Inflammation ( # 2014) DOI: 10.1007/s10753-014-9929-7
Protective Effects of Chelerythrine Against Lipopolysaccharide-Induced Endotoxic Shock in Mice Xiaofeng Niu,1 Qingli Mu,1 Weifeng Li,1,2 Huimin Huang,1 Huan Yao,1 and Huani Li1
Abstract—Chelerythrine (CHE), a quaternary benzo[c]phenanthridine alkaloid, exhibits a wide spectrum of pharmacological effects. Although CHE has been used to treat various diseases, the protective effects of CHE on lipopolysaccharide (LPS)-induced endotoxic shock have not been explored. The aims of the study were to investigate the protective effects of CHE on LPS-induced endotoxic shock in mice and clarify the mechanism of the effects. We found that pretreatment with CHE (1, 5, and 10 mg/kg, po) at 1 and 12 h before injected intraperitoneally with 1 mg/kg LPS markedly decreased the production of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and myeloperoxidase (MPO) and attenuated the lung histopathological changes. Meanwhile, the effects were dependent on the inhibition of the expression of p65 nuclear factor κB (NF-κB). The protective effects of CHE on LPS-induced endotoxic shock can be attributed to attenuating inflammatory cytokines and inhibition of the expression of NF-κB. KEY WORDS: chelerythrine; lipopolysaccharide; endotoxic shock; inflammatory cytokines; p65 NF-κB.
INTRODUCTION Inflammation, a response to mechanical stress, infection, and shock, is mediated by series inflammation mediators produced by macrophages and leukocytes [1]. Lipopolysaccharide, a glycolipid constituent of the outer membrane of Gram-negative bacteria, is an endotoxin-induced acute inflammation reaction septic shock and sepsis [2]. Sepsis, associated with high morbidity and mortality, is characterized by hypotension, organ perfusion, and multiorgan dysfunction [3]. Endotoxic shock, involved in the secretion of cytokines and chemokines, will trigger series of symptoms such as increased blood flow, leukocyte infiltration, and release of proteases [4]. Most inflammation reaction induced by (LPS) is triggered by endogenous mediators such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α); among these cytokines, TNF-α plays a
1
School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, 710061( Shaanxi Province, People’s Republic of China 2 To whom correspondence should be addressed at School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, 710061( Shaanxi Province, People’s Republic of China. E-mail: [email protected]
significant role in endotoxic shock [5, 6]. TNF-α, regarded as the first-line cytokine, can induce most characteristics of endotoxic shock and decreases the production of inflammatory cytokines including IL-6 and IL-12 [7–9]. The balance of the circulating levels of pro-inflammatory effect and anti-inflammatory effect of IL-6, which is involved in the pathophysiology of septic shock, determines the severity of disease [10, 11]. Nuclear factor κB (NF-κB), which plays a critical role in the response to cytokines and stress, is responsible for the transcription of TNF-α and IL-6 cytokines [12]. Thus, inhibition of these pro-inflammatory cytokines especially TNF-α may serve as therapeutic target to therapy septic shock [13]. Chelerythrine (CHE), a quaternary benzo[c]phenanthridine alkaloid, derives from the great plant species Chelidonium majus and other Fumaria. It indicated a broad range of biological effects such as anti-microbial, anti-tumor, and anti-inflammatory activities. Although CHE has been widely used on various diseases, the protective effects on LPSinduced endotoxic shock have not been investigated; the aims of the study are to explore the protective effects of CHE on the endotoxic shock inflammatory model and provide safe options for inflammationmediated diseases.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Niu, Mu, Li, Huang, Yao, and Li MATERIALS AND METHODS Reagents CHE was purchased from Xi’an Honson Biotechnology Co., Ltd. (Shannxi, China) and identified by the Pharmacognosy Laboratory, School of Pharmacy, Xi’an Jiaotong University (Xi’an, China). LPS was purchased from Sigma (St. Louis, MO, USA). The enzyme immunosorbent assay (ELISA) kits for mouse TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA). The kits for biochemical analysis of myeloperoxidase (MPO) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Histostain-Plus Kits, DAB (3, 3′-diaminobenzidine) staining Kit, and p65 NF-κB polyclonal antibody were purchased from Beijing Boisynthesis Biotechnology Co., Ltd. Other reagents were of commercially available analytical grade. Animals Male (25–30 g) Kunming mice were purchased from the Experimental Animal Centre, Xi’an Jiaotong University (Xi’an, China). Mice under free conditions with a 12-h light/dark cycle were acclimatized for at last 1 week prior to the experiments with food and water ad libitum. All experiments were conducted according to the ethical guidelines for investigations of experimental pain in conscious animals. Endotoxic Shock Model Induced by LPS All male mice were randomly divided into five groups (n=6 per group): Normal control group (group 1) received equal volume of vehicle. Challenge LPS control group (group 2) was injected intraperitoneally with 1 mg/kg LPS in sterile saline. Three treatment groups (groups 3– 5) received two intragastric administration of CHE dissolved in 1 % CMC solution at various doses (1, 5, and 10 mg/kg) at 12 and 1 h before LPS injection. Detection of IL-6 and TNF-α in Serum The mice were fasted after CHE administration or LPS injection and sacrificed by cervical dislocation. The production of TNF-α and IL-6 was analyzed by commercial ELISA kits according to the manufacturer’s instructions. Blood samples were immediately collected from retroorbital plexus before sacrifice and centrifuged at 12,000 rpm for 10 min. Plasma was stored at −20 °C until the quantification of cytokine. Then, 100-μL samples and standard sample buffer were added to 96-well plate and
