|Year : 2019 | Volume
| Issue : 4 | Page : 263-268
Jian-Pi-Yi-Fei granule suppresses airway inflammation in mice induced by cigarette smoke condensate and lipopolysaccharide
Cui-Ying Tang1, Si-Li Tang2, Kai-Feng Huang1, Jian Xu1, Hui Yu1, Lin Lin1
1 Department of Respiratory, Guangdong Hospital of Traditional Chinese Medicine, Guangzhou, China
2 Department of Pharmacochemistry, College of Pharmaceutical Science, Guangzhou Medical University, Guangzhou, China
|Date of Submission||28-Feb-2018|
|Date of Acceptance||19-Aug-2019|
|Date of Web Publication||13-Sep-2019|
Dr. Lin Lin
Department of Respiratory, Guangdong Hospital of Traditional Chinese Medicine, 111 Dade Road, Guangzhou, 510120
Source of Support: None, Conflict of Interest: None
INTRODUCTION: As a chronic, progressive, and lethal pulmonary disease, chronic obstructive pulmonary disease (COPD) is lacking effective treatment. Chronic inflammatory processes, including inflammatory cytokines, play an important role with in its pathogenesis. Jianpiyifei (JPYF) granule is a traditional Chinese herbal formula historically used to strengthen the spleen and tonify the lung. JPYF is used clinically to treat stable COPD. However, whether the purported anti-inflammatory effect of JPYF in COPD involves regulation of key inflammatory cytokines is not clear.
MATERIALS AND METHODS: The mice model of pulmonary inflammation was induced by lipopolysaccharide (LPS) and cigarette smoke condensate (CSC). The influence of JPYF on airway inflammation in vivo was investigated. Mice were divided into three groups: control, model, and treatment groups. In the CSC + LPS model group and JPYF treatment group, intratracheal injection of CSC and LPS was used to induce airway inflammation for 5 days. JPYF group animals were also orally administered 5.5 g/kg JPYF granule for 12 days.
RESULTS: The number of neutrophils and total cells in bronchoalveolar lavage fluid of the JPYF group were markedly lower than in the model group. The levels of interleukin (IL)-1 β and IL-6 were lower; tumor necrosis factor-alpha was downregulated, and IL-10 was higher in the JPYF group than the model group. In the JPYF group, histone deacetylase 2 (HDAC2) activity and protein expression were restored.
CONCLUSION: The anti-inflammatory activity of JPYF involves the suppression of pro-inflammatory cytokines, enhanced IL-10 secretion, and the restoration of HDAC2 activity.
Keywords: Histone deacetylase, inflammation, Jianpiyifei granule, lung
|How to cite this article:|
Tang CY, Tang SL, Huang KF, Xu J, Yu H, Lin L. Jian-Pi-Yi-Fei granule suppresses airway inflammation in mice induced by cigarette smoke condensate and lipopolysaccharide. Indian J Pharmacol 2019;51:263-8
|How to cite this URL:|
Tang CY, Tang SL, Huang KF, Xu J, Yu H, Lin L. Jian-Pi-Yi-Fei granule suppresses airway inflammation in mice induced by cigarette smoke condensate and lipopolysaccharide. Indian J Pharmacol [serial online] 2019 [cited 2019 Nov 21];51:263-8. Available from: http://www.ijp-online.com/text.asp?2019/51/4/263/266817
| » Introduction|| |
Chronic obstructive pulmonary disease (COPD), a chronic, progressive, and lethal lung disease, is characterized by complex inflammation and sustained obstruction of airflow. COPD is currently a major health problem lacking effective treatments. Although COPD is a preventable and treatable common disease, its morbidity and mortality are predicted to increase as population age in many countries.
