key: cord-0970756-05ttk6ir
authors: Liu, Chun-Ping; Liu, Jian-Xing; Gu, Jiangyong; Liu, Fang; Li, Jin-Hua; Bin-Yang,; Yuan-Zheng,; Jie-Li,; Wu, Shou-hai; Wu, Qing-he; Xian-Zhang,; Li, Long-Mei; Yang, Hai-Long; Wang, Lei; Li, Xiong
title: Combination Effect of Three Main Constituents From Sarcandra glabra Inhibits Oxidative Stress in the Mice Following Acute Lung Injury: A Role of MAPK-NF-κB Pathway
date: 2021-01-11
journal: Front Pharmacol
DOI: 10.3389/fphar.2020.580064
sha: 3741e93457a364a9bd7a6c6c81e7732ba768e822
doc_id: 970756
cord_uid: 05ttk6ir

Caffeoylquinic acids, coumarins and dicaffeoyl derivatives are considered to be three kinds of the most abundant bioactive components in Sarcandra glabra, an anti-inflammatory herb mainly found in Southern Asia. The combined anti-inflammatory effect of three typical constituents C + R + I (chlorogenic acid + rosmarinic acid + isofraxidin) from this plant has been investigated. The result implies that targeting the MAPK-NF-κB pathway would be one of the major mechanisms involved, using LPS stimulated RAW 264.7 cells as in vitro model and LPS-induced acute lung injury in mice as in vivo model. C + R + I can significantly suppress the levels of nitric oxide (NO), pro-inflammatory cytokines, and inhibit iNOS and COX-2 expression in LPS-treated RAW264.7 macrophage cells. Western blot analysis showed that C + R + I suppressed phosphorylation of NF-κB and MAPK, including phosphorylation of p65-NF-κB, IKB, ERK, JNK and P38. Besides, C + R + I suppressed MPO protein expression, but promoted SOD and HO-1 expression, and the related targets for C, R, and I were also predicted by molecular docking. This indicated that C + R + I could alleviate oxidative stress induced by LPS, which were further verified in the in vivo model of mice with acute lung injury through the measurement of corresponding inflammatory mediators and the analysis of immunehistochemistry.

Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a central complication during the pathological process in the patients with severe coronary virus disease 2019 . It is found that the patients diagnosed with ALI/ARDS have poorer prognosis and higher mortality (Li et al., 2020; van de Veerdonk et al., 2020) . ALI and its severe form, ARDS, can lead to rapid loss of lung function, characterized by overwhelming inflammatory responses in the lung (Rebetz and Semple, 2018) . Lipopolysaccharide (LPS) from gram-negative bacteria can promote inflammatory responses to mammalians, and has been chosen as the inflammatory inducer to develop the experimental model of ALI/ARDS . As indicated by previous research, MAPK-NF-κB signaling is a key pathway which is involved in the regulation of ALI/ARDS (Lv et al., 2017; de Oliveira et al., 2019) . Likewise, free radicals can trigger inflammatory signaling, resulting in an increased level of circulating pro-inflammatory cytokines, which are known to induce ALI/ARDS (Zielińska et al., 2017) . Thus, the treatments aimed at the inhibition of NF-κB and MAPKs, as well as the direct inhibition of oxidative stress, may have potential therapeutic advantages in curing ALI/ARDS. Anti-inflammatory active ingredients have a widespread presence in Chinese medicine, which have exhibited great therapeutic effect through multicomponent and multi-target approach. The study of the main anti-inflammatory ingredients and their synergistic mechanism will help us to understand the nature of the role of Chinese medicine (Chen et al., 2019a; Chen et al., 2019b) .

