# Qi-Dong-Huo-Xue-Yin Inhibits Inflammation in Acute Lung Injury in Mice via Toll-Like Receptor 4/Caveolin-1 Signaling

Authors: Li-Ying Xu; Wan-Ru Cai; Chun-Fang Ma; Qi-Yang Shou; Jing-Li Qian; Turan S. Huseyin
Journal: Evidence-Based Complementary and Alternative Medicine
 (2018)

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## Abstract

Acute lung injury (ALI) is a critical illness with no current effective treatment. Caveolin-1 indirectly activates inflammation-associated signaling pathways by inhibiting endothelial nitric oxide synthase (eNOS). This induces an imbalance between pro- and anti-inflammatory cytokine levels, which are involved in the pathogenesis of ALI. The compound Chinese prescription Qi-Dong-Huo-Xue-Yin (QDHXY) is efficacious for ALI treatment via an anti-inflammatory effect; however, the exact underlying mechanism is unknown. Therefore, we explored the protective effect of QDHXY against lipopolysaccharide- (LPS-) induced ALI in mice. Histopathological changes in mouse lung tissues were studied. Furthermore, alterations in the serum levels of pro- and anti-inflammatory cytokines were investigated. The levels of tumor necrosis factor- (TNF-)α, interleukin- (IL-) 6, IL-1β, and interferon-γ-induced protein 10 in bronchoalveolar lavage fluid were measured. Additionally, the expression levels of myeloid differentiation factor 88 (MyD88), caveolin-1, and eNOS were assessed. QDHXY significantly reduced lung infiltration with inflammatory cells and the production of serum pro- and anti-inflammatory cytokines and inhibited the expression of TNF-α, IL-1β, caveolin-1, and MyD88 but not eNOS. These indicate that QDHXY significantly improved the balance between pro- and anti-inflammatory cytokine levels, possibly by inhibiting the caveolin-1 signaling pathway. Therefore, QDHXY may be a potential treatment for ALI.


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## Body

## 1. Introduction

Acute lung injury (ALI) is an emergency and severe respiratory disease that results from various direct and indirect insults such as severe infection, trauma, shock, acidosis, and inhalation of harmful gases. It is a leading cause of morbidity and mortality in critically ill patients. ALI is characterized by acute hypoxemic respiratory failure with bilateral pulmonary infiltrates [1, 2]. The precise mechanisms underlying the cellular activation that results in ALI are poorly understood, which hinders the discovery of effective treatments [3].Furthermore, increasing evidence shows that an imbalance between pro- and anti-inflammatory cytokine levels affect the severity of ALI [3, 4]. Therefore, focusing on inhibiting inflammation may be a potential strategy in the prevention and treatment of ALI. Caveolin-1 is a cytoskeleton protein associated with the cell membrane that plays an important role in ALI-related inflammation [5]. It upregulates proinflammatory cytokine levels and inhibits nuclear factor- (NF-) κB and p38 mitogen-activated protein kinase (MAPK) signaling pathways via interaction with endothelial nitric oxide synthase (eNOS).Traditional Chinese Medicine has been used in the treatment of diseases in China and other Asian countries for thousands of years. Qi-Dong-Huo-Xue-Yin (QDHXY), which is composed ofAstragalus membranaceus,Radix Ophiopogonis,Polygonum cuspidatum,Angelica sinensis, andRheum officinale,has been shown to have a notable effect on ALI. A previous study reported that this decoction protected the lung tissue of mice with ALI from inflammation [6]. However, the molecular mechanisms underlying this effect are unclear.In the present study, the effects of QDHXY on the secretion of proinflammatory cytokines (such as granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage CSF [GM-CSF], interferon- [IFN-]γ, interleukin- [IL-] 1β, IL-5, IL-6, IL-7, IL-15, IL-17, IFN-γ-induced protein 10 [IP-10], growth-regulated oncogene α [GROα]/keratinocyte chemoattractant [KC], monocyte chemotactic protein- [MCP-] 1, macrophage inflammatory protein- [MIP-] 1α, MIP-1β, regulated on activation normal T cell expressed and secreted [RANTES], tumor necrosis factor- [TNF-] α) and anti-inflammatory cytokines (such as IL-10 and IL-13) were investigated in mice with lipopolysaccharide- (LPS-) induced ALI. The expression levels of myeloid differentiation factor 88 (MyD88) and caveolin-1 were also evaluated.


