Regulatory Effect of Shenge Yifei Capsule on TGF-β1/Smad Signaling Pathway in Rats with Chronic Obstructive Pulmonary Disease

Ying HAO1, Hongwei ZHONG*, Yuanyun GU, Hui CHEN

1. Southwest Medical University, Luzhou 646000, China; 2. Hospital of Traditional Chinese Medicine Affiliated to the Southwest Medical University, Luzhou 646000, China

Abstract [Objectives] This study aimed to study the effects of Shenge Yifei capsule on the TGF-β1/Smad signaling pathway in rats with chronic obstructive pulmonary disease (COPD). [Methods] Ten rats were randomly selected as the control group, and the other 40 rats were selected for modeling by fumigation combined with Klebsiella pneumoniae infection. A total of 38 rats were successfully modeled. They were randomly divided into model group (8 rats), low-dose Shenge Yifei capsule group (10 rats), high-dose Shenge Yifei capsule group (10 rats) and theophylline group (10 rats) in accordance with the principle of half male and half female. The rats in the model and control groups were given with distilled water by gavage, and the rats in the drug administration groups were given with corresponding drugs. The TGF-β1 level in the serum, and the expression levels of TGF-β1, Smad2, Smad3 and Smad7 and TGF-β1, Smad3 and Smad7 in airway tissues were detected. [Results] After 12 weeks, the serum TGF-β1 levels of the theophylline group and high-dose Shenge Yifei capsule group were lower than that of the low-dose Shenge Yifei capsule group (P<0.05). The expression levels of TGF-β1 and Smad3 in the theophylline group and high-dose Shenge Yifei capsule group were lower than that in the low-dose Shenge Yifei capsule group (P<0.05). The expression levels of TGF-β1 and Smad3 in the high-dose Shenge Yifei capsule group were lower than those in the low-dose Shenge Yifei capsule group and theophylline group (P<0.05). The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups (P<0.05). The expression levels of Smad7 in the theophylline group and high-dose Shenge Yifei capsule group were higher than that in the low-dose Shenge Yifei capsule group (P<0.05). After 18 weeks, no significant difference was found in serum TGF-β1 level among the theophylline group and low and high-dose Shenge Yifei capsule groups (P>0.05). The expression levels of Smad7 and the proteins in the model group were lower than those in the other groups. The expression level of Smad7 in the high-dose Shenge Yifei capsule group was lower than that in the theophylline group (P<0.05). [Conclusions] Shenge Yifei capsule can regulate the TGF-β1/Smads signaling pathway. They can down-regulate the expression of TGF-β1, Smad2 and Smad3 and up-regulate the expression of Smad7, reducing the degree of airway modeling, delaying the development of COPD disease. Conventional high-dose Shenge Yifei capsule is more effective in inhibiting the expression of Smad2.

Key words Chronic obstructive pulmonary disease (COPD), Shenge Yifei capsule, Tansforming growth factor-β1, Signaling pathway, Rat

1 Introduction

Chronic obstructive pulmonary disease (COPD) is a common disease characterized by persistent respiratory symptoms and restricted airflow, and is caused by abnormalities in airway and (or) alveoli caused by toxic particles or gases[1]. The global incidence is high, about 4%-10%. WHO estimates that by 2030, COPD will become the third leading cause of death after ischemic heart disease and sudden death[2]. At present, traditional Chinese medicine has shown good curative effects and application prospects in clinical and experimental studies of COPD[3]. Shenge Yifei capsule is a traditional Chinese medicine preparation in the Hospital of Traditional Chinese Medicine Affiliated to the Southwest Medical University. The purpose of this experiment is to observe the effect of Shenge Yifei capsule on the TGF-β1/Smad signaling pathway in COPD rats at stable stage and to explain the therapeutic mechanism.