Chinese herbal medicine is clinically used to treat COPD. Jianpiyifei (JPYF) granule is a Traditional Chinese Medicine (TCM) preparation, containing six Chinese herbs. JPYF granule is used to strengthen the spleen and tonify the lung and is used in clinical practice for stable COPD. Our previous clinical trials have demonstrated that JPYF granule can ameliorate symptoms, pulmonary function, and airway inflammation in COPD patients.,,
Chronic inflammation plays an essential pathophysiological role during the progression of COPD, involving various inflammatory cytokines. Furthermore, chronic inflammation has been associated with genetic regulation such as histone modification. Acetylation of core histones is involved in regulating pro-inflammatory gene expression, allowing RNA polymerase and transcription factor binding to DNA, thus initiating gene transcription. In comparison to healthy controls, significant alterations of histone acetyltransferase (HAT) and histone deacetylase (HDAC) were observed in COPD patient lung tissues.,,
However, whether JPYF exerts this anti-inflammatory efficacy in COPD by regulating key inflammatory cytokines and HDAC2 levels remains unclear. The impact of JPYF granule was investigated in an in vivo model of lung inflammation induced by intratracheal injection of lipopolysaccharide (LPS) and cigarette smoke condensate (CSC).
| » Materials and Methods|| |
Compounds and reagents
Antibodies directed toward HDAC2 (55 kDa) were acquired from Abcam (Cambridge, UK) and antibodies directed toward β-actin (43 kDa) were supplied by Santa Cruz Biotechnology (Santa Cruz, USA). LPS was purchased from Sigma-Aldrich (St. Louis, USA). Shuangxi brand cigarettes (11 mg tar, 1 mg nicotine) were acquired from China Tobacco Guangdong Industrial Co., Ltd (Guangzhou, China). Enhanced chemiluminescence reagent was acquired from Amersham/GE Healthcare (Buckinghamshire, UK); Diff-Quik staining reagent was acquired from IMEB Inc. (Deerfield, IL, USA); and interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were detected with enzyme-linked immunosorbent assay (ELISA) kits which were acquired from R and D System (Minneapolis, USA) and Bender MedSystems (Vienna, Austria), respectively, and ELISA kits for IL-10 and IL-1 β were acquired from eBioScience (San Diego, CA). Polyvinylidene difluoride membranes were acquired from Roche (Basel, Switzerland). Fluorometric HDAC2 activity assay kit was supplied by BioVision (Mountain View, CA, USA), and the calycosin-7-O-β-D-glucoside standard was acquired from Nanjing Spring and Autumn Biological Engineering Co., Ltd. (Nanjing, China).
The JPYF granule contains six traditional Chinese herbs [Table 1]. Fluid extracts of herbs were concentrated and dried to obtain the final granule product. The standardized package of JPYF granule was 10 g. JPYF granule quality is assessed for consistency according to the industry standards. The JPYF granule was prepared and provided by the Department of Pharmacy in Guangdong Provincial Hospital of TCM (Guangzhou, China), production batch number: 20140203.
High-performance liquid chromatography analysis
The chromatographic fingerprint representative of calycosin-7-glucoside was investigated by high-performance liquid chromatography (HPLC). JPYF granule (2 g) was dissolved in 25 mL 50% methanol and was extracted by ultrasonic extraction (100 w, 40 kHz, 30 min), and the subsequent filtrate was used. Reference standards were prepared to obtain 50 μg/mL solution of calycosin-7-O-β-D-glucoside. HPLC analysis was performed by LC-20A HPLC (Shimadzu, Kyoto, Japan) with SPD-20A detector. Chromatographic separation was carried out via Phenomenex, Prodigy 5 μM ODS3 100A (250 mm × 4.60 mm, 5 μM) with a column temperature of 30°C and ultraviolet wavelength of 260 nm. The mobile phase contained methanol and water-formic acid (100:0.2, v/v). The volume per injection was 10 μL, and the velocity was 1 mL/min. The gradient program was as follows: 20% B for 0–20 min and 40% B for 20–30 min.
Cigarette smoke condensate preparation
CSC was prepared as previously described. Briefly, CSC was collected on a 92-mm Cambridge filter pad. The solvent of methanol extract was vacuum evaporated and freeze-dried. The lyophilized cigarette smoke crude extract was stored at −60°C until use.