## 2. Materials and Methods

### 2.1. Reagents and Antibodies

Fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit anti-mouse MyD88, caveolin-1, and eNOS antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were purchased from OriGene (Beijing, China). All primers were synthesized at Tsingke (Hangzhou, China). Ethylenediaminetetraacetic acid and phosphate-buffered saline (PBS) were purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Enzyme-linked immunosorbent assay (ELISA) kits for detecting mouse IL-6, IL-1β, IP-1, and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Luminex liquid chip was purchased from EMD Millipore (Billerica, MA, USA).


### 2.2. Preparation of QDHXY Decoction

QDHXY, comprising 20 gAstragalus membranaceus, 12 gRadix Ophiopogonis, 20 gPolygonum cuspidatum, 12 gAngelica sinensis, and 9 gRheum officinale, was purchased from the Pharmacy of The First Affiliated Hospital of Zhejiang Chinese Medical University. The herbs were boiled in water (8–10 times the weight of the herbs) for 30 min and steeped for 30 min to obtain the decoction, which was then concentrated for 25 min. Dilute solutions were subsequently prepared at concentrations of 1 and 2 g/mL in 3–5 times the weight of water.


### 2.3. Animals

Specific-pathogen-free BALB/c mice (female; weight: 20 g) were obtained from Slaccas (Shanghai, China) for the study. Protocols for the animal experiments were approved by the Animal Care and Use Committee of Zhejiang Chinese Medicine University. The animals were divided into the following groups and treated as indicated: normal control (0.9% sodium chloride [NaCl]), ALI (LPS and 0.9% NaCl), ALI + LDQ (LPS and low-dose [1 g/mL] QDHXY), ALI + HDQ (LPS and high-dose [2 g/mL] QDHXY), and ALI + DEX (LPS and dexamethasone).The rats were administered 0.9% NaCl, QDHXY, or dexamethasone (each 0.4 mL once daily) intragastrically on days 1–6, whereas LPS was administered by endotracheal instillation. LPS-induced ALI was established on day 5 as follows: At 2 h after intragastric administration of the various treatments, the mice were anesthetized, their tracheas were exposed, and 50μg LPS (5 mg/kg) was instilled. Mice in the normal control group received 50 μg 0.9% NaCl (5 mL/kg) instead of LPS and analepsia occurred after 1-2 min.All the mice were euthanized 24 h after ALI was induced and the bronchoalveolar lavage fluid (BALF) from the right lung was collected for ELISA. The left lung was examined by immunohistochemical analysis, hematoxylin and eosin (H&E) staining, and quantitative polymerase chain reaction (qPCR). Blood samples were collected via the orbital vein after removing the eyeball. Serum was then obtained and analyzed using Luminex assay.


### 2.4. H&E Staining

One-third of the left lung tissue of each mouse was excised, fixed in 4% formaldehyde for 24 h, embedded in paraffin, cut into 5-μm-thick sections, and H&E-stained. The nuclei and cytoplasm were stained with alum hematoxylin and eosin, respectively, and the sections were analyzed under an inverted microscope to study pathomorphological tissue changes.


## 2.1. Reagents and Antibodies

Fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit anti-mouse MyD88, caveolin-1, and eNOS antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were purchased from OriGene (Beijing, China). All primers were synthesized at Tsingke (Hangzhou, China). Ethylenediaminetetraacetic acid and phosphate-buffered saline (PBS) were purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Enzyme-linked immunosorbent assay (ELISA) kits for detecting mouse IL-6, IL-1β, IP-1, and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Luminex liquid chip was purchased from EMD Millipore (Billerica, MA, USA).


## 2.2. Preparation of QDHXY Decoction

QDHXY, comprising 20 gAstragalus membranaceus, 12 gRadix Ophiopogonis, 20 gPolygonum cuspidatum, 12 gAngelica sinensis, and 9 gRheum officinale, was purchased from the Pharmacy of The First Affiliated Hospital of Zhejiang Chinese Medical University. The herbs were boiled in water (8–10 times the weight of the herbs) for 30 min and steeped for 30 min to obtain the decoction, which was then concentrated for 25 min. Dilute solutions were subsequently prepared at concentrations of 1 and 2 g/mL in 3–5 times the weight of water.


