Inflammation ( # 2014) DOI: 10.1007/s10753-014-9945-7
Tetrahydrocoptisine Protects Rats from LPS-Induced Acute Lung Injury Weifeng Li,1 Huimin Huang,1 Xiaofeng Niu,1,2 Ting Fan,1 Hua Hu,1 Yongmei Li,1 Huan Yao,1 Huani Li,1 and Qingli Mu1
Abstract—Recent studies show that nuclear factor-kappa B (NF-κB) signaling pathway plays a key role in contributing to the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Tetrahydrocoptisine is one of the main active components of Chelidonium majus L. and has been described to be effective in suppressing inflammation. The aim of the present study is to evaluate the protective effect of tetrahydrocoptisine on LPS-induced ALI in rats and clarify its underlying mechanisms of action. We found that in vivo pretreatment with tetrahydrocoptisine to rats 30 min before inducing ALI by LPS markedly decreased the mortality rate, lung wet weight to dry weight ratio, and ameliorated lung pathological changes. Meanwhile, tetrahydrocoptisine significantly inhibited the increase of the amounts of inflammatory cells, total protein content, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in the bronchoalveolar lavage fluids (BALFs). Furthermore, tetrahydrocoptisine inhibited myeloperoxidase (MPO) accumulation in lung tissue and alleviated TNF-α and IL-6 production in serum. Additionally, immunohistochemistry showed that tetrahydrocoptisine efficiently reduced nuclear factorkappa B (NF-κB) activation by inhibiting the translocation of NF-κBp65. In conclusion, our results demonstrate that tetrahydrocoptisine possesses a protective effect on LPS-induced ALI through inhibiting of NF-κB signaling pathways, which may involve the inhibition of pulmonary inflammatory process. KEY WORDS: tetrahydrocoptisine; lipopolysaccharide; acute lung injury; anti-inflammation; cytokine; nuclear factor-kappa B.
INTRODUCTION Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) occur in the setting of an acute severe illness complicated by systemic inflammation [1]. ALI is associated with the excessive production of inflammatory mediators, such as cytokines, chemokines, multiple families of cell-specific adhesion molecules and bioactive lipid products [2–4], and often show as severe hypoxemia, alveolar–capillary barrier damage, high-permeability pulmonary edema and neutrophil accumulation in the lung [5–8]. Because of the serious morbidity and mortality, increasing insights into ALI/ ARDS pathobiology are needed for its therapeutics. 1
School of Pharmacy, Xi’an Jiaotong University, Number 76 Western Yanta Road, Xi’an City, Shaanxi 710061, China 2 To whom correspondence should be addressed at School of Pharmacy, Xi’an Jiaotong University, Number 76 Western Yanta Road, Xi’an City, Shaanxi 710061, China. E-mail: [email protected]
The release of lipopolysaccharide (LPS) from the outermost membrane of gram-negative bacteria has been recognized as a principal pathogen in the pulmonary inflammation and sepsis leading to ALI/ARDS [9, 10]. Acute exposure to LPS provokes the innate immune system, leading to the activation of monocytes, macrophages, neutrophils, and lymphocytes [11, 12], which initiate a cascade of inflammatory cell influx, increase of cytokine release and lung capillary permeability, resulting in pulmonary edema [13, 14]. Previous studies have shown that the participation of nuclear factor-kappa B (NF-κB) signaling pathway is involved in the mechanism of LPS-induced ALI [9, 15]. The activation of NF-κB signaling pathway leads to the up-regulation of many genes responsible for the release of inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, iNOS and monocyte chemotactic protein-1 (MCP-1) [8, 16, 17]. Thus, agents that can inhibit the production of inflammatory factors may be beneficial in decreasing damage of LPS-induced ALI.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Li, Huang, Niu, Fan, Hu, Li, Yao, Li, and Mu Tetrahydrocoptisine (THC; 6,7,12b,13-tetrahydro4H-bis[1, 3]benzodioxolo[5,6-a:4′,5′-g]quinolizine) is a protoberberine compound present in the roots, shoots and leaves of Chelidonium majus L. and other phytomedicinal plants, including Corydalis impatiens. The chemical structure of THC is shown in Fig. 1. It has been reported that THC exhibits allosteric modulation of the gammaaminobutyric acid type A (GABAA) receptor, detoxification of xenobiotics and anti-inflammatory properties [18, 19]. Further study indicates that THC can suppress the production of PGE2, TNF-α, IL-1β and IL-6 by downregulation of cyclooxygenase-2 (COX-2) [18]. We therefore hypothesized that tetrahydrocoptisine might also exert protective effect on lung injury depending on inhibiting the expression of inflammatory cytokines and myeloperoxidase (MPO) production. The aim of this study was designed to investigate whether THC could ameliorate LPS-induced ALI in rats and further revealed its underlying mechanism.