Female BALB/c mice (specific-pathogen-free; 8 week old; 19–21 g) were obtained from the Laboratory Animal Center (Guangdong, China). All mice were kept in the Laboratory Animal Center of Guangzhou Medial University and were kept in a temperature controlled rooms with a cycle of 12-h dark/12-h light. The mice were maintained with standard chow and water ad libitum. This study was permitted by the Ethical Committee for Experimental Work of Guangzhou Medical University (No. 2015-205). Mice were divided into three groups: (i) mice received water in control group; (ii) CSC + LPS model group mice received CSC and LPS; and (iii) JPYF group mice received CSC + LPS and were administered JPYF granule orally (5.5 g/kg JPYF granule in distilled water for 12 continuous days) (n = 8). Pulmonary inflammation was induced in model and JPYF groups by intratracheal injections of CSC (50 μL, 10 mg/mL in phosphate-buffered saline [PBS]) and LPS (50 μL, 50 μg/mL in PBS) for 5 days (day 8–day 12) after intraperitoneal administration of anesthetics (pentobarbital: 50 mg/kg). All mice were sacrificed on day 13. Moreover, lung tissues and bronchoalveolar lavage (BAL) fluid were acquired.
Bronchoalveolar lavage fluid and cell number calculation
Lungs were lavaged with 0.5 mL PBS (pH 7.4) for three times, 1.5 mL BAL fluid in total for each mouse. After centrifuging at 5000 rpm for 10 min, the supernatant of BAL fluid was stored in aliquots at −60°C before the analysis. The cells in BAL fluid were resuspended; Diff-Quik staining reagent was used to fix and stain the cells in BAL fluid. The smears were air-dried and were fixed in “Diff-Quik” for 30 s. The samples were stained with “Diff-Quik” solution II for 30 s, counterstained with “Diff-Quik” solution I for 30 s. For removing excess stain, the samples were rinsed in tap water. Finally, the samples were dehydrated in absolute alcohol. The number of neutrophils and total cells in the BAL fluid were calculated blindly by two investigators.
Measurements of interleukin-6, interleukin-10, interleukin-1 beta, and tumor necrosis factor-alpha
BAL fluid content of IL-6, IL-10, IL-1 β, and TNF-α was determined using ELISA kits following the manufacturer's instructions. The antibodies specific for mouse cytokines were coated on a 96 well plates. The BAL fluid was pipetted into the wells and immobilized antibodies were bound to cytokines to be tested. After washing away unbound antibodies, the Biotinylated Anti-Mouse Antibodies were added. The wells were washed again and conjugated streptavidin was pipetted to the wells. A Tetramethylbenzidine (TMB) substrate solution was added to the wells after washing the wells. The amount of cytokines bound was in proportion to the intensity of the color. The intensity of the color was measured with microplate reader.
Lung tissues were homogenized. Extracted proteins (30 μg) were separated by electrophoresis containing 10% sodium dodecyl sulfate polyacrylamide gel. Then, the proteins were transferred onto polyvinylidene difluoride membranes. Rabbit monoclonal anti-HDAC2 (55 kDa) antibody (1:1000 dilution) or β-actin (43 kDa) antibody (1:5000 dilution) was used to incubate the membranes. The molecular band intensity was determined relative to β-actin.
Measurement of histone deacetylase 2 activity
HDAC2 activity was evaluated using the fluorometric HDAC2. Bicinchoninic acid protein assay was used to determine the protein content. The HDAC2 activity was detected according to the detailed instructions of the activity assay kit. The activity of HDAC2 was represented as the relative fluorescence units per mg protein.
Histopathologic analysis for lungs
Histopathologic changes were evaluated with hematoxylin and eosin staining. The inflated lungs were fixed with 4% paraformaldehyde for at least 72 h. Five μm sections of paraffin-embedded lungs were stained using hematoxylin and eosin. Images were captured at ×100 magnification for morphologic assay.
All data were represented as mean ± standard error of mean and were analyzed with Prism 5.01 software (GraphPad Software, California, USA). One-way analysis of variance with Tukey's test was applied. P < 0.05 was required to indicate statistical differences.
| » Results|| |
The chromatographic fingerprint of Jianpiyifeigranule
Calycosin-7-glucoside is one active compound of JPYF granule, and it is the standard detected by HPLC for quality control of relative Chinese herbs. The chromatographic fingerprint of calycosin-7-glucoside was observed in JPYF preparation. As shown in [Figure 1], the retention time of calycosin-7-O-β-D-glucoside was at 8.195 min.