## 2.3. Animals

Specific-pathogen-free BALB/c mice (female; weight: 20 g) were obtained from Slaccas (Shanghai, China) for the study. Protocols for the animal experiments were approved by the Animal Care and Use Committee of Zhejiang Chinese Medicine University. The animals were divided into the following groups and treated as indicated: normal control (0.9% sodium chloride [NaCl]), ALI (LPS and 0.9% NaCl), ALI + LDQ (LPS and low-dose [1 g/mL] QDHXY), ALI + HDQ (LPS and high-dose [2 g/mL] QDHXY), and ALI + DEX (LPS and dexamethasone).The rats were administered 0.9% NaCl, QDHXY, or dexamethasone (each 0.4 mL once daily) intragastrically on days 1–6, whereas LPS was administered by endotracheal instillation. LPS-induced ALI was established on day 5 as follows: At 2 h after intragastric administration of the various treatments, the mice were anesthetized, their tracheas were exposed, and 50μg LPS (5 mg/kg) was instilled. Mice in the normal control group received 50 μg 0.9% NaCl (5 mL/kg) instead of LPS and analepsia occurred after 1-2 min.All the mice were euthanized 24 h after ALI was induced and the bronchoalveolar lavage fluid (BALF) from the right lung was collected for ELISA. The left lung was examined by immunohistochemical analysis, hematoxylin and eosin (H&E) staining, and quantitative polymerase chain reaction (qPCR). Blood samples were collected via the orbital vein after removing the eyeball. Serum was then obtained and analyzed using Luminex assay.


## 2.4. H&E Staining

One-third of the left lung tissue of each mouse was excised, fixed in 4% formaldehyde for 24 h, embedded in paraffin, cut into 5-μm-thick sections, and H&E-stained. The nuclei and cytoplasm were stained with alum hematoxylin and eosin, respectively, and the sections were analyzed under an inverted microscope to study pathomorphological tissue changes.


## 3. Immunohistochemical Analysis

Each lung tissue sample was segmented into three random sections and analyzed immunohistochemically to evaluate the expression of MyD88, caveolin-1, and eNOS. The sections were dewaxed in xylene and anhydrous alcohol, cultured in 0.3% hydrogen peroxide to eliminate intrinsic peroxidase, and incubated at 18–20°C in the dark for 10 min. After antigen retrieval, the sections were incubated with the following antibodies: anti-MyD88 (1 : 200, Cell Signaling Technology, ab2068), anti-caveolin-1 (1 : 600, Cell Signaling Technology, 3267s), and anti-eNOS (1 : 200, Cell Signaling Technology, 9586s) for 1 h at 18–20°C, using PBS as the control.The samples were incubated with biotinylated goat anti-mouse secondary antibody for 30 min at 18–20°C. 3,3′-Diaminobenzidine and Mayer’s hematoxylin were used for color development and counterstaining, respectively. The slides were analyzed using an Olympus BX41 microscope at ×400 magnification. The intensity of brown-positive staining in lung tissues was analyzed by the integrated optical density (IOD) method using Image-Pro® Plus software (version 5.1; Media Cybernetics, Inc., Rockville, MD, USA). All images were captured using the same light microscope and camera system (Leica Microsystems GmbH, Wetzlar, Germany). The average IOD per stained area (IOD/area,μm2) was calculated for the positively stained tissues.


### 3.1. ELISA

The concentrations of TNF-α, IL-6, IL-1β, and IP-10 in mouse BALF were measured using ELISA kits. BALF samples were collected from the left lung of each mouse using PBS at 24 h after the last treatment and stored at −80°C until analysis. ELISA was performed using the corresponding kits according to the manufacturer’s instructions.


### 3.2. Luminex Assay

Luminex magnetic bead assay (Bio-Techne, Minneapolis, MN, USA) was performed to detect the following 18-plex mouse cytokines in serum: G-CSF, GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-10, IL-13, IL-15, IL-17, IP-10/C-X-C motif chemokine 10 (CXCL10), KC, MCP-1/chemokine (C-C motif) ligand (CCL) 2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, and TNF-α. The cytokine levels were assessed using Luminex xMAP technology with multianalyte profiling beads. Data were analyzed using Bio-Plex software (Bio-Rad, Hercules, CA, USA). All samples were run in triplicate.