2 Materials and methods

2.1ExperimentalanimalFifty SD rats of clean grade (half male and female), (230 ± 20) g for female and (290 ± 20) g for male, were purchased from the Animal Experimental Center of Southwest Medical University[license No. SYXK (Sichuan) 2013-065].

2.2DrugsandreagentsShenge Yifei capsule (Hospital of Traditional Chinese Medicine Affiliated to the Southwest Medical University, batch No. Chuan Yao Zhi Zi Z20080756); theophylline sustained-release tablets (Guangzhou Maite Xinghua Pharmaceutical Factory, batch No. H44023791).

Cigarettes of Huangguoshu brand were produced by China Tobacco Guizhou Industrial Co., Ltd. The fumigation box (45 cm×30 cm×20 cm) used was self-prepared, and the top cover of the box had ventilation holes. Liquid ofKlebsiellapneumoniae(6×108CFU/mL) was provided by the laboratory of Hospital of Traditional Chinese Medicine Affiliated to the Southwest Medical University.

Transforming growth factor-β1 (TGF-β1) ELISA kit (Shanghai Qiaodu Biological Technology Co., Ltd., China); SYBR Green kit (TOYOBO, Japan); RT Kit (TOYOBO, Japan); Marker III (Solarbio, China); Ripa lysate (Solarbio, China); TBST (Biodder); western blocking solution (Biodder); western primary antibody diluent (Biodder); western secondary antibody diluent (Biodder).

2.3ModelestablishmentTen rats were randomly selected as the control group, and the other 40 rats were selected for modeling by fumigation combined withK.pneumoniaeinfection. The specific method was as follows. The rats were subjected to fumigation treatment, four cigarettes for 30 min each time. From week 1 to week 2, fumigation was performed twice a day; and from week 3 to week 6, fumigation was performed third times a day. The interval between every two times of fumigation within one day was greater than 3 h. The fumigation lasted for a total of six weeks. From week 1 to week 3, the nasal cavity of each rat was instilled with 0.1 mL ofK.pneumoniaesolution (6×108CFU/mL), once every 5 d. After 6 weeks, one and two rats were randomly selected from the control group and modeling group, and their right lower lobes were sampled for hematoxylin-eosin (HE) staining. Morphological changes in the lung tissues were observed to judge whether models were established successfully.

2.4GroupingandadministrationThirty-eight rats successfully modeled were randomly divided into model group (8 rats), low-dose Shenge Yifei capsule group (10 rats), high-dose Shenge Yifei capsule group (10 rats) and theophylline group (10 rats) in accordance with the principle of half male and half female. All the rats were numbered and marked with 3% picric acid solution.

Drugs began to be administered from the 1st d of the 7th week since the beginning of the modeling. In the low-dose Shenge Yifei capsule group, the rats were administered with 0.42 g/( kg·d) of Shenge Yifei capsule by gavage; in the high-dose Shenge Yifei capsule group, the rats were administered with 0.62 g/( kg·d) of Shenge Yifei capsule by gavage; in the theophylline group, the rats were administered with 0.02 g/( kg·d) of theophylline by gavage; and in the control group and model group, the rats were given with equal amount of distilled water. The administration was continued until the end of the 18th week. During the administration period, the rats in all the groups except those in the control group were still subjected to fumigation treatment, four cigarettes for 30 min, and twice a day. The interval between every two times of fumigation was longer than 3 h.

2.5SpecimencollectionandprocessingOn the 1st d after the end of the 12th week and the 18th week, four, four, five and five rats were randomly selected from the control group, model group and low and high-dose Shenge Yifei capsule groups, and their abdominal aortic blood was collected. After standing at 4 ℃ for 4 h, the blood samples were centrifuged at 3 000 r/min for 10 min, and the serum was collected and stored at -80 ℃ for future use. Then, the chest of each of the rats was opened. The lungs were taken out, and the right lower lobes were separated for HE staining. A portion of the lung and airway tissues was quickly sampled and placed into freezer tubes and stored at -80 ℃.