|Figure 1: The chromatographic fingerprint of Jianpiyifei granule with the representative of calycosin-7-glucoside. (a) Standard of calycosin-7-glucoside at 8.195 min|
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Effects of Jianpiyifei granule on the neutrophil count and total cell in bronchoalveolar lavage fluid of cigarette smoke condensate- and lipopolysaccharide-induced mice
Only a small number of neutrophils and other cells can be seen in the control group. In mouse models of CSC- and LPS-induced pulmonary inflammation, the neutrophil count increased and total cell count was significantly higher than in the control group. The BAL fluid's total cell and neutrophil counts were significantly lower in JPYF granule-treated animals than the model group [P< 0.01, [Figure 2]. JPYF granule could reduce the total number of cells and neutrophil count by about half.
|Figure 2: Effect of JPYF on neutrophil numbers in bronchoalveolar lavage fluid of cigarette smoke condensate- and lipopolysaccharide-induced mice. (a) Total cell number in BAL fluid of cigarette smoke condensate- and lipopolysaccharide-induced mice. (b) Neutrophil number in BAL fluid of cigarette smoke condensate- and lipopolysaccharide-induced mice. Data are presented as mean ± SEM (n =8). *P < 0.01 versus control group; #P < 0.01 versus CSC and LPS group. LPS: Lipopolysaccharide, CSC: Cigarette smoke condensate, SEM: Standard error of mean, JPYF: Jianpiyifei granule|
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Effects of Jianpiyifei granule on cytokines in cigarette smoke condensate- and lipopolysaccharide-induced mice
The levels of inflammatory cytokines were very low, close to the background level. As shown in [Figure 3], cytokines were all induced by intratracheal injections of LPS and CSC (P< 0.01). The levels of TNF-α (3A) were downregulated and IL-1 β (3B) and IL-6 (3C) in BAL fluid were significantly lower in animals administered JPYF granule (P< 0.01), while the IL-10 (3D) concentration was higher (P< 0.01).
|Figure 3: Effect of JPYF on cytokines in bronchoalveolar lavage fluid of CSC and LPS-induced mice. (a) Tumor necrosis factor-alpha; (b) interleukin-1 β; (c) interleukin-6; (d) and interleukin-10 levels in bronchoalveolar lavage fluid of cigarette smoke condensate- and lipopolysaccharide-induced mice. Data are presented as mean ± SEM (n=8). *P < 0.01 versus control group; #P < 0.01 versus CSC and LPS group. LPS: Lipopolysaccharide, CSC: Cigarette smoke condensate, SEM: Standard error of mean, JPYF: Jianpiyifei granule|
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Effects of Jianpiyifei granule on the expression and activity of histone deacetylase 2 in cigarette smoke condensate- and lipopolysaccharide-induced mice
HDAC2 expression was significantly lower in CSC- and LPS-induced mice than controls (P< 0.01). In JPYF granule-treated mice, the levels of HDAC2 protein were significantly higher than in the model mice [P< 0.01, [Figure 4]a and [Figure 4]b. HDAC2 activity was also lower in model animals than controls but was restored in JPYF granule-treated animals [P< 0.01, [Figure 4]c.
|Figure 4: Western blotting for histone deacetylase 2 and histone deacetylase 2 activity in lung tissue lysates from each group. (a) Western blotting for histone deacetylase 2, β-actin was used as an internal control. (b) Relative density analysis of histone deacetylase 2 bars indicate the mean density ratio ± standard error of mean. (c) Histone deacetylase 2 activity (n = 8). *P < 0.01 versus control group; #P < 0.01 versus CSC and LPS group. LPS: Lipopolysaccharide, CSC: Cigarette smoke condensate, JPYF: Jianpiyifei granule|
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Effect of Jianpiyifei granule on cigarette smoke condensate- and lipopolysaccharide -induced lung histological damage
[Figure 5] shows the histopathological changes of lungs observed in model mice induced by CSC and LPS. There were obvious inflammation reaction, inflammatory cell infiltration, and exudation of inflammatory cells in the connective tissue of the alveolar septum. Particularly, the neutrophils around pulmonary vessels and bronchi at all levels were obviously retained and aggregated. In addition, congestion, edema, and alveolar diaphragm thickening were also observed in the lung tissue. Compared with CSC- and LPS-induced mice, the degree of infiltration of inflammatory cells was ameliorated in animals treated with JPYF. Inflammatory reaction, neutrophil retention and aggregation, and alveolar diaphragm thickening were also observed in the treatment group, but the inflammatory reaction was mild, alveolar wall thickening was not obvious, and a small amount of congestion and edema could be seen in the lung tissue.