### 3.3. qPCR

Total RNA was extracted from tissues and cells using TRIzol reagent and reverse transcribed using Superscript Reverse Transcription kit (Thermo Fisher). Real-time qPCR was performed using SYBR Green Master Mix kit (Bio-Rad) and a Bio-Rad C1000 thermal cycler. The following primers were used: TNF-α forward, 5′-GCCTCCCTCTCATCAGTTCTA-3′; TNF-α reverse, 5′-GGCAGCCTTGTCCCTTG-3′; IL-1β forward, 5′-ACCTGTGTCTTTCCCGTGG-3′; IL-1β reverse, 5′-TCATCTCGGAGCCTGTAGTG-3′; IL-6 forward, 5′-AGTTGTGCAATGGCAATTCTGA-3′; IL-6 reverse, 5′-AGGACTCTGGCTTTGTCTTTCT-3′; caveolin-1 forward, 5′-ACAGCCCAGGGAAACCTC-3′; caveolin-1 reverse, 5′-GATGGGAACGGTGTAGAGATG-3′; β-actin forward, 5′-TGCTGTCCCTGTATGCCTCT-3′; and β-actin reverse, 5′-TTTGATGTCACGCACGATTT-3.′ mRNA expression was normalized to β-actin mRNA levels.


### 3.4. Statistical Analysis

Data were analyzed using the Statistical Package for the Social Sciences for Windows (version 22; IBM Corporation, Chicago, IL, USA). All the data have been expressed as mean ± standard deviation (SD).P values < 0.05 were considered statistically significant. Each experiment was repeated at least three times.


## 3.1. ELISA

The concentrations of TNF-α, IL-6, IL-1β, and IP-10 in mouse BALF were measured using ELISA kits. BALF samples were collected from the left lung of each mouse using PBS at 24 h after the last treatment and stored at −80°C until analysis. ELISA was performed using the corresponding kits according to the manufacturer’s instructions.


## 3.2. Luminex Assay

Luminex magnetic bead assay (Bio-Techne, Minneapolis, MN, USA) was performed to detect the following 18-plex mouse cytokines in serum: G-CSF, GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-10, IL-13, IL-15, IL-17, IP-10/C-X-C motif chemokine 10 (CXCL10), KC, MCP-1/chemokine (C-C motif) ligand (CCL) 2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, and TNF-α. The cytokine levels were assessed using Luminex xMAP technology with multianalyte profiling beads. Data were analyzed using Bio-Plex software (Bio-Rad, Hercules, CA, USA). All samples were run in triplicate.


## 3.3. qPCR

Total RNA was extracted from tissues and cells using TRIzol reagent and reverse transcribed using Superscript Reverse Transcription kit (Thermo Fisher). Real-time qPCR was performed using SYBR Green Master Mix kit (Bio-Rad) and a Bio-Rad C1000 thermal cycler. The following primers were used: TNF-α forward, 5′-GCCTCCCTCTCATCAGTTCTA-3′; TNF-α reverse, 5′-GGCAGCCTTGTCCCTTG-3′; IL-1β forward, 5′-ACCTGTGTCTTTCCCGTGG-3′; IL-1β reverse, 5′-TCATCTCGGAGCCTGTAGTG-3′; IL-6 forward, 5′-AGTTGTGCAATGGCAATTCTGA-3′; IL-6 reverse, 5′-AGGACTCTGGCTTTGTCTTTCT-3′; caveolin-1 forward, 5′-ACAGCCCAGGGAAACCTC-3′; caveolin-1 reverse, 5′-GATGGGAACGGTGTAGAGATG-3′; β-actin forward, 5′-TGCTGTCCCTGTATGCCTCT-3′; and β-actin reverse, 5′-TTTGATGTCACGCACGATTT-3.′ mRNA expression was normalized to β-actin mRNA levels.


## 3.4. Statistical Analysis

Data were analyzed using the Statistical Package for the Social Sciences for Windows (version 22; IBM Corporation, Chicago, IL, USA). All the data have been expressed as mean ± standard deviation (SD).P values < 0.05 were considered statistically significant. Each experiment was repeated at least three times.