Sarcandra glabra (Thunb) Nakai (S. glabra) is an herbal medicine distributed in Southern Asia. According to Chinese Pharmacopoeia, the entire plant of S. glabra and its extract can be used to treat respiratory tract inflammation related diseases such as acute respiratory infections, pneumonia, and so on (National Commission of Chinese Pharmacopoeia, 2015) . Furthermore, this herb has also been recommended to prevent and treat COVID-19 by the Chinese government (Liang et al., 2020) . Several types of components in S. glabra have been purified and identified by the LC-MS technique (Li et al., 2011) . 17 representative compounds were further accurately determined by the HPLC-ESI-MS/MS method (Li et al., 2014) , including caffeoylquinic acids (3-O-caffeoyl quinic acid, 4-O-caffeoyl quinic acid and chlorogenic acid), dicaffeoyl derivatives (rosmarinic acid and rosmarinic acid-4-O-β-D-glucoside), and coumarins (isofraxidin and eleutheroside B1). Among them, eleutheroside B 1 , isofraxidin and rosmarinic acid-4-O-β-D-glucoside are rare natural products (Li et al., 2014; Liu et al., 2017) . An integral analysis of absorbed constituents and their metabolites of S. glabra reveals that chlorogenic acid (C), isofraxidin (I) and rosmarinic acid (R) all exist as the parent absorbed forms in rat plasma (Xiong et al., 2016) , although these polyphenol components may have low oral bioavailability . It is known that using the entire plant or the mixture of major bioactive components to treat diseases is one of the typical characteristics of Chinese medicine. However, the potential synergistic effects existing in the major bioactive compounds are unknown. This study aims to investigate whether C + R + I have protective effects in suppressing LPS-induced ALI and attempts to identify the mechanisms in the NF-κB and MAPK signaling pathways. The potential combined anti-inflammatory effect mechanisms between them have been studied and discussed.

Chlorogenic acid and rosmarinic acid were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), isofraxidin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and their purities were more than 98%. RAW 264.7 macrophages cell line was purchased from American Type Culture Collection (Rockville, MD, United States). Antibodies, including anti-NF-κB, anti-IκB, anti-INOS, anti-COX-2, anti-P38, anti-JNK, anti-ERK and anti-GAPDH were purchased from Cell Signaling Technologies (Pickering, ON, CAN). Lipopolysaccharide (LPS) was purchased from Sigma (Sigma, MO, United States). Fetal bovine serum, Dulbecco's Modified Eagle's Medium and penicillin/streptomycin solution were purchased from HyClone (Logan, UT, United States). Cytometric Bead Array (CBA) Kits were purchased from eBioscience (San Diego, CA, United States). Enhanced Chemiluminescence (ECL) Solution Kit and secondary antibodies was purchased from Millipore (Billerica, MA, United States).

Adult female and male BALB/c mice (8-10 weeks old, 18-22 g weight) were purchased from Guangdong Laboratory Animal Medicine Center. Mice were allowed to acclimatize to the laboratory for at least 7 days in a standard laboratory with controlled temperature (22 ± 2°C), relative humidity (45-55%), artificial light with a 12 h light and 12 h darkness cycle, and provided free access to food and water under a specific pathogenfree environment. Animal Experimental Ethics Committee of The Second Affiliated Hospital of Guangzhou University of Chinese Medicine approved the protocols.

The procedures for induction of ALI by LPS in mice were performed as previously described in previous reports (Niu et al., 2012) . Briefly, mice were randomly divided into six groups, including control, model, dexamethasone (DEX), and three experimental groups C + R + I high dose (50, 20, and 20 mg/kg), C + R + I middle dose (25, 10, and 10 mg/kg), C + R + I low dose (12.5, 5, and 5 mg/kg). The experimental groups were intraperitoneally (i.p.) injected with 50, 25, 12.5 mg/kg of chlorogenic acid, 20, 10, and 5 mg/kg of rosmarinic acid, and 20, 10, and 5 mg/kg of isofraxidin once a day, respectively. The administration dose for in vivo investigation was chosen according to previous studies (Jiang et al., 2009; Niu et al., 2012; Meng et al., 2014) . In contrast, the groups of control, model, and dexamethasone (DEX) received an i. p. injection of saline solution. Three days later, the mice fasted for about 12 h and then received 50 μL LPS solution (10 μg LPS) through intratracheal (i.t.) instillation. Each group was dosed once again after the first administration for 1 h, whereas the positive control group was injected with 2 mg/kg DEX.

Upon LPS stimulation for 24 h, the mice were sacrificed with 50 mg/kg pentobarbital. Blood and lung samples were collected from ten animals, individually. The blood was then used for iNOS and TNF-α analysis, the lung samples were used for MPO, SOD, IL-1β and COX-2 determination, histopathological and immunohistochemistry studies. Bronchopleural lavage fluid was collected from the other six mice for leukocytes analysis.