|Figure 5: The effects of JPYF on the histopathological change in the lungs of CSC-and LPS-induced mice. Representative section of lung tissues from each experimental group was stained with hematoxylin and eosin (×100, bar = 200 μm). LPS: Lipopolysaccharide, CSC: Cigarette smoke condensate, JPYF: Jianpiyifei granule|
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| » Discussion|| |
COPD, a chronic pulmonary disease, mainly affects the lung, and in TCM, it is considered to lead to Ben-root deficiency, characterized by Qi deficiency syndrome related to the lung, spleen, or kidney. In TCM, JPYF granule is formulated to strengthen the Spleen (Pi) and tonify Lung (Fei) principle, designed to improve the function of the lung to protect stable COPD patients from disease progression.,, Inflammation plays a causal role in COPD; hence, new anti-inflammatory strategies are urgently sought to treat COPD. We showed that JPYF granule could inhibit lung inflammation and elevate HDAC2 expression in a mouse model of CSC- and LPS-induced pulmonary inflammation.
Here, we observed that JPYF granule reduced lung inflammation and upregulated the HDAC2 expression in the mice of pulmonary inflammation induced with CSC and LPS. Within the BAL fluid of these mice, the total cell count was significantly reduced in JPYF-treated mice, suggesting that JPYF granule significantly reduced inflammatory cell infiltration into the airway. We also found that JPYF granule suppressed the levels of cytokines, IL-6, IL-1 β, and TNF-α which encourage inflammation development, in BAL fluid. These cytokines are overexpressed in COPD patients and animal models. IL-6 is an important mediator of lung inflammation in COPD. Compared to healthy volunteers, the markedly increased IL-6 levels were detected in patients with stable COPD. TNF-α and IL-1 β are multifunctional cytokines produced by alveolar macrophages. Higher levels of these cytokines can stimulate endothelial and epithelial cells to produce additional inflammatory mediators. Cigarette-smoke-induced lung damage is mediated by TNF-α. JPYF granule significantly downregulated the level of TNF-α. Therefore, we believed that it could decrease several inflammatory mediators of IL-1 β and IL-6, by partly decreasing TNF-α. As a strong anti-inflammatory cytokine, IL-10 can suppress the release of pro-inflammatory cytokines., JPYF granule induced IL-10 secretion in CSC- and LPS-induced mice thus may inhibit the development of lung inflammation.
Although COPD is a chronic inflammatory disease, glucocorticosteroids (GCs), the main treatment for asthma, are not effective against COPD-associated inflammation. Recruitment of HDAC2 is a key mechanism related to the inflammatory gene regulated by glucocorticosteroids (GCs). Corticosteroid resistance may be caused by downregulated HDAC2 activity. HDAC2, a class I HDAC, was found to be anti-inflammatory and mainly localized in the nucleus. It has been suggested that histone acetylation and deacetylation are essential regulators of pro-inflammatory genes. The activities of transcription factors of pro-inflammatory genes are modulated by HDACs and HATs. In patients with COPD, the expression of inflammatory genes is significantly increased in lung tissues, an effect that can be suppressed by HDAC2. Restoring HDAC2 activity reduced inflammation in patients with COPD. JPYF granule treatment clearly exhibited anti-inflammatory effects on CSC- and LPS-induced mice, with concomitant restoration of HDAC2 activity. This suggests that JPYF granule may restore the anti-inflammatory effect of HDAC2 and thus control the underlying pathological development of COPD. Consistently, histopathologic changes in the lung tissues including inflammatory cell infiltration were improved by JPYF granule.
The molecular mechanism and profound cell signaling pathway involved in the anti-inflammatory effects by JPYF granule were not investigated in our current study. These aspects will be studied in future experiments using cell and animal models.
| » Conclusion|| |
Our current study demonstrated that JPYF granule inhibited CSC- and LPS-induced pulmonary inflammation in mice. The effects of JPYF granule on airway inflammation may involve inhibition of pro-inflammatory cytokines, enhancement of IL-10, and the restoration of HDAC2 activity.
Financial support and sponsorship
This work was funded by NSFC (No. 81373566 to Dr. L Lin) and Guangdong S and T Planning Project (No. 2014KT1510 to Dr. TY Tang).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]