## 4. Results

### 4.1. QDHXY Attenuates Inflammation in ALI

Dexamethasone, which is used clinically to treat inflammation, was used as the positive control drug in this study. H&E staining was used to assess inflammatory cell infiltration in the lungs of the mice. The mice in the ALI group showed the following signs: destroyed, edematous, and small alveoli with hemorrhagic or dilated capillaries; pneumatocele; and thickened alveolar septum. In addition, there were numerous inflammatory cells including neutrophils and macrophages in the lung parenchyma (Figure1). These signs indicated that the ALI model was established successfully. After treatment with QDHXY and dexamethasone, the inflammatory cell infiltration was markedly reduced, and the alveolar structure was maintained. Furthermore, the effect of the decoction was more marked in the ALI + HDQ group than it was in the ALI + LDQ group.Figure 1
Effects of QDHXY on pathological changes in the lung tissues of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days. They were then administered 50 μL of 1 mg/mL LPS by endotracheal instillation on day 5. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). Changes in lung tissue were observed under a microscope. The red and black arrows show thickening of the alveolar septum and pulmonary congestion, respectively. The yellow and blue arrows show the lung parenchyma with alveolar atrophy or expansion and pulmonary bullae formation due to alveolar fusion and expansion, respectively. The green arrows show inflammatory cell infiltration. Magnification: ×200.


### 4.2. QDHXY Reduces Inflammatory Cytokine Secretion in BALF of Mice with ALI

The levels of TNF-α, IL-1β, IL-6, and CXCL10/IP-10 in BALF were significantly higher in the ALI group than they were in the normal control group. However, the levels of these cytokines were inhibited by QDHXY and dexamethasone. The high-dose QDHXY showed a stronger effect than the low-dose QDHXY did on the levels of all the cytokines except IL-1β (Figure 2).Figure 2
Effects of QDHXY on the expression of inflammatory cytokines in the BALF of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The levels of cytokines (TNF-α, IL-1β, IL-6, and CXCL10) in BALF were assayed by ELISA. All the data are presented as ratios against a blank control and as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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### 4.3. QDHXY Inhibits mRNA Expression of Inflammatory Cytokines in Mice with LPS-Induced ALI

qPCR was performed to evaluate the effects of QDHXY on ALI in mice and investigate whether QDHXY functions by interacting with caveolin-1. As shown in Figure3, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in lung tissues were greatly increased and higher in the ALI group than they were in the normal control group. However, the levels were reduced by QDHXY and dexamethasone. Furthermore, we found that the decoction and dexamethasone reduced LPS-induced increase in caveolin-1 levels. Taken together, the results show that QDHXY exerted its anti-inflammatory effect by downregulating the expression of caveolin-1 in the mice.Figure 3
Effects of QDHXY on the expression levels of inflammatory cytokines and caveolin-1 in the lungs of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The mRNA levels of IL-6, TNF-α, IL-1β, and caveolin-1 in lung tissue were assayed by real-time PCR using SYBR Green. β-Actin was used as the endogenous control. All the data are ratios against a normal control and have been presented as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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### 4.4. Effects of QDHXY on the Balance between Serum Pro- and Anti-Inflammatory Cytokine Expression Levels in Mice with ALI

In order to further investigate the anti-inflammatory effect of QDHXY on ALI-induced changes in cytokine levels, blood was sampled from the eyes of the mice and serum cytokine levels were evaluated by the Luminex assay. As shown in Figure4, after endotracheal instillation of LPS, the serum levels of GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-10, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, and IL-13 significantly increased. However, the levels of these cytokines in the ALI + LDQ and ALI + HDQ groups were significantly lower than they were in the ALI group. A similar trend was observed in the ALI + DEX group.Figure 4
Inhibitory effect of QDHXY on the expression of cytokines in the serum of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The levels of proinflammatory cytokines [(a) IL-5, IL-7, IL-17; (b) IL-1β; (c) IL-6, G-CSF, GM-CSF; (d) IL-15, TNF-α, IFN-γ; (e) MCP-1, MIP-1α, MIP-1β; and (f) IP-10, KC, RANTES] and anti-inflammatory cytokines [(b) IL-10 and IL-13] in serum were assayed using a MILLIPLEX MAP kit. All data are ratios of a normal control and expressed as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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(f)Furthermore, the levels of GM-CSF, IL-5, IL-6, IL-7, IL-10, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, and IL-13 in the ALI + LDQ group were lower than those in the ALI + HDQ group. However, a contrasting trend was observed for IFN-γ, IL-1β, and IL-15 levels in the two groups.


