Effective treatment for chronic obstructive pulmonary disease (COPD) and knowledge of the underlying mechanism are urgently required. Xiaoqinglong decoction (XQL) is widely used to treat COPD in Traditional Chinese Medicine, but the mechanism remains unclear. In this study, we tested the hypothesis that XQL ameliorates COPD via inhibition of autophagy in lung tissue on a rat model. Rats were divided into five groups, namely, Control group, COPD group, COPD + XQL group, COPD + Rapamycin group, and COPD + XQL + Rapamycin group. Pathological changes on cellular and molecular levels, apoptosis reflected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, and autophagy represented by LC3II/LC3I ratio and p62 level were investigated for each group. Compared with the Control group, COPD rats exhibited structural changes and activated inflammation in the lung tissue, together with enhanced apoptosis and elevated autophagy biomarkers. XQL treatment significantly ameliorated these changes, while rapamycin augmented them. These data altogether confirmed the involvement of autophagy in the pathogenesis of COPD and suggested that XQL attenuates COPD via inhibition of autophagy.
COPD is an obstructive, long-term lung disease characterized by airway remodeling, chronic airway inflammation, and excessive mucus secretion [
Smoking is the major cause of COPD [
Autophagy is a mechanism that cell disassembles unnecessary or dysfunctional cytoplasmic material of endogenous or exogenous origin within lysosomes [
The involvement of autophagy in lung diseases was hypothesized in 2000 when autophagic vacuoles were observed in the liver tissue of a patient with
Xiaoqinglong decoction (XQL), which was recorded in Treatise on Febrile Diseases in Han Dynasty of China, is used to treat diseases of respiratory system including cough, asthma, and COPD [
In this study, we hypothesized that XQL exhibited its anti-COPD effect via suppression of autophagy in lung tissue. To test the hypothesis, we firstly observed the autophagy indicators together with COPD symptoms in smoke-induced COPD rat model to confirm if the animal model was successful and whether autophagy was involved in COPD. Secondly, we examined the changes of the abovementioned index when these animals were given XQL and the classic autophagy inducer rapamycin, respectively, or simultaneously, to demonstrate the effectiveness of XQL in attenuating COPD and its association with autophagy.
XQL was prepared based on the traditional method. Specifically, 14 g of ephedra herb, 16 g of cassia twig, 14 g of Radix Paeoniae Alba, 18 g of
Thirty SD rats aged 6−8 weeks and weighed 230−250 g were equally randomized into five groups (
All rats were kept in a temperature-controlled room (21°C) in 12 h/12 h light/dark cycles and had free access to tap water and normal chow. The handling and treatment of animals were performed in accordance with the guidelines of the Experimental Animal Care and Institutional Animal Ethical Committee of West China Hospital of Sichuan University.
COPD model was established via combinational use of cigarette smoke, lipopolysaccharide (LPS), and cold stimuli. Specifically, on day 1 and day 14, 200
From day 29 to day 43, 2 mL of XQL was orally administered to each rat in the COPD + XQL group and the COPD + XQL + Rapamycin group. Rats in the Control group, the COPD group, and the COPD + Rapamycin group received equivalent volume of water by gavage. In addition, the COPD + Rapamycin group and the COPD + XQL + Rapamycin group were intraperitoneally injected with 0.2 mg/kg of rapamycin, while rats in the Control, COPD, and COPD + XQL groups were injected with 0.2 mL of saline. Body weights of all rats were recorded daily. On day 44, blood samples were collected from the rats’ hearts after anesthesia. After the rats stopped breathing, the lungs were collected, perfused with cold (4°C) saline, transferred into liquid nitrogen immediately, and stored at −80°C until analysis. Serum was obtained by centrifuging coagulated blood samples at 3000 g and 4°C for 5 min.
The lower right lung tissue was fixed by 4% paraformaldehyde for 24 h, dehydrated in alcohol for 12 h, embedded in paraffin, cut into 4
The mean linear intercept (MLI), an indicator of alveolar size, and mean alveolar number (MAN), an indicator for density of alveoli, were determined to assess the degree of lung emphysema. The number of linings (NS) and the total alveolar count (Na) were calculated, and the area of the image (S) was measured in a HE photograph. The MLI and MAN were calculated according to the formula MLI = L/NS and MAN = Na/S. For each indicator, the average value was calculated from six randomly selected fields, each from the lung of one rat [
Apoptosis of the lung was evaluated using TUNEL assay with TUNEL Apoptosis Assay Kit E607172 purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), following the manufacturer’s instructions. Briefly, the paraffin sections described above were deparaffinized with xylene and alcohol, permeabilized with proteinase K at 37°C for 15 min, and incubated consecutively with TDT enzyme solution and antibodies. The processed sections were observed and photographed under a CKX41SF fluorescence microscope (Olympus Corporation, Tokyo, Japan).