MATERIALS AND METHODS Animals Adult male Sprague–Dawley rats (200–250 g weight) purchased from the Experimental Animal Center, Xi’an Jiao tong University (Xi’an, China) were housed in a temperature-controlled (24±2 °C) and light-controlled room (12 h light/dark cycle),with free access to water and food. All animals were acclimated to housing conditions for at least 1 week prior to experiment. The animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health. Materials
Dexamethasone (DEX) was supplied by Xi’an Lijun Pharmaceutical Company Limited (Shaanxi, China). LPS was obtained from Sigma (St. Louis, MO, USA). The enzyme-linked immunosorbent assay (ELISA) kits for rats TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA). The kit for determination of MPO content was obtained from Jiancheng Bioengineering Institute (Nanjing, China). NF-κBp65 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Reagents were analytical grade.
Survival Study For the assessment of mortality rate, rats were given a vena caudalis injection of LPS (20 mg/kg) with different doses of 0.5 ml THC (10 and 20 mg/kg, i.p.) and DEX (5 mg/kg) pretreatment 30 min before LPS injection. The control rats were only given an equal volume of 0.9 % saline without LPS. The mortality of rat was recorded every 12 h for 3 days after the LPS challenge in each treated group. Experiments were performed with littermate rats and each group contains ten animals.
Model and Grouping After adjusting to the environment, rats were randomly divided into five groups: control group (n=10), LPS group (n=10), DEX group (n=10), LPS–10 mg/kg THC group (n=10) and LPS–20 mg/kg THC group (n=10). Rats were pretreated i.p. with 0.5 ml of THC (10 mg/kg or 20 mg/kg), DEX or vehicle (0.9 % saline) 30 min prior to LPS administration by intratracheal instillation (5 mg/ kg). In all groups, measurements were made at 24 h after LPS or saline administration. The doses of THC we chose were on the basis of our preliminary experiments.
Tetrahydrocoptisine (THC) was purchased from Xi'an Honson Biotechnology Co., Ltd. (Shaanxi, China). Collection of Bronchoalveolar Lavage Fluid
Fig. 1. The chemical structure of tetrahydrocoptisine.
The rats were anesthetized and provided with a plastic cannula inserted into the trachea. Bronchoalveolar lavage fluid (BALF) was obtained by washing the airways three times with a total of 5 ml PBS (pH7.2). BALF samples were centrifuged (1,500 rpm, 4 °C) for 10 min. BALF supernatants were stored at −80 °C and harvested for total protein analysis using the method of BCA and cytokines assay. The pellets were prepared for differential cell counts by Wright–Giemsa staining method.