incubated for 40 min at 37 °C. After incubation, the wash buffer was used to wash the plate for five times. After washing, 50-μL distilled water and biotinylated antibody were allowed to react with samples in each well at 37 °C for 20 min. Then, 100-μL enzyme conjugate was added to microplate well for 10 min at 37 °C. After this procedure, the 96-well plate was put in darkness for 15 min at 37 °C to react with 100-μL TMB solution. The reaction was ended by the addition of 100-μL stop solution, and the optical density was read by microplate reader at 450 nm within 30-min range. Detection of IL-6 and TNF-α in the Lung The lung tissues were quickly removed and refrigerated at −20 °C for cytokine detection. Frozen tissue samples were placed into sterile saline at a ratio of 100 mg per 600 mL. Samples were homogenized and centrifuged at 12,000 rpm for 10 min. The supernatants were added to 96well plate for the detection of TNF-α and IL-6 using commercial ELISA kits according to the manufacturer’s instructions. Histopathology Evaluation The lung tissue was put into 4 % neutral formaldehyde for 24 h and dehydrated in increased concentration gradient of ethanol for the corresponding hours. The dehydrated tissues were transparentized in xylene and embedded in paraffin. Sections (5 μm) were cut by microtome and mounted on slides stained with hematoxylin and eosin reagent for the evaluation of leukocyte accumulation under light microscopy. In order to minimize the bias, the observer did not know which group was analyzed. Immunohistochemical Study The lung tissues were put into 4 % neutral formaldehyde for 24 h and dehydrated in increased concentration gradient of ethanol for the corresponding hours. The dehydrated tissues were transparentized in xylene and embedded in paraffin. Sections (5 μm) were cut by microtome and mounted on slides stained with hematoxylin and eosin reagent. For immunohistochemical study, after deparaffination and rehydration of tissue sections, hot fix antigen was performed in microwave oven, and then, endogenous peroxidase activity was quenched with 3.0 % hydrogen peroxide in methanol for 10 min at room temperature. After washing three times with
Protective Effects of Chelerythrine Against Endotoxic Shock phosphate-buffered saline (PBS), samples were blocked at room temperature using goat serum working solution for 20 min. Then, all the sections were incubated with polyclonal antibody for NF-κB p65 at a dilution of 1:100 in PBS (v/v) at 4 °C overnight, followed by washing with PBS solution and incubated with rabbit anti-mouse second antibody at 37 °C for 30 min. After washing with PBS for three times, the samples were incubated with horseradish peroxides at room temperature for 20 min and stained with 3,3′-diaminobenzidine for 2 min. The last procedure was dehydration and transparence with grade concentration ethanol and xylene, respectively. Then, the sections were mounted with neutral gum. The slides were visualized under light microscope to evaluate the positive immunity cells in the selected fields. Myeloperoxidase Assay The leukocyte infiltration was marked by MPO activity which was analyzed by MPO detection kit (Nanjing Jiancheng Bioengineering Institute). Frozen tissues were
homogenized into buffer application solution at a ratio of 50 mg per 950 μL. MPO activity was measured by ultraviolet spectrophotometer (756pc Shanghai Spectrum Instrument Co. Ltd., China) at 460 nm. One MPO activity unit was defined as the decomposition of 1 μmol hydrogen peroxide per gram weight of wet tissue at 37 °C; the value was calculated by the following formula. MPO activityðμ=gÞ ¼ X =ð11:3 weight of taken tissuesÞ
X equals sample OD value minus control OD value. Statistical Analysis All data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using SPSS 17.0 statistical software. The statistical significance of any difference in each parameter among the groups was evaluated by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. P
Protective Effects of Chelerythrine Against Lipopolysaccharide-Induced Endotoxic Shock in Mice Xiaofeng Niu,1 Qingli Mu,1 Weifeng Li,1,2 Huimin Huang,1 Huan Yao,1 and Huani Li1
Abstract—Chelerythrine (CHE), a quaternary benzo[c]phenanthridine alkaloid, exhibits a wide spectrum of pharmacological effects. Although CHE has been used to treat various diseases, the protective effects of CHE on lipopolysaccharide (LPS)-induced endotoxic shock have not been explored. The aims of the study were to investigate the protective effects of CHE on LPS-induced endotoxic shock in mice and clarify the mechanism of the effects. We found that pretreatment with CHE (1, 5, and 10 mg/kg, po) at 1 and 12 h before injected intraperitoneally with 1 mg/kg LPS markedly decreased the production of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and myeloperoxidase (MPO) and attenuated the lung histopathological changes. Meanwhile, the effects were dependent on the inhibition of the expression of p65 nuclear factor κB (NF-κB). The protective effects of CHE on LPS-induced endotoxic shock can be attributed to attenuating inflammatory cytokines and inhibition of the expression of NF-κB. KEY WORDS: chelerythrine; lipopolysaccharide; endotoxic shock; inflammatory cytokines; p65 NF-κB.