### 4.5. Effects of QDHXY on Immunohistochemical Expression of Caveolin-1, eNOS, and MyD88 in Lung Tissues of Mice with ALI

The immunohistochemical expression of inflammation-related proteins such as caveolin-1, eNOS, and MyD88 in the cytoplasm and nuclei of epithelial, alveolar, and smooth muscle cells were seen as yellow or brown grains (Figure5). In the ALI group, the expression levels of caveolin-1 and MyD88 increased, whereas that of eNOS decreased.Figure 5
Effects of QDHXY on caveolin-1, MyD88, and eNOS expression in the lungs of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were administered 50 μL of 1 mg/mL LPS via endotracheal instillation. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). Lung tissues were studied immunohistochemically. Changes in the expression levels of (a) caveolin-1, (b) MyD88, and (c) eNOS in the lung were observed under a microscope (original magnification: ×400). The positively stained cells are seen as yellow or brown granules in the cytoplasm and nuclei. (d) IOD/area of caveolin-1, MyD88, and eNOS in the lung were calculated. Data were analyzed using Image-Pro Plus software. All data are ratios of the normal control and expressed as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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(d)Similar to the trend of the immunohistochemical expression of the proteins, the IOD/area results indicated that QDHXY decreased the expression of caveolin-1 and eNOS. However, it increased the expression of eNOS in the lung tissues more than dexamethasone did (Figure5).


## 4.1. QDHXY Attenuates Inflammation in ALI

Dexamethasone, which is used clinically to treat inflammation, was used as the positive control drug in this study. H&E staining was used to assess inflammatory cell infiltration in the lungs of the mice. The mice in the ALI group showed the following signs: destroyed, edematous, and small alveoli with hemorrhagic or dilated capillaries; pneumatocele; and thickened alveolar septum. In addition, there were numerous inflammatory cells including neutrophils and macrophages in the lung parenchyma (Figure1). These signs indicated that the ALI model was established successfully. After treatment with QDHXY and dexamethasone, the inflammatory cell infiltration was markedly reduced, and the alveolar structure was maintained. Furthermore, the effect of the decoction was more marked in the ALI + HDQ group than it was in the ALI + LDQ group.Figure 1
Effects of QDHXY on pathological changes in the lung tissues of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days. They were then administered 50 μL of 1 mg/mL LPS by endotracheal instillation on day 5. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). Changes in lung tissue were observed under a microscope. The red and black arrows show thickening of the alveolar septum and pulmonary congestion, respectively. The yellow and blue arrows show the lung parenchyma with alveolar atrophy or expansion and pulmonary bullae formation due to alveolar fusion and expansion, respectively. The green arrows show inflammatory cell infiltration. Magnification: ×200.


## 4.2. QDHXY Reduces Inflammatory Cytokine Secretion in BALF of Mice with ALI

The levels of TNF-α, IL-1β, IL-6, and CXCL10/IP-10 in BALF were significantly higher in the ALI group than they were in the normal control group. However, the levels of these cytokines were inhibited by QDHXY and dexamethasone. The high-dose QDHXY showed a stronger effect than the low-dose QDHXY did on the levels of all the cytokines except IL-1β (Figure 2).Figure 2
Effects of QDHXY on the expression of inflammatory cytokines in the BALF of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The levels of cytokines (TNF-α, IL-1β, IL-6, and CXCL10) in BALF were assayed by ELISA. All the data are presented as ratios against a blank control and as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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## 4.3. QDHXY Inhibits mRNA Expression of Inflammatory Cytokines in Mice with LPS-Induced ALI

qPCR was performed to evaluate the effects of QDHXY on ALI in mice and investigate whether QDHXY functions by interacting with caveolin-1. As shown in Figure3, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in lung tissues were greatly increased and higher in the ALI group than they were in the normal control group. However, the levels were reduced by QDHXY and dexamethasone. Furthermore, we found that the decoction and dexamethasone reduced LPS-induced increase in caveolin-1 levels. Taken together, the results show that QDHXY exerted its anti-inflammatory effect by downregulating the expression of caveolin-1 in the mice.Figure 3
Effects of QDHXY on the expression levels of inflammatory cytokines and caveolin-1 in the lungs of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The mRNA levels of IL-6, TNF-α, IL-1β, and caveolin-1 in lung tissue were assayed by real-time PCR using SYBR Green. β-Actin was used as the endogenous control. All the data are ratios against a normal control and have been presented as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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## 4.4. Effects of QDHXY on the Balance between Serum Pro- and Anti-Inflammatory Cytokine Expression Levels in Mice with ALI