Tetrahydrocoptisine Protects Rats from Acute Lung Injury Measurement of TNF-α and IL-6 Levels in BALF Levels of TNF-α and IL-6 in BALF supernatant were measured with mouse TNF-α and IL-6 by enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions. The absorbance was measured at 450 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). A standard curve was run on each assay plate using recombinant IL-6 and TNF-α in serial dilution. The levels of IL-6 and TNF-α in the samples were calculated based on the standard curve generated from recombinant mice IL-6 and TNF-α. The results were expressed as pg/ml BALF. Measurement of TNF-α and IL-6 Levels in Serum Twenty-four hours after LPS injection, blood samples were collected from the retro-orbital plexus of each animal and were then centrifuged for 10 min at 2,500×g, 4 °C, to obtain clear sera which were stored at −80 °C and used within 48 h for the determination of IL-6 and TNF-α. The levels of cytokines (IL-6 and TNF-α) in the serum were analyzed by enzyme- linked immunosorbent assay using ELISA kits for rats according to the manufacturer’s instructions. The results were expressed as pg/ml serum. Lung Wet/Dry (W/D) Weight Ratio Calculation The rats were killed by cervical dislocation 24 h after LPS challenge. The right lungs were excised and weighed immediately after removal to obtain the “wet” weight, and then placed in an oven at 80 °C for 48 h to obtain the “dry” weight. The ratio of the wet lung to the dry lung was calculated to the magnitude of pulmonary edema [20]. Myeloperoxidase Activity Assay The accumulation of neutrophils in the lung tissue was assessed by MPO activity. Briefly, frozen lung tissues were thawed and homogenized in homogenate medium. The activity of MPO enzyme in the homogenates was assessed spectrophotometrically using an MPO detection kit according to the manufacturer’s instructions. MPO activity was determined according to the absorbance measured at 460 nm [21], and MPO activity was expressed as units per gram tissue. Histological Study Lung samples were obtained at 24 h after LPS administration and fixed with 10 % formalin. After fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. Histology of the lung was
examined using hematoxylin–eosin staining and the slides were histopathologically evaluated using a semi-quantitative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: interstitial inflammation, inflammatory cell infiltration, congestion, and edema. The total lung injury score was calculated by adding up the individual scores of each category [15, 22].
Immunohistochemistry for NF-κBp65 For NF-κBp65 immunohistochemistry assay, the streptavidin–biotin–peroxidase method was used. In brief, the lung tissues were fixed in 10 % neutral formalin, dehydrated with increasing concentrations of ethanol, embedded in paraffin, and sectioned. Paraffin sections for different animal groups were deparaffinized, dehydrated in xylol and ethanol, and immersed in 0.1 M citrate buffer (pH 6.0) in microwave for antigen retrieval. After cooling, the sections were washed with PBS, and endogenous peroxidase activity was quenched with 3.0 % hydrogen peroxide in methanol for 30 min at room temperature. Slides were incubated with polyclonal antibody of NFκBp65 (diluted to 1:100) overnight at 4 °C. After being washed thrice in phosphate-buffered saline (PBS), sections were incubated with the biotinylated polyclonal rabbit anti-mouse secondary antibody (Dako, Carpinteria, CA, USA) at room temperature for 30 min, and then again with avidin–biotin–horseradish peroxidase conjugate. NF-κB immunostainings were visualized with the chromogen 3,3′-diaminobenzidine (DAB). Then the sections were dehydrated with increasing concentration of ethanol and mounted with neutral gum. The results were evaluated semiquantitatively according to the percentage of positive cells in ten randomly selected fields under 100-fold and 400-fold magnification.
Statistical Analysis The results are presented as mean±SEM. The statistical significance of differences for each parameter among the groups was evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s t test. Survival data were presented by the Kaplan–Meier method and comparisons were made by the log rank test. Kruskal–Wallis test and the Dunn's tests were used for analysis of the histological scores. A P value less than 0.05 was considered statistically significant.
Li, Huang, Niu, Fan, Hu, Li, Yao, Li, and Mu RESULTS
Effect of THC on LPS-Induced Mortality in Rats To evaluate the protective effect of THC on rats with endotoxemia, THC (10 or 20 mg/kg) was administrated i.p. 30 min prior to LPS injection (20 mg/kg) to induce endotoxemia, survival was assessed for 72 h. As shown in Fig. 2, the accumulative mortalities during 3 days in THC (10 and 20 mg/kg) and DEX pretreatment groups were 60 %, 30 % and 20 %, respectively, which were significantly lower than that in the LPS group (80 %). Kaplan–Meier survival analysis indicated that pretreatment with THC could significantly protect rats with endotoxemia from death.