Serum levels of the inflammation indicators IL-8 and TNF-
Western blotting was applied to quantify LC3 and p62 proteins in the lung tissue. The left lung was weighed and protein extracted. Approximately 5 mg of tissue was homogenized and centrifuged at 12000
All statistics were based on the six animals in each group. Data were expressed as mean ± SD and compared using one-way ANOVA and following Least Significant Difference test by SPSS 16.0 (IBM Corporation, New York).
Compared with the Control rats, the COPD rats displayed changes in microstructure of bronchus, namely, narrowed airways and airway wall inflammation, the latter featured by increased infiltration of inflammatory cells in the airway walls. XQL administration partially reversed these changes, as shown in the COPD + XQL group. Rapamycin administration in the COPD + Rapamycin and COPD + XQL + Rapamycin groups worsened the pathological changes as compared with the COPD and COPD + XQL groups, respectively. The comparison between the COPD + Rapamycin and COPD + XQL + Rapamycin group showed that the latter experienced less structural changes than the former (Figure
HE staining of lung tissue. The bar graphs show semiquantification of mean linear intercept (MLI) and mean alveolar number (MAN).
Semiquantitative measurements showed that MLI increased significantly in the COPD rats. Rapamycin augmented the change while XQL attenuated it. Likewise, MAN decreased significantly in the COPD rats. This trend was amplified by rapamycin but inhibited by XQL (Figure
ELISA results (Figure
Serum IL-8 and TNF-
Body weight of each rat was recorded throughout the study (Supplementary Materials, Table
TUNEL test revealed that, compared with the Control group, the rats in COPD group exhibited higher levels of apoptosis (Figure
TUNEL results of lung tissue. Intensity of fluorescence reflects the apoptosis level.
Figure
Western blotting of LC3I, LC3II, p62, and
In regard to p62, the levels in all four COPD groups were significantly lower than the Control group. The COPD + XQL group showed higher p62 levels than the COPD group, while the COPD + Rapamycin group showed lower p62 levels than the COPD group. Besides, the COPD + XQL + Rapamycin group displayed higher p62 levels than the COPD + Rapamycin group.
These results indicated that the LC3II/LC3I ratio was upregulated and p62 level downregulated in COPD rats and that XQL could attenuate or reverse these changes. The protective effects induced by XQL were counteracted by rapamycin, the autophagy activator, suggesting opposite effects of XQL and rapamycin.
The COPD mice in this study exhibited typical symptoms and characteristics of the disease. XQL treatment ameliorated these symptoms, while rapamycin worsened them. The autophagy biomarker, LC3II/LC3I ratio, was elevated while p62 concentration was decreased in the COPD rats; these changes were enlarged by rapamycin administration but attenuated by XQL treatment. These data altogether exhibited the effect of XQL, which was opposite to the autophagy activator rapamycin.
Rapamycin activates autophagy via inhibition of the mammalian target of rapamycin mTOR [
Involvement of autophagy in COPD has been confirmed by a number of studies. For example, Chen et al. found that microtubule-associated protein 1 light chain 3 beta (LC3B) activated apoptosis in the cigarette smoke-induced emphysema mice model, suggesting autophagy is responsible for the apoptosis in lung diseases [
Herbal medicine has been used to treat lung diseases for a long time. Apart from XQL, other herbal medicines, such as
Hence, it raises a question of which component in XQL is responsible for its effect on COPD and autophagy. XQL is traditional Chinese medication prepared from eight herbs, among which only one plant,
A summary of the abovementioned studies on herbal medicine and autophagy shows that all of the herbal medicine exhibited protective effect. However, XQL and
It should be noted that, considering that XQL is a combination of eight herbs and each one contains a variety of components, the ingredients and compounds responsible for the pharmacological effects observed in this study remain unclear, which is a challenge in all studies on herbal medicine.
Another limitation of the study is that no sample was collected between completion of modeling and commencement of XQL treatment. Thus, it was unclear whether XQL prevented or reversed the progress of COPD. Nevertheless, the study clearly shows that XQL possesses protective effect on COPD in rats, which is associated with the inhibition of autophagy.
To summarize, this study supports the hypothesis that XQL inhibits the progress of COPD via attenuating the autophagy process. Further studies are required to investigate the key components in the decoction that are responsible for this effect.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors acknowledge the Supporting Program of Sichuan Provincial Department of Science and Technology (2013SZ0001-5) for financial support.
Table S1: the body weights of the rats in each group and week, which indicates general wellbeing of the rats. Body weights (grams) of the rats in each group and week of treatment.