore, the suppression of NF-κB is regarded to be a critical treatment strategy for controlling COPD. In this study, LTE effectively inhibited the activation of NF-κB in CSC+LPS exposed mice, which led to a reduction in inflammatory cytokines. These results indicate that administration of LTE suppressed pulmonary inflammation induced by CSC+LPS exposure via the inhibition of NF-κB.
Exposure to CS also induces oxidative damage to an organism when ROS is produced. Healthy organisms protect against oxidative damage through the enhancement of antioxidant systems, such as reduced glutathione, glutathione reductase, superoxide dismutase, and catalase production [35]. However, repeated exposure to CS continuously produces excessive ROS, which eventually causes an imbalance between ROS and the antioxidant system, leading to a pathophysiological alteration of the respiratory tract [36]. Therefore, the enhancement of the antioxidant system is an aspect for controlling COPD. Nrf2 is an antioxidant transcription factor triggered by oxidative stress induced by various stimuli [35], and binds to Kelch-like ECH associated protein1 (Keap1) under homeostatic condition. However, under oxidative conditions, Nrf2 released from Keap1, translocates into nuclei and binds to antioxidant response elements, leading to the transcription of several genes, such as hemeoxygenase-1 and nicotinamide adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1 [37]. These events eventually protect organisms against oxidative stress via the enhancement of their antioxidant statuses. Nrf2 is also involved in the progression of inflammation as it suppresses the activation of NF-κB by reducing intracellular ROS levels and reducing the degradation of IκB-α and translocation of NF-κB into nuclei, leading to a decrease in the generation of inflammatory cytokines, including TNF-α and IL-1β [35,38,39]. In this study, LTE induced the translocation of Nrf2 into the nucleus, with a reduction in NF-κB translocation into the nucleus in CSC exposed cells and CSC+LPS exposed mice. This eventually decreased inflammatory cytokines and inflammatory responses in the lung tissues of CSC+LPS exposed mice. Therefore, the therapeutic effects of LTE against CSC+LPS caused pulmonary inflammation were intimately linked with the modulation of Nrf2 and NF-κB signaling.
Therapeutic effects of the components of LTE on CSC induced pulmonary inflammation occurred owing to its various pharmacological properties, e.g., anti-inflammatory and anti-oxidative properties.

Conclusions
Collectively, our study provides evidence that LTE inhibited inflammatory responses in CSC+LPS-exposed mice in vivo and CSC-exposed H292 cells in vitro. These effects may be involved in the activation of Nrf2 and suppression of NF-κB. Therefore, we considered that LTE has potential as a therapeutic agent for controlling pulmonary inflammation induced by CS.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11101885/s1, Figure S1. Purity information of four single compounds by HPLC chromatogram and extracted-ion chromatogram (XIC). Table S1: Information of small molecules and genes in Loranthus tanakae; Table S2: COPD-related genes in Gene Cards database.  Institutional Review Board Statement: The procedure of animal experiment was animal study was obtained approval from the Institutional Animal Care and Use Committee of the Chonnam National University (CNU IACUC-YB-2021-39).