Effects of THC on TNF-α and IL-6 Secretion in the Serum of LPS-Treated Rats Concentrations of TNF-α and IL-6 in serum were measured by ELISA kits for rats, according to the manufacturer's instructions. As shown in Fig. 4a and b, the serum levels of TNF-α and IL-6 rised markedly in the LPS group (P
Tetrahydrocoptisine Protects Rats from LPS-Induced Acute Lung Injury Weifeng Li,1 Huimin Huang,1 Xiaofeng Niu,1,2 Ting Fan,1 Hua Hu,1 Yongmei Li,1 Huan Yao,1 Huani Li,1 and Qingli Mu1
Abstract—Recent studies show that nuclear factor-kappa B (NF-κB) signaling pathway plays a key role in contributing to the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Tetrahydrocoptisine is one of the main active components of Chelidonium majus L. and has been described to be effective in suppressing inflammation. The aim of the present study is to evaluate the protective effect of tetrahydrocoptisine on LPS-induced ALI in rats and clarify its underlying mechanisms of action. We found that in vivo pretreatment with tetrahydrocoptisine to rats 30 min before inducing ALI by LPS markedly decreased the mortality rate, lung wet weight to dry weight ratio, and ameliorated lung pathological changes. Meanwhile, tetrahydrocoptisine significantly inhibited the increase of the amounts of inflammatory cells, total protein content, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in the bronchoalveolar lavage fluids (BALFs). Furthermore, tetrahydrocoptisine inhibited myeloperoxidase (MPO) accumulation in lung tissue and alleviated TNF-α and IL-6 production in serum. Additionally, immunohistochemistry showed that tetrahydrocoptisine efficiently reduced nuclear factorkappa B (NF-κB) activation by inhibiting the translocation of NF-κBp65. In conclusion, our results demonstrate that tetrahydrocoptisine possesses a protective effect on LPS-induced ALI through inhibiting of NF-κB signaling pathways, which may involve the inhibition of pulmonary inflammatory process. KEY WORDS: tetrahydrocoptisine; lipopolysaccharide; acute lung injury; anti-inflammation; cytokine; nuclear factor-kappa B.
INTRODUCTION Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) occur in the setting of an acute severe illness complicated by systemic inflammation [1]. ALI is associated with the excessive production of inflammatory mediators, such as cytokines, chemokines, multiple families of cell-specific adhesion molecules and bioactive lipid products [2–4], and often show as severe hypoxemia, alveolar–capillary barrier damage, high-permeability pulmonary edema and neutrophil accumulation in the lung [5–8]. Because of the serious morbidity and mortality, increasing insights into ALI/ ARDS pathobiology are needed for its therapeutics. 1
School of Pharmacy, Xi’an Jiaotong University, Number 76 Western Yanta Road, Xi’an City, Shaanxi 710061, China 2 To whom correspondence should be addressed at School of Pharmacy, Xi’an Jiaotong University, Number 76 Western Yanta Road, Xi’an City, Shaanxi 710061, China. E-mail: [email protected]
The release of lipopolysaccharide (LPS) from the outermost membrane of gram-negative bacteria has been recognized as a principal pathogen in the pulmonary inflammation and sepsis leading to ALI/ARDS [9, 10]. Acute exposure to LPS provokes the innate immune system, leading to the activation of monocytes, macrophages, neutrophils, and lymphocytes [11, 12], which initiate a cascade of inflammatory cell influx, increase of cytokine release and lung capillary permeability, resulting in pulmonary edema [13, 14]. Previous studies have shown that the participation of nuclear factor-kappa B (NF-κB) signaling pathway is involved in the mechanism of LPS-induced ALI [9, 15]. The activation of NF-κB signaling pathway leads to the up-regulation of many genes responsible for the release of inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, iNOS and monocyte chemotactic protein-1 (MCP-1) [8, 16, 17]. Thus, agents that can inhibit the production of inflammatory factors may be beneficial in decreasing damage of LPS-induced ALI.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Li, Huang, Niu, Fan, Hu, Li, Yao, Li, and Mu Tetrahydrocoptisine (THC; 6,7,12b,13-tetrahydro4H-bis[1, 3]benzodioxolo[5,6-a:4′,5′-g]quinolizine) is a protoberberine compound present in the roots, shoots and leaves of Chelidonium majus L. and other phytomedicinal plants, including Corydalis impatiens. The chemical structure of THC is shown in Fig. 1. It has been reported that THC exhibits allosteric modulation of the gammaaminobutyric acid type A (GABAA) receptor, detoxification of xenobiotics and anti-inflammatory properties [18, 19]. Further study indicates that THC can suppress the production of PGE2, TNF-α, IL-1β and IL-6 by downregulation of cyclooxygenase-2 (COX-2) [18]. We therefore hypothesized that tetrahydrocoptisine might also exert protective effect on lung injury depending on inhibiting the expression of inflammatory cytokines and myeloperoxidase (MPO) production. The aim of this study was designed to investigate whether THC could ameliorate LPS-induced ALI in rats and further revealed its underlying mechanism.