INTRODUCTION Inflammation, a response to mechanical stress, infection, and shock, is mediated by series inflammation mediators produced by macrophages and leukocytes [1]. Lipopolysaccharide, a glycolipid constituent of the outer membrane of Gram-negative bacteria, is an endotoxin-induced acute inflammation reaction septic shock and sepsis [2]. Sepsis, associated with high morbidity and mortality, is characterized by hypotension, organ perfusion, and multiorgan dysfunction [3]. Endotoxic shock, involved in the secretion of cytokines and chemokines, will trigger series of symptoms such as increased blood flow, leukocyte infiltration, and release of proteases [4]. Most inflammation reaction induced by (LPS) is triggered by endogenous mediators such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor alpha (TNF-α); among these cytokines, TNF-α plays a
1
School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, 710061( Shaanxi Province, People’s Republic of China 2 To whom correspondence should be addressed at School of Pharmacy, Xi’an Jiaotong University, No. 76 Western Yanta Road, Xi’an, 710061( Shaanxi Province, People’s Republic of China. E-mail: [email protected]
significant role in endotoxic shock [5, 6]. TNF-α, regarded as the first-line cytokine, can induce most characteristics of endotoxic shock and decreases the production of inflammatory cytokines including IL-6 and IL-12 [7–9]. The balance of the circulating levels of pro-inflammatory effect and anti-inflammatory effect of IL-6, which is involved in the pathophysiology of septic shock, determines the severity of disease [10, 11]. Nuclear factor κB (NF-κB), which plays a critical role in the response to cytokines and stress, is responsible for the transcription of TNF-α and IL-6 cytokines [12]. Thus, inhibition of these pro-inflammatory cytokines especially TNF-α may serve as therapeutic target to therapy septic shock [13]. Chelerythrine (CHE), a quaternary benzo[c]phenanthridine alkaloid, derives from the great plant species Chelidonium majus and other Fumaria. It indicated a broad range of biological effects such as anti-microbial, anti-tumor, and anti-inflammatory activities. Although CHE has been widely used on various diseases, the protective effects on LPSinduced endotoxic shock have not been investigated; the aims of the study are to explore the protective effects of CHE on the endotoxic shock inflammatory model and provide safe options for inflammationmediated diseases.