The targets for chlorogenic acid, rosmarinic acid, and isofraxidin were predicted by molecular docking. The crystal structures of ligand-protein complexes were downloaded from the RSCB Protein Data Bank. The PDB ID for SOD, MPO, HO-1, ERK, iNOS, COX-2, JNK, NF-kB, and p38 MAPK were 5O3Y (Wright et al., 2013) , 4C1M (Forbes et al., 2013) , 6EHA (Mucha et al., 2019) , 4QTB (Chaikuad et al., 2014) , 3E7G (Garcin et al., 2008) , 5IKR (Orlando and Malkowski, 2016) , 4QTD (Chaikuad et al., 2014) , 6NV2 (Wolter et al., 2020) and, 3ZS5 (Azevedo et al., 2012) , respectively. The binding site for each protein was defined by the occupied space of the corresponding ligand in the crystal structure. The calculations were performed by Autodock 4.2. 6 [22,89] with default parameters. The binding energy was used to evaluate the binding affinity between each compound and each protein.

RAW 264.7 macrophages were maintained in DMEM supplemented with 10% heat-inactivated FBS, 10,000 U/ml penicillin and 10,000 μg/ml streptomycin at 37°C in an incubator with a humidified atmosphere and 5% CO 2 . RAW 264.7 macrophages were cultured in the presence of LPS (100 ng/ ml) with C + R + I (0.03, 0.06, and 0.06 mmol). The choice of concentration for C + R + I was also according to reference Shan et al., 2009; Niu et al., 2012) and CCK8 pre-experiment.

Evaluation of TNF-α, COX-2, iNOS, and IL-6 Inflammatory Cytokines

The levels of inflammatory cytokines, including TNF-α, COX-2, iNOS, and IL-6 of the lung in LPS-induced pulmonary injury were measured using a CBA assay kit. Then, these concentrations were calculated via the standard curves of TNF-α, iNOS, COX-2, and IL-6.

To analyze the production of nitric oxide, RAW264.7 cells in 6well plates were cultured for 3 h with or without C + R + I and then incubated with LPS for 24 h. NO production was then evaluated by quantifying the nitrite in the culture medium using the Griess reagent. Briefly, 100 μL of the collected supernatant was mixed with Griess reagent and incubated at room temperature for 15 min. Absorbance values at 540 nm were recorded, and the levels of nitric oxide were determined according to the standard curve of sodium nitrite ranging from 0.1 to 10 μM.

After treatments, RAW264.7 cells were washed with pre-cooling PBS and lyzed with RIPA (containing proteinase inhibitor and phospho-protein inhibitor), then centrifuged at 12,000 rpm for 10 min at 4°C and the lyzed supernatant was collected. Protein content of the lyzed cells was analyzed by BCA assay kit. Protein samples were loaded, separated by sodium dodecyl and transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes. 0.5% BSA in 1× Tris-buffered saline (TBS) containing 0.05% Tween-20 was used to block membranes by 1 h incubation. Protein samples were then separated by sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels. Membranes were treated with primary antibodies at 4°C overnight and then treated with horseradish peroxidasecoupled secondary antibody for 2 h at 4°C. Finally, the membranes were washed with TBST after each antibodybinding reaction. Enhanced chemiluminescence (ECL) kit was used to detect the protein.

The lung tissues were fixed in 4% paraformaldehyde and then embedded in paraffin for sectioning. Several sections were stained with hematoxylin and eosin to examine the histological differences.

The lung samples were fixed with 4% paraformaldehyde, followed by washes with PBS. Then tissues were permeabilized with 0.1% Triton-X 100 for 10 min, blocked with 5% donkey serum for 1 h and incubated with P-p65, P-p38, and COX-2 antibody separately overnight at 4°C. Following incubation with goat serum (secondary antibody) for 1 h at room temperature, the samples were then incubated with hematoxylin stain.

Statistical analysis was performed using Student t test, or one-way ANOVA followed by Tukey's post-test. The results are expressed as mean ± standard error of the mean (s.e.m.) and significance was set as p < 0.05.

To evaluate the effect of C + R + I on ALI, we examined the degree of damage that occurred in the lung of LPS-administered mice pretreated with or without C + R + I by H&E staining. No histopathological changes were observed from the control group under the optical microscope ( Figure 1A) , while changes in lung structure, pulmonary edema, and the accumulation of inflammatory cells were observed in animals without C + R + I pretreatment ( Figure 1B) . As expected, 1 h of C + R + I pretreatment reduced the LPS-induced lung damage at all low, middle and high doses ( Figure 1D -F-F), similar results were observed in mice pretreated with DEX ( Figure 1C ). To sum up, these results suggest that C + R + I can improve the condition of ALI induced by LPS.

Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 4 ALI is characterized by the infiltration of polynuclear neutrophil, white corpuscles and bronchialveolar. To investigate whether C + R + I could reduce the inflammatory cells infiltration in vivo, we detected the changes in inflammatory cells infiltration occurring in the lung of LPS-induced mice pretreated with or without C + R + I. As showed in Figure 2A -C, significant reductions in the inflammatory cell infiltration were observed in the BALF with C + R + I pretreatment. The wet/dry lung weight ratio was also obviously decreased compared with the LPS group ( Figure 2D) . Results demonstrated that C + R + I at the middle dose had protective effect on ALI mice by decreasing inflammatory cells infiltration.

Oxidative stress has a critical role in the pathophysiology of ALI. The anti-oxidative enzyme SOD is consumed during amelioration of ALI. In this study, we found that SOD secretion was significantly inhibited in LPS-induced mice, while the pretreatment with C + R + I recovered the LPSinduced reduction in a concentration-dependent manner ( Figure 3A) .

We further measured the MPO influence of C + R + I treatment, which is the marker for macrophage infiltration. We found that MPO secretion was increased in the lung tissue of LPS-induced mice. As expected, the up-regulation of MPO was Values are expressed as the mean 7 SD (n1⁄ 43 or 4 in each group). *p < 0.05, **p < 0.01 as compared to the LPS group without C + R + I, #p < 0.05, ##p < 0.01 as compared to the normal control group without C + R + I. Effects of C + R + I on TNF-α and IL-1β, COX-2 and iNOS production in LPS-induced ALI. Values are expressed as the mean 7 SD (n1⁄ 43 or 4 in each group). *p < 0.05, **p < 0.01 as compared to the LPS group without C + R + I, #p < 0.05, ##p < 0.01 as compared to the normal control group without C + R + I. Effects of C + R + I on inhibition of MAPK-NF-κB in LPS-induced ALI.

Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 5 FIGURE 5 | Effects of C + R + I on the protein of P-p65, P-p38 and COX-2 in LPS-induced ALI. Immunostaining of; (A) activated P-p65; (B) activated P-p38; (C) COX-2. Effects of C + R + I on NO, HO-1, SOD and MPO in LPS-induced ALI.

Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 6 prevented in mice pretreated with C + R + I ( Figure 3B) . The results showed that C + R + I could protect LPS-induced ALI mice by reducing macrophage activation.

Pro-inflammatory cytokines, including TNF-α, iNOS, COX-2 and IL-6 are major mediators involved in recruitment of macrophages into the lungs during LPS-induced pulmonary injury. In this study, the TNF-α, iNOS, COX-2,, and IL-6 levels in the serum of ALI were tested via Liquid chip method. LPS induced TNF-α, iNOS, COX-2 and IL-6 production by the macrophages in ALI, while the productions were decreased by C + R + I in a dose-dependent manner (p < 0.05 or 0.01, Figure 4A-D-D) . The results demonstrated that C + R + I decreased the expression of pro-inflammatory cytokines.

In other tissue contexts, cooperativity of p65-NF-κB, p-JNK and iNOS has been shown to reflect cross talk on signaling pathways, particularly NF-κB signaling. To detect.

Whether MAPK-NF-κB signaling pathway was involved, the immune cytochemical analyses were used to measure protein expression of the lung in LPS-induced ALI with or without C + R + I pretreatment. LPS-induced mice resulted in phosphorylation of JNK, ERK and p38 MAPK in lung tissue. Herein, we found that pretreatment with C + R + I decreased the phosphorylation of NF-κB p65, and p38 in the lung of LPS-induced ALI. (Figure 5A-C-C) .

The anti-oxidative protein HO-1 is expressed during the relieving of ALI. The effect of C + R + I on HO-1 expression in LPS-stimulated RAW 264.7 cells were detected by westernblot. As shown in Figure 6B , C + R + I increased LPS-stimulated HO-1 expression, suggesting that C + R + I can decrease LPSstimulated oxidative stress (p < 0.05). The ant-oxidative enzyme SOD is consumed during amelioration of ALI. In this study, we found that SOD is significantly inhibited in LPS-induced mice compared to the control group, while the pretreatment with C + R + I increased the anti-oxidative enzymes' activity ( Figure 6C) . MPO activity is a marker for macrophage and neutrophil infiltration and activation. We measured the ability of C + R + I in influence of MPO activity and found that MPO was significantly downregulated in lung tissue of ALI ( Figure 6D ). In addition, it is well known that NO are important active molecules with key roles in macrophage. In this study, we found that pre-treatment with C + R + I also significantly reduced the production of NO in lung tissue of ALI (p < 0.05) ( Figure 6A ). These results demonstrated that C + R + I had a protective effect on LPS-induced ALI mice by inhibiting macrophage infiltration and activation.