MATERIALS AND METHODS Animals Adult male Sprague–Dawley rats (200–250 g weight) purchased from the Experimental Animal Center, Xi’an Jiao tong University (Xi’an, China) were housed in a temperature-controlled (24±2 °C) and light-controlled room (12 h light/dark cycle),with free access to water and food. All animals were acclimated to housing conditions for at least 1 week prior to experiment. The animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institute of Health. Materials
Dexamethasone (DEX) was supplied by Xi’an Lijun Pharmaceutical Company Limited (Shaanxi, China). LPS was obtained from Sigma (St. Louis, MO, USA). The enzyme-linked immunosorbent assay (ELISA) kits for rats TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA). The kit for determination of MPO content was obtained from Jiancheng Bioengineering Institute (Nanjing, China). NF-κBp65 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Reagents were analytical grade.
Survival Study For the assessment of mortality rate, rats were given a vena caudalis injection of LPS (20 mg/kg) with different doses of 0.5 ml THC (10 and 20 mg/kg, i.p.) and DEX (5 mg/kg) pretreatment 30 min before LPS injection. The control rats were only given an equal volume of 0.9 % saline without LPS. The mortality of rat was recorded every 12 h for 3 days after the LPS challenge in each treated group. Experiments were performed with littermate rats and each group contains ten animals.
Model and Grouping After adjusting to the environment, rats were randomly divided into five groups: control group (n=10), LPS group (n=10), DEX group (n=10), LPS–10 mg/kg THC group (n=10) and LPS–20 mg/kg THC group (n=10). Rats were pretreated i.p. with 0.5 ml of THC (10 mg/kg or 20 mg/kg), DEX or vehicle (0.9 % saline) 30 min prior to LPS administration by intratracheal instillation (5 mg/ kg). In all groups, measurements were made at 24 h after LPS or saline administration. The doses of THC we chose were on the basis of our preliminary experiments.
Tetrahydrocoptisine (THC) was purchased from Xi'an Honson Biotechnology Co., Ltd. (Shaanxi, China). Collection of Bronchoalveolar Lavage Fluid
Fig. 1. The chemical structure of tetrahydrocoptisine.
The rats were anesthetized and provided with a plastic cannula inserted into the trachea. Bronchoalveolar lavage fluid (BALF) was obtained by washing the airways three times with a total of 5 ml PBS (pH7.2). BALF samples were centrifuged (1,500 rpm, 4 °C) for 10 min. BALF supernatants were stored at −80 °C and harvested for total protein analysis using the method of BCA and cytokines assay. The pellets were prepared for differential cell counts by Wright–Giemsa staining method.
