Inflammation ( # 2014) DOI: 10.1007/s10753-014-9833-1
Protective Effect of Carvacrol on Acute Lung Injury Induced by Lipopolysaccharide in Mice Xiaosheng Feng1,2 and Aiqing Jia1
Abstract—Carvacrol, the major component of Plectranthus amboinicus, has been known to exhibit antiinflammatory activities. The aim of this study was to investigate the effects of carvacrol on lipopolysaccharide (LPS)-induced endotoxemia and acute lung injury (ALI) in mice. Mice were injected intraperitoneally (i.p.) with LPS and the mortality of mice for 7 days were observed twice a day. Meanwhile, the protective effect of carvacrol (20, 40 or 80 mg/kg) on LPS-induced endotoxemia were detected. Using an experimental model of LPS-induced ALI, we examined the effect of carvacrol in resolving lung injury. The results showed that carvacrol could improve survival during lethal endotoxemia and attenuate LPS-induced ALI in mice. The anti-inflammatory mechanisms of carvacrol may be due to its ability to inhibit NF-κB and MAPKs signaling pathways, thereby inhibiting inflammatory cytokines TNF-α, IL-6 and IL-1β production. KEY WORDS: carvacrol; acute lung injury; LPS; NF-κB; MAPK.
INTRODUCTION The acute respiratory distress syndrome (ARDS), a common clinical catastrophe following acute lung injury (ALI), is a significant cause of morbidity and mortality in critically ill patients [1–4]. ALI/ARDS is characterized by non-cardiogenic pulmonary edema, pulmonary and systemic inflammation with a spectrum of increasing severity of lung injury resulting in respiratory failure [5]. Lipopolysaccharide (LPS) is the major stimulus for the release of inflammatory mediators [6], which is released during sepsis. Administration of LPS in experimental animals causes the pathological condition of ongoing sepsis and concomitant ARDS-like lung injury, including polymorphonuclear neutrophil sequestration and lung edema [7]. Inflammatory stimuli from microbial pathogens, such as endotoxin (LPS), are well recognized for their ability to induce pulmonary inflammation, and experimental administration of LPS, both systemically and intratracheally, has been used
to induce pulmonary inflammation in animal models of ALI. Carvacrol (Fig. 1), a monoterpenic phenol which occurs in many essential oils, has been reported to have a number of pharmacological activities such as antioxidant, anti-titumor and anti-inflammatory effects. Carvacrol was found to inhibit COX-2 expression in LPS-activated U937 cells. In addition, some reports have demonstrated that carvacrol suppresses the expression of inflammatory marker genes in D-galactosamine-hepatotoxic rats. However, whether carvacrol can attenuate LPS-induced ALI and its underlying molecular mechanisms remains unclear. In this study, we sought to assess the preventive effects of carvacrol in a LPS-induced mouse ALI model and elucidated the potential anti-inflammatory mechanism.
MATERIALS AND METHODS Materials and Chemicals
X. Feng an A. Jia contributed equally to this work 1
Guangdong Haid Institute of Animal Husbandry and Veterinary, Guangzhou, Guangdong 510000, China 2 To whom correspondence should be addressed at Guangdong Haid Institute of Animal Husbandry and Veterinary, Guangzhou, Guangdong 510000, China. E-mail: [email protected]
Carvacrol (purity >98 %) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Dexamethasone (purity >99.6 %) was purchased from Changle Pharmaceutical Co. (Xinxiang, Henan, China). Mouse TNF-α, IL-6 and IL-1β
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Feng, and Jia
Fig. 1. Chemical structure of carvacrol.
enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (California, USA). Mouse mAb phospho-NF-κBp65, mouse mAb phospho-IκBα and rabbit mAb IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Mouse mAb TLR4 was purchased from Gene Tex. HRP-conjugated goat anti-rabbit and goat–mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade. Animals Male BALB/c mice, 6–8 weeks, weighing approximately 18–20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). This study was approved by the Jilin University Animal Care and Use Committee. The protocols were reviewed and approved by the committee. The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24±1 °C, and relative humidity was 40–80 %. Mice were housed for 2–3 days to adapt the environment before experimentation. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
40 mg/kg, i.p.). The mortality of mice was observed twice a day for 7 days. In drug testing, the effect of carvacrol (20, 40 and 80 mg/kg) on LPS-induced mortality was assessed by giving carvacrol 1 h before LPS challenge. Survival in each group was assessed every 12 h for 7 days. LPS-Induced ALI in Mice The 72 healthy male BALB/c mice were randomly classified into six groups: Control, LPS, LPS+ Carvacrol (20, 40 and 80 mg/kg) and LPS+Dexamethasone (LPS+DEX). LPS+Dex group serves as positive control. Carvacrol (20, 40 and 80 mg/kg) and DEX (5 mg/kg) were given intraperitoneally. Mice from the control and LPS groups received an equal volume of PBS instead of carvacrol or DEX. One hour later, after the BALB/c mice were diethyl ether-anaesthetized, 10 μg LPS was instilled intranasally in 50 μl PBS to induce lung injury [8]. Control mice were given 50 μl PBS without LPS. All the mice were alive after 7 h LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 ml. The chosen doses of these drugs were based on our previous studies and preliminary experiments (data not provided). Lung Wet to Dry Weight (W/D) Ratio Measurement
LPS-Induced Endotoxemia in Mice The 48 healthy male BALB/c mice were randomly classified into four groups and challenged with LPS (5–
After the mice were euthanized, the lungs were removed and the wet weight was determined. The lung tissue was placed in an oven at 60 °C for 24 h to obtain the dry weight.