0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York
Niu, Mu, Li, Huang, Yao, and Li MATERIALS AND METHODS Reagents CHE was purchased from Xi’an Honson Biotechnology Co., Ltd. (Shannxi, China) and identified by the Pharmacognosy Laboratory, School of Pharmacy, Xi’an Jiaotong University (Xi’an, China). LPS was purchased from Sigma (St. Louis, MO, USA). The enzyme immunosorbent assay (ELISA) kits for mouse TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA). The kits for biochemical analysis of myeloperoxidase (MPO) were purchased from Jiancheng Bioengineering Institute (Nanjing, China). Histostain-Plus Kits, DAB (3, 3′-diaminobenzidine) staining Kit, and p65 NF-κB polyclonal antibody were purchased from Beijing Boisynthesis Biotechnology Co., Ltd. Other reagents were of commercially available analytical grade. Animals Male (25–30 g) Kunming mice were purchased from the Experimental Animal Centre, Xi’an Jiaotong University (Xi’an, China). Mice under free conditions with a 12-h light/dark cycle were acclimatized for at last 1 week prior to the experiments with food and water ad libitum. All experiments were conducted according to the ethical guidelines for investigations of experimental pain in conscious animals. Endotoxic Shock Model Induced by LPS All male mice were randomly divided into five groups (n=6 per group): Normal control group (group 1) received equal volume of vehicle. Challenge LPS control group (group 2) was injected intraperitoneally with 1 mg/kg LPS in sterile saline. Three treatment groups (groups 3– 5) received two intragastric administration of CHE dissolved in 1 % CMC solution at various doses (1, 5, and 10 mg/kg) at 12 and 1 h before LPS injection. Detection of IL-6 and TNF-α in Serum The mice were fasted after CHE administration or LPS injection and sacrificed by cervical dislocation. The production of TNF-α and IL-6 was analyzed by commercial ELISA kits according to the manufacturer’s instructions. Blood samples were immediately collected from retroorbital plexus before sacrifice and centrifuged at 12,000 rpm for 10 min. Plasma was stored at −20 °C until the quantification of cytokine. Then, 100-μL samples and standard sample buffer were added to 96-well plate and
incubated for 40 min at 37 °C. After incubation, the wash buffer was used to wash the plate for five times. After washing, 50-μL distilled water and biotinylated antibody were allowed to react with samples in each well at 37 °C for 20 min. Then, 100-μL enzyme conjugate was added to microplate well for 10 min at 37 °C. After this procedure, the 96-well plate was put in darkness for 15 min at 37 °C to react with 100-μL TMB solution. The reaction was ended by the addition of 100-μL stop solution, and the optical density was read by microplate reader at 450 nm within 30-min range. Detection of IL-6 and TNF-α in the Lung The lung tissues were quickly removed and refrigerated at −20 °C for cytokine detection. Frozen tissue samples were placed into sterile saline at a ratio of 100 mg per 600 mL. Samples were homogenized and centrifuged at 12,000 rpm for 10 min. The supernatants were added to 96well plate for the detection of TNF-α and IL-6 using commercial ELISA kits according to the manufacturer’s instructions. Histopathology Evaluation The lung tissue was put into 4 % neutral formaldehyde for 24 h and dehydrated in increased concentration gradient of ethanol for the corresponding hours. The dehydrated tissues were transparentized in xylene and embedded in paraffin. Sections (5 μm) were cut by microtome and mounted on slides stained with hematoxylin and eosin reagent for the evaluation of leukocyte accumulation under light microscopy. In order to minimize the bias, the observer did not know which group was analyzed. Immunohistochemical Study The lung tissues were put into 4 % neutral formaldehyde for 24 h and dehydrated in increased concentration gradient of ethanol for the corresponding hours. The dehydrated tissues were transparentized in xylene and embedded in paraffin. Sections (5 μm) were cut by microtome and mounted on slides stained with hematoxylin and eosin reagent. For immunohistochemical study, after deparaffination and rehydration of tissue sections, hot fix antigen was performed in microwave oven, and then, endogenous peroxidase activity was quenched with 3.0 % hydrogen peroxide in methanol for 10 min at room temperature. After washing three times with
Protective Effects of Chelerythrine Against Endotoxic Shock phosphate-buffered saline (PBS), samples were blocked at room temperature using goat serum working solution for 20 min. Then, all the sections were incubated with polyclonal antibody for NF-κB p65 at a dilution of 1:100 in PBS (v/v) at 4 °C overnight, followed by washing with PBS solution and incubated with rabbit anti-mouse second antibody at 37 °C for 30 min. After washing with PBS for three times, the samples were incubated with horseradish peroxides at room temperature for 20 min and stained with 3,3′-diaminobenzidine for 2 min. The last procedure was dehydration and transparence with grade concentration ethanol and xylene, respectively. Then, the sections were mounted with neutral gum. The slides were visualized under light microscope to evaluate the positive immunity cells in the selected fields. Myeloperoxidase Assay The leukocyte infiltration was marked by MPO activity which was analyzed by MPO detection kit (Nanjing Jiancheng Bioengineering Institute). Frozen tissues were
homogenized into buffer application solution at a ratio of 50 mg per 950 μL. MPO activity was measured by ultraviolet spectrophotometer (756pc Shanghai Spectrum Instrument Co. Ltd., China) at 460 nm. One MPO activity unit was defined as the decomposition of 1 μmol hydrogen peroxide per gram weight of wet tissue at 37 °C; the value was calculated by the following formula. MPO activityðμ=gÞ ¼ X =ð11:3 weight of taken tissuesÞ
X equals sample OD value minus control OD value. Statistical Analysis All data were expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using SPSS 17.0 statistical software. The statistical significance of any difference in each parameter among the groups was evaluated by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. P