Tetrahydrocoptisine Protects Rats from Acute Lung Injury Measurement of TNF-α and IL-6 Levels in BALF Levels of TNF-α and IL-6 in BALF supernatant were measured with mouse TNF-α and IL-6 by enzyme-linked immunosorbent assay kits according to the manufacturer’s instructions. The absorbance was measured at 450 nm with an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). A standard curve was run on each assay plate using recombinant IL-6 and TNF-α in serial dilution. The levels of IL-6 and TNF-α in the samples were calculated based on the standard curve generated from recombinant mice IL-6 and TNF-α. The results were expressed as pg/ml BALF. Measurement of TNF-α and IL-6 Levels in Serum Twenty-four hours after LPS injection, blood samples were collected from the retro-orbital plexus of each animal and were then centrifuged for 10 min at 2,500×g, 4 °C, to obtain clear sera which were stored at −80 °C and used within 48 h for the determination of IL-6 and TNF-α. The levels of cytokines (IL-6 and TNF-α) in the serum were analyzed by enzyme- linked immunosorbent assay using ELISA kits for rats according to the manufacturer’s instructions. The results were expressed as pg/ml serum. Lung Wet/Dry (W/D) Weight Ratio Calculation The rats were killed by cervical dislocation 24 h after LPS challenge. The right lungs were excised and weighed immediately after removal to obtain the “wet” weight, and then placed in an oven at 80 °C for 48 h to obtain the “dry” weight. The ratio of the wet lung to the dry lung was calculated to the magnitude of pulmonary edema [20]. Myeloperoxidase Activity Assay The accumulation of neutrophils in the lung tissue was assessed by MPO activity. Briefly, frozen lung tissues were thawed and homogenized in homogenate medium. The activity of MPO enzyme in the homogenates was assessed spectrophotometrically using an MPO detection kit according to the manufacturer’s instructions. MPO activity was determined according to the absorbance measured at 460 nm [21], and MPO activity was expressed as units per gram tissue. Histological Study Lung samples were obtained at 24 h after LPS administration and fixed with 10 % formalin. After fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. Histology of the lung was
examined using hematoxylin–eosin staining and the slides were histopathologically evaluated using a semi-quantitative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: interstitial inflammation, inflammatory cell infiltration, congestion, and edema. The total lung injury score was calculated by adding up the individual scores of each category [15, 22].
Immunohistochemistry for NF-κBp65 For NF-κBp65 immunohistochemistry assay, the streptavidin–biotin–peroxidase method was used. In brief, the lung tissues were fixed in 10 % neutral formalin, dehydrated with increasing concentrations of ethanol, embedded in paraffin, and sectioned. Paraffin sections for different animal groups were deparaffinized, dehydrated in xylol and ethanol, and immersed in 0.1 M citrate buffer (pH 6.0) in microwave for antigen retrieval. After cooling, the sections were washed with PBS, and endogenous peroxidase activity was quenched with 3.0 % hydrogen peroxide in methanol for 30 min at room temperature. Slides were incubated with polyclonal antibody of NFκBp65 (diluted to 1:100) overnight at 4 °C. After being washed thrice in phosphate-buffered saline (PBS), sections were incubated with the biotinylated polyclonal rabbit anti-mouse secondary antibody (Dako, Carpinteria, CA, USA) at room temperature for 30 min, and then again with avidin–biotin–horseradish peroxidase conjugate. NF-κB immunostainings were visualized with the chromogen 3,3′-diaminobenzidine (DAB). Then the sections were dehydrated with increasing concentration of ethanol and mounted with neutral gum. The results were evaluated semiquantitatively according to the percentage of positive cells in ten randomly selected fields under 100-fold and 400-fold magnification.
Statistical Analysis The results are presented as mean±SEM. The statistical significance of differences for each parameter among the groups was evaluated by one-way analysis of variance (ANOVA), followed by Dunnett’s t test. Survival data were presented by the Kaplan–Meier method and comparisons were made by the log rank test. Kruskal–Wallis test and the Dunn's tests were used for analysis of the histological scores. A P value less than 0.05 was considered statistically significant.
Li, Huang, Niu, Fan, Hu, Li, Yao, Li, and Mu RESULTS
Effect of THC on LPS-Induced Mortality in Rats To evaluate the protective effect of THC on rats with endotoxemia, THC (10 or 20 mg/kg) was administrated i.p. 30 min prior to LPS injection (20 mg/kg) to induce endotoxemia, survival was assessed for 72 h. As shown in Fig. 2, the accumulative mortalities during 3 days in THC (10 and 20 mg/kg) and DEX pretreatment groups were 60 %, 30 % and 20 %, respectively, which were significantly lower than that in the LPS group (80 %). Kaplan–Meier survival analysis indicated that pretreatment with THC could significantly protect rats with endotoxemia from death.
Effects of THC on TNF-α and IL-6 Secretion in the Serum of LPS-Treated Rats Concentrations of TNF-α and IL-6 in serum were measured by ELISA kits for rats, according to the manufacturer's instructions. As shown in Fig. 4a and b, the serum levels of TNF-α and IL-6 rised markedly in the LPS group (P