Protective Effect of Carvacrol on Acute Lung Injury The ratio of the wet lung to the dry lung was calculated to assess tissue edema. Inflammatory Cell Counts of BALF The fluid recovered from each sample was centrifuged (4 °C, 3,000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. Cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method. Cytokine Assays The concentrations of TNF-α, IL-6, and IL-1β in the supernatants of the BALF were measured using sandwich ELISA kits (Biolegend, USA) according to the protocol recommended by the manufacturer. Pulmonary MPO Activity in Acute Lung Injury Mice MPO activity, reflecting the parenchymal infiltration of neutrophils and macrophages, was
determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions. Mice under diethyl ether anesthesia were killed 7 h after LPS treatment, and then the right lungs were excised. Lung tissues of 100 mg were homogenized and fluidized in extraction buffer to obtain 5 % of homogenate. The sample including 0.9 ml homogenate and 0.1 ml of reaction buffer was heated to 37 °C in water for 15 min, on which occasion the enzymatic activity was determined by measuring the change in absorbance at 460 nm using a 96-well plate reader.
Histopathologic Evaluation of the Lung Tissue Histopathologic examination was performed on mice that were not subjected to BALF collection. Lungs were fixed with 10 % buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of lung tissues were observed under a light microscope.
Fig. 2. Effects of carvacrol on LPS-induced lethality in mice. Mice were given a intraperitoneal injection of carvacrol (20, 40 and 80 mg/kg) 1 h prior to LPS challenged. a The survival rate of mice challenged with LPS of different doses. b Effect of carvacrol (20, 40 and 80 mg/kg) treatment on LPS-induced lethality. The survival was monitored every 12 h for 7 days. #p
Protective Effect of Carvacrol on Acute Lung Injury Induced by Lipopolysaccharide in Mice Xiaosheng Feng1,2 and Aiqing Jia1
Abstract—Carvacrol, the major component of Plectranthus amboinicus, has been known to exhibit antiinflammatory activities. The aim of this study was to investigate the effects of carvacrol on lipopolysaccharide (LPS)-induced endotoxemia and acute lung injury (ALI) in mice. Mice were injected intraperitoneally (i.p.) with LPS and the mortality of mice for 7 days were observed twice a day. Meanwhile, the protective effect of carvacrol (20, 40 or 80 mg/kg) on LPS-induced endotoxemia were detected. Using an experimental model of LPS-induced ALI, we examined the effect of carvacrol in resolving lung injury. The results showed that carvacrol could improve survival during lethal endotoxemia and attenuate LPS-induced ALI in mice. The anti-inflammatory mechanisms of carvacrol may be due to its ability to inhibit NF-κB and MAPKs signaling pathways, thereby inhibiting inflammatory cytokines TNF-α, IL-6 and IL-1β production. KEY WORDS: carvacrol; acute lung injury; LPS; NF-κB; MAPK.
INTRODUCTION The acute respiratory distress syndrome (ARDS), a common clinical catastrophe following acute lung injury (ALI), is a significant cause of morbidity and mortality in critically ill patients [1–4]. ALI/ARDS is characterized by non-cardiogenic pulmonary edema, pulmonary and systemic inflammation with a spectrum of increasing severity of lung injury resulting in respiratory failure [5]. Lipopolysaccharide (LPS) is the major stimulus for the release of inflammatory mediators [6], which is released during sepsis. Administration of LPS in experimental animals causes the pathological condition of ongoing sepsis and concomitant ARDS-like lung injury, including polymorphonuclear neutrophil sequestration and lung edema [7]. Inflammatory stimuli from microbial pathogens, such as endotoxin (LPS), are well recognized for their ability to induce pulmonary inflammation, and experimental administration of LPS, both systemically and intratracheally, has been used
to induce pulmonary inflammation in animal models of ALI. Carvacrol (Fig. 1), a monoterpenic phenol which occurs in many essential oils, has been reported to have a number of pharmacological activities such as antioxidant, anti-titumor and anti-inflammatory effects. Carvacrol was found to inhibit COX-2 expression in LPS-activated U937 cells. In addition, some reports have demonstrated that carvacrol suppresses the expression of inflammatory marker genes in D-galactosamine-hepatotoxic rats. However, whether carvacrol can attenuate LPS-induced ALI and its underlying molecular mechanisms remains unclear. In this study, we sought to assess the preventive effects of carvacrol in a LPS-induced mouse ALI model and elucidated the potential anti-inflammatory mechanism.