In order to further investigate the anti-inflammatory effect of QDHXY on ALI-induced changes in cytokine levels, blood was sampled from the eyes of the mice and serum cytokine levels were evaluated by the Luminex assay. As shown in Figure4, after endotracheal instillation of LPS, the serum levels of GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-10, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, and IL-13 significantly increased. However, the levels of these cytokines in the ALI + LDQ and ALI + HDQ groups were significantly lower than they were in the ALI group. A similar trend was observed in the ALI + DEX group.Figure 4
Inhibitory effect of QDHXY on the expression of cytokines in the serum of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The levels of proinflammatory cytokines [(a) IL-5, IL-7, IL-17; (b) IL-1β; (c) IL-6, G-CSF, GM-CSF; (d) IL-15, TNF-α, IFN-γ; (e) MCP-1, MIP-1α, MIP-1β; and (f) IP-10, KC, RANTES] and anti-inflammatory cytokines [(b) IL-10 and IL-13] in serum were assayed using a MILLIPLEX MAP kit. All data are ratios of a normal control and expressed as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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(f)Furthermore, the levels of GM-CSF, IL-5, IL-6, IL-7, IL-10, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, and IL-13 in the ALI + LDQ group were lower than those in the ALI + HDQ group. However, a contrasting trend was observed for IFN-γ, IL-1β, and IL-15 levels in the two groups.


## 4.5. Effects of QDHXY on Immunohistochemical Expression of Caveolin-1, eNOS, and MyD88 in Lung Tissues of Mice with ALI

The immunohistochemical expression of inflammation-related proteins such as caveolin-1, eNOS, and MyD88 in the cytoplasm and nuclei of epithelial, alveolar, and smooth muscle cells were seen as yellow or brown grains (Figure5). In the ALI group, the expression levels of caveolin-1 and MyD88 increased, whereas that of eNOS decreased.Figure 5
Effects of QDHXY on caveolin-1, MyD88, and eNOS expression in the lungs of mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were administered 50 μL of 1 mg/mL LPS via endotracheal instillation. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). Lung tissues were studied immunohistochemically. Changes in the expression levels of (a) caveolin-1, (b) MyD88, and (c) eNOS in the lung were observed under a microscope (original magnification: ×400). The positively stained cells are seen as yellow or brown granules in the cytoplasm and nuclei. (d) IOD/area of caveolin-1, MyD88, and eNOS in the lung were calculated. Data were analyzed using Image-Pro Plus software. All data are ratios of the normal control and expressed as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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(d)Similar to the trend of the immunohistochemical expression of the proteins, the IOD/area results indicated that QDHXY decreased the expression of caveolin-1 and eNOS. However, it increased the expression of eNOS in the lung tissues more than dexamethasone did (Figure5).