Next, we investigated whether C, R, and I used alone could modulate the expression of key molecules in MAPK-NF-κB signaling pathway in RAW264.7 cells stimulated by LPS. Briefly, cells were pretreated with C, R or I alone for 3 h, following exposure to LPS for 24 h. Western blot analysis was then used to measure the protein expression of p38-MAPK, Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 P-JNK, and P-ERK. After treatment with LPS, the protein expression of MAPK-NF-κB pathway, including P-p38, P-JNK, and P-ERK significantly increased. As shown in Figure 7A , pretreatment with C alone reduced the expression of P-JNK protein by 38%, but not obviously influenced the expression of P-p38 and P-ERK protein. In addition, the expression of P-p38, P-JNK and P-ERK protein was inhibited but not significantly influenced by R and I alone ( Figure 7B , C).

We further investigated whether C + R + I could synergistically increase and modulate the expression of key molecules in MAPK-NF-κB signaling pathway. Combined pretreatment with C + R + I, with the same concentration used alone for 3 h following exposure to LPS for 24 h significantly decreased the expressions of IκB, COX-2, iNOS, NF-κB P-p65, P-p38, P-JNK, and P-ERK (p < 0.05 or p < 0.01) (Figure 8) . Through comprehensive analysis of the data, an obvious synergistic effect was found for combination use of C + R + I. The binding affinity between each compound and nine proteins in these pathways were calculated by molecular docking. The results demonstrated that the three compounds can interact with multiple targets, and similar phenomena have been reported Chen et al., 2019a; Chen et al., 2019b) . C had a good affinity with the inhibitor binding site of iNOS (the binding energy was -8.39 kcal/mol), MPO (-7.34 kcal/mol) and p38 MAPK (-6.94 kcal/mol) ( Figure 9A-C) . R can target iNOS (-7.28 kcal/mol), ERK (-7.11 kcal/mol) and COX-2 (-6.96 kcal/ mol) ( Figure 9D-E) . I can interact with iNOS (-6.28 kcal/mol), MPO (-7.38 kcal/mol) and HO-1 (-6.76 kcal/mol) ( Figure 9G-I) . It is interesting that the three compounds had a common target (iNOS), which indicated an inhibitory effect on this pro-inflammatory cytokine. Therefore, the combined effect of C + R + I on suppressing MAPK-NF-κB signaling pathway would attribute to the interactions through multiple targets. . LPS-induced ALI in mice is a widely used animal model to examine ALI in vivo; this method can mimic the pathological characteristics of human ALI (Matthay et al., 2019; Zhou et al., 2019) . In this study, LPS was delivered to mice through intratracheal instillation to develop ALI. Pulmonary cells and alveolar macrophages of ALI release cytotoxic and inflammatory mediators, such as NO, and secrete proinflammatory cytokines, including iNOS, tumor necrosis factor TNF-α, interleukin (IL)-6 and IL-1β. The pro-inflammatory cytokines TNF-α and IL-1β stimulate the neighboring cells to generate more effective pro-inflammatory cytokines such as IL-6, and subsequently mediate the recruitment of PMNs, macrophages, and lymphocytes. S. glabra could reduce the FIGURE 8 | Combined effects of C + R + I (1 + 2+3) on suppressing the activation of MAPK-NF-κB signaling pathway in RAW264.7 cells stimulated by LPS. Lungs were harvested and analyzed by Western blotting. Values are indicated as the mean of four mice per group. *p < 0.05, **p < 0.01 as compared to the LPS group without C + R + I, #p < 0.05, ##p < 0.01 as compared to the normal control group without C + R + I. C, R, and I have multiple targets.

Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 generation of IL-6 and TNF-α in LPS-treated RAW264.7 cells Yang et al., 2019) , and in acute lung damage in LPS-intoxicated rats . In this paper, we studied the combined effect of three main constituents in S. glabra (chlorogenic acid, rosmarinic acid and isofraxidin) in treating and preventing ARDS. Our data showed that C + R + I greatly decreased the secretion of TNF-α, IL-1β, and IL-6 in serum and leukocyte in the lung of LPS-induced ALI, which suggested that C + R + I might reduce inflammatory cells infiltration into lung by reducing the production of pro-inflammatory cytokines (Liu, 2016) . NF-κB pathways is key for regulating inflammatory response by decreasing the inflammatory cells infiltration in ALI (Arcambal et al., 2019) . Phosphorylation of p65 plays central role in the NF-κB signaling cascade, and IκB is associated with p65. Our study found that C + R + I decreased the levels of LPSinduced phosphorylated NF-κB protein expression. To further evaluate whether IκB participated in this process, we detected the cytoplasmic levels of IκB protein. Our data demonstrated that C + R + I can suppress the expression and degradation of IκB in LPSstimulated RAW264.7 cells.

Heat shock protein (HO-1) has indispensable effects on restraining the NF-κB pathway. HO-1 is an enzyme catalyzing substrate to carbon monoxide and biliverdin, which participates in anti-oxidant and anti-inflammatory processes (Kim et al., 2016) . Our results indicated high expression of HO-1 in mice treated with LPS group. This data revealed that LPS activates NF-κB in macrophages, which could be effectively depressed by C + R + I. Meanwhile, we found that pretreatment with C + R + I inhibited the expression of INOS and COX-2 in LPS-induced RAW 264.7 cells.

Furthermore, MAPK is one of the upstream kinases in the NF-κB p65 phosphorylation pathway, in which MAPK activation is necessary at threonine and tyrosine residues within the kinase's activation loop. Phosphorylation of p38 MAPK and JNK induced by LPS can be inhibited by S. glabra . Our study demonstrated that LPS stimulation resulted in phosphorylation of ERK, p38 MAPK, and JNK in lung tissue, while C + R + I pretreatment prevented these manifestations. Parallel trends found that C + R + I inhibit the phosphorylation of MAPK and NF-κB p65. Western-blot results showed that C + R + I could decrease the expression of p-ERK, P-p38 and p-JNK. In addition, MAPK pathway has an important role in macrophage activation and infiltration (Shu et al., 2018) . Superoxide dismutase (SOD) is an antioxidant metal enzyme in organisms and is related to the activation of P38 signaling pathway. Our study showed that pretreated with C + R + I was able to promote the activation of SOD.

MPO is a heme-containing peroxidase abundantly expressed in macrophage (Li et al., 2015; Wu et al., 2019) . It catalyzes the Frontiers in Pharmacology | www.frontiersin.org January 2021 | Volume 11 | Article 580064 generation of hypochlorous acid by consuming hydrogen peroxide, which in turn decreases the secretion of TNF-α, IL-6, and IL-1β (Fernandez and Walters, 1996; Wu et al., 2019) . In this study, we found that C + R + I could alleviate LPS-induced acute lung injury of mice. It decreased LPS-induced ALI effectively by inhibiting infiltration of leukocytes and histopathological changes in the lungs. MPO was obviously decreased after the treatment with C + R + I, which induced over-production of NO from iNOS that is important mediator in the pathogenesis of inflammation. Results showed that IL-1β, IL-6, and TNF-αsuppressed downstream signaling pathways, thus decreasing iNOS and COX-2 protein expression. These results indicated that the main components (chlorogenic acid, isofraxidin and rosmarinic acid) from Sarcandra glabra exerted anti-inflammatory effect through multiple targets. In summary, the action mechanism of the combination of three main constituents C + R + I from S. glabra were studied. The result ravels that C + R + I target MAPK-NF-κB pathway in LPS stimulated RAW 264.7 cells, as well as in mice with LPS-induced acute lung injury. C + R + I extracted from S. glabra decreased oxidative stress and inflammatory response by inhibiting the activation of the macrophages resulting in decreasing the secretion of NO, IL-6, and TNF-α ( Figure 10) . In conclusion, this study provides new views into the potential treatment with S. glabra and shows a promising candidate for treating ALI/ARDS caused by pneumonia, COVID-19, appendicitis, and so on.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Ethics Committee of Experimental Animals of Guangdong Hospital of Traditional Chinese Medicine.

C-PL and J-HL mainly for the experiment carrying out and article writing, XL mainly for experiment design, J-XL data sorting and statistics, JG for molecular docking, FL, BY, JL, and the QW mainly to modify articles; XZ, L-ML, H-LY, and LW focuses on the language of the article polish. funding sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

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