MATERIALS AND METHODS Materials and Chemicals
X. Feng an A. Jia contributed equally to this work 1
Guangdong Haid Institute of Animal Husbandry and Veterinary, Guangzhou, Guangdong 510000, China 2 To whom correspondence should be addressed at Guangdong Haid Institute of Animal Husbandry and Veterinary, Guangzhou, Guangdong 510000, China. E-mail: [email protected]
Carvacrol (purity >98 %) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Jilin, China). Dexamethasone (purity >99.6 %) was purchased from Changle Pharmaceutical Co. (Xinxiang, Henan, China). Mouse TNF-α, IL-6 and IL-1β
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Feng, and Jia
Fig. 1. Chemical structure of carvacrol.
enzyme-linked immunosorbent assay (ELISA) kits were purchased from Biolegend (California, USA). Mouse mAb phospho-NF-κBp65, mouse mAb phospho-IκBα and rabbit mAb IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). Mouse mAb TLR4 was purchased from Gene Tex. HRP-conjugated goat anti-rabbit and goat–mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade. Animals Male BALB/c mice, 6–8 weeks, weighing approximately 18–20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). This study was approved by the Jilin University Animal Care and Use Committee. The protocols were reviewed and approved by the committee. The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24±1 °C, and relative humidity was 40–80 %. Mice were housed for 2–3 days to adapt the environment before experimentation. All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
40 mg/kg, i.p.). The mortality of mice was observed twice a day for 7 days. In drug testing, the effect of carvacrol (20, 40 and 80 mg/kg) on LPS-induced mortality was assessed by giving carvacrol 1 h before LPS challenge. Survival in each group was assessed every 12 h for 7 days. LPS-Induced ALI in Mice The 72 healthy male BALB/c mice were randomly classified into six groups: Control, LPS, LPS+ Carvacrol (20, 40 and 80 mg/kg) and LPS+Dexamethasone (LPS+DEX). LPS+Dex group serves as positive control. Carvacrol (20, 40 and 80 mg/kg) and DEX (5 mg/kg) were given intraperitoneally. Mice from the control and LPS groups received an equal volume of PBS instead of carvacrol or DEX. One hour later, after the BALB/c mice were diethyl ether-anaesthetized, 10 μg LPS was instilled intranasally in 50 μl PBS to induce lung injury [8]. Control mice were given 50 μl PBS without LPS. All the mice were alive after 7 h LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 ml. The chosen doses of these drugs were based on our previous studies and preliminary experiments (data not provided). Lung Wet to Dry Weight (W/D) Ratio Measurement
LPS-Induced Endotoxemia in Mice The 48 healthy male BALB/c mice were randomly classified into four groups and challenged with LPS (5–
After the mice were euthanized, the lungs were removed and the wet weight was determined. The lung tissue was placed in an oven at 60 °C for 24 h to obtain the dry weight.
Protective Effect of Carvacrol on Acute Lung Injury The ratio of the wet lung to the dry lung was calculated to assess tissue edema. Inflammatory Cell Counts of BALF The fluid recovered from each sample was centrifuged (4 °C, 3,000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. Cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method. Cytokine Assays The concentrations of TNF-α, IL-6, and IL-1β in the supernatants of the BALF were measured using sandwich ELISA kits (Biolegend, USA) according to the protocol recommended by the manufacturer. Pulmonary MPO Activity in Acute Lung Injury Mice MPO activity, reflecting the parenchymal infiltration of neutrophils and macrophages, was
determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions. Mice under diethyl ether anesthesia were killed 7 h after LPS treatment, and then the right lungs were excised. Lung tissues of 100 mg were homogenized and fluidized in extraction buffer to obtain 5 % of homogenate. The sample including 0.9 ml homogenate and 0.1 ml of reaction buffer was heated to 37 °C in water for 15 min, on which occasion the enzymatic activity was determined by measuring the change in absorbance at 460 nm using a 96-well plate reader.
Histopathologic Evaluation of the Lung Tissue Histopathologic examination was performed on mice that were not subjected to BALF collection. Lungs were fixed with 10 % buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of lung tissues were observed under a light microscope.
Fig. 2. Effects of carvacrol on LPS-induced lethality in mice. Mice were given a intraperitoneal injection of carvacrol (20, 40 and 80 mg/kg) 1 h prior to LPS challenged. a The survival rate of mice challenged with LPS of different doses. b Effect of carvacrol (20, 40 and 80 mg/kg) treatment on LPS-induced lethality. The survival was monitored every 12 h for 7 days. #p