## 5. Discussion

ALI is a complex clinical syndrome involving acute inflammation and microvascular damage to the lungs. It is also characterized by increased pulmonary vascular and epithelial permeability. ALI frequently results in acute respiratory failure, which leads to potentially fatal acute respiratory distress syndrome [1–3]. It is very important to understand the mechanism underlying the pathogenesis of ALI to prevent disease progression and choose an effective treatment.Latest studies showed that the severity of ALI derives from the cumulative effects of several risky factors. Dysregulated accumulation of leukocytes into the alveolar space, vascular hyperpermeability, impairment of edema clearance, and cell death are all associated with lung injury [6]. Treatment options on ALI with inflammatory disorder are limited; therefore, ALI is proved to be a major cause of mortality in adult and pediatric critical care medicine [7]. In addition, there are also study reports that showed that Toll-like receptor 4 (TLR4) mutant mice display natural resistance to acid-induced ALI, which indicates that TLR4-MyD88 signaling is a key disease pathway controlling the severity of ALI [8]. Therefore, canonical TLR4-MyD88 pathway seems to be “druggable” target, representing putative specific therapies for patients with ALI.QDHXY has been used to effectively treat ALI for three decades because of its anti-inflammatory effect. However, its mechanism of action is not fully understood [9]. The results of our previous study showed that the protein and mRNA expression levels of TLR4 and NF-κB p65 increased in rats with ALI; however, they were reduced by QDHXY in a dose-dependent manner. In the present study, we found that QDHXY suppressed the expression of caveolin-1 and MyD88 but enhanced the expression of eNOS, which mediates anti-inflammatory signaling pathways. Consequently, the expression levels of pro- and anti-inflammatory cytokines were affected. The data obtained are consistent with those of previous studies [10, 11].The findings of the present study show that QDHXY inhibited inflammatory cell secretion and ameliorated lung injury. In addition, it inhibited the secretion of G-CSF, GM-CSF, IFN-γ, IL-1β, IL-5, IL-6, IL-7, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α. However, we found that it also inhibited the secretion of anti-inflammatory cytokines such as IL-10 and IL-13.In order to evaluate the effect of QDHXY on the balance between pro- and anti-inflammatory cytokine levels, IL-6/IL-10 and GM-CSF/IL-13 ratios were analyzed (Figure6). The results showed statistically significant correlations in the ratios between the normal control and ALI groups (P< 0.05). After treatment with the decoction, IL-6/IL-10 and GM-CSF/IL-13 ratios were significantly reduced. Similar trends in the ratios were observed in the ALI + DEX group. The results indicate that QDHXY restored the ALI-induced imbalance between pro- and anti-inflammatory responses.Figure 6
Regulation of the balance between serum pro- and anti-inflammatory cytokine levels by QDHXY in mice with LPS-induced ALI. Mice were pretreated with QDHXY (0.4 or 0.8 g) or dexamethasone (0.1 mg) daily for 6 days, after which they were treated with 50 μL of 1 mg/mL LPS. The five groups were, respectively, labeled as normal control, ALI, ALI + LDQ (LPS and low-dose QDHXY), ALI + HDQ (LPS and high-dose QDHXY), and ALI + DEX (LPS and dexamethasone). The serum ratios of (a) IL-6 to IL-10 and (b) GM-CSF to IL-13 were calculated. All data are ratios of the normal control and expressed as mean ± SD of three independent experiments. # indicates P < 0.01 and ## indicates P < 0.05 when compared to the normal control and ALI groups, respectively.
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(b)The key mechanism involved in the pathogenesis of ALI is inflammatory cell infiltration, which results in an imbalance between pro- and anti-inflammatory cytokine levels [3]. Early inflammatory factors such as IL-1β, IL-6, and TNF-α are synthesized and secreted by neutrophils, granulocytes, and macrophages following stimulation by GM-CSF and G-CSF [12–16]. However, IL-7 enhances macrophage activity, which generates IL-15 and IL-17. IL-15 activates T and natural killer cells to produce IFN-γ, which suppresses the production of anti-inflammatory cytokines such as IL-13 and IL-10. Moreover, IL-17 effectively mediates neutrophil mobilization during inflammatory responses. The production of chemokines such as MCP-1, MIP-1, IP-10, and RANTES is induced by TNF-α and IFN-γ in macrophages and endothelial cells [5, 17–19]; however, the relationship between IL-5 and ALI is currently unknown [20].TLR4 is considered a pattern-recognition receptor in ALI that binds to LPS on the cytomembrane. This results in the secretion of cytokines that are regulated by NF-κB, p38 MAPK, and c-Jun N-terminal kinase (JNK) through the MyD88 signaling pathway [21–29]. Caveolin-1, a principle transmembrane cytoskeletal protein located on neutrophils, granulocytes, macrophages, and endothelial cells, mediates inflammatory responses. It is reported to have an important role in ALI [9, 30–37]. Caveolin-1 binds to TLR4 and inhibits LPS-induced production of proinflammatory cytokines such as TNF-α and IL-6. In contrast, eNOS suppresses the NF-kB, p38MAPK, and JNK signaling pathways to inhibit inflammatory responses [38] (Figure 7).Figure 7
Speculated mechanism involved in the mediation of inflammatory responses in ALI and the related signaling pathways (⊥: inhibition; ↓: facilitation).Our findings confirmed that QDHXY inhibited inflammation in the serum, BALF, and lung tissues of the mice. This was mediated by the TLR4/caveolin-1 signal transduction pathway via inhibition of caveolin-1 and MyD88 expression and enhancement of eNOS expression. Moreover, we found that the decoction showed stronger effects at a high dose. Further studies are needed to elucidate the specific p38MAPK- and JNK-related mechanisms underlying the effects of QDHXY.


## 6. Conclusion

Our results demonstrate that QDHXY suppresses inflammation by enhancing eNOS expression and inhibiting the expression of caveolin-1 and MyD88, which might facilitate its clinical applications in the management of ALI.



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