2.6ObservationanddeterminationThe fur, behavior, spirit, respiration, body weight, food intake, water intake and feces of the rats were observed. The pulmonary index (lung mass/body mass) of the groups was compared. The histopathology of the lung and airway tissues was observed according to the conventional HE staining procedure. The concentration was determined with the TGF-β ELISA kit. The expression levels ofTGF-β1,Smad2,Smad3 andSmad7 in the lung and airway tissues were detected by real-time fluorescence quantitative PCR method. The expression levels of TGF-β1, Smad3 and Smad7 in the lung and airway tissues were detected by western-blot.

2.7StatisticsStatistical analysis was performed using SPSS 20.0. Quantitative data are expressed as mean±standard deviation. Comparisons between groups were performed using independent samplet-test (between two groups) or one-way analysis of variance. The pairwise comparisons between groups were performed using theLSDmethod. The significance level of differences was set at α=0.05. WhenP<0.05, differences were considered significant.

3 Results and analysis

3.1ComparisonsofgeneralindicesbeforeandaftermodelingBefore modeling, the rats in each group had shiny skin, normal behavior, stable breathing, good mental state, normal good intake and water intake, and normal defecation. After modeling, the rats in all the groups except those in the control group preferred lying still, wheezed with open mouth and sweated, with secretions from the nose and mouth and reduced activity, and they recovered 15 min after removal from the fumigation box. The general situation of the control group was not significantly different from that before the experiment. The rats in the model group and the drug administration groups showed yellow fur, significantly reduced activity, short breath, frequent coughing, unresponsiveness and debility. There were no significant changes in their food intake, water intake or defecation. The drug administration groups and the model group showed some improvement compared with before administration. Among them, the improvement in the theophylline group and high-dose Shenge Yifei capsule group was more significant.

3.2PathologicalchangesinlungtissuesofratsineachgroupThe structure of trachea, bronchi and alveoli in the control group were basically intact, and no filtration of inflammatory cells was seen. After 6 weeks of modeling, the bronchial cilia of various levels were dumped and disordered and some of them had fallen off; some of the mucosal epithelium had squamous metaplasia; the number of goblet cells increased; the glands were hypertrophic; the bronchial wall was thicker than the control group; a large number of inflammatory cells infiltrated the bronchi at various levels; the alveolar wall was thinner than the control group; the alveolar cavity was enlarged; and some of the alveoli were ruptured to form bullae (Fig.1). After 12 weeks of treatment, the pathological changes of the drug administration groups were mitigated to varying degrees compared with the model group, and the mitigation in the high-dose Shenge Yifei capsule group was more obvious (Fig.2). After 18 weeks of treatment, the pathological changes of the drug administration groups were mitigated to varying degrees compared with the model group, and the mitigation in the theophylline group and high-dose Shenge Yifei capsule group was more obvious (Fig.3).

Note: A. control group; B. modeling group.

Fig.1Morphologyoflungtissuesofratsinthecontrolgroupandmodelinggroupatweek6ofmodeling(HE, ×200)

Note: A. control group; B. model group; C. theophylline group; D. low-dose Shenge Yifei capsule group; E. high-dose Shenge Yifei capsule group.

Fig.2Morphologyoflungtissuesofratsinvariousgroupsatweek12ofadministration(HE, ×200)

Note: A. control group; B. model group; C. theophylline group; D. low-dose Shenge Yifei capsule group; E. high-dose Shenge Yifei capsule group.

Fig.3Morphologyoflungtissuesofratsinvariousgroupsatweek18ofadministration(HE, ×200)

3.3Comparisonsofpulmonaryindexamongdifferentgroupsofrats12and18weeksafterdrugadministrationThe specific results are shown in Table 1.

Table1Comparisonsofpulmonaryindexamongdifferentgroupsatweeks12and18ofadministration

GroupAt week 12At week 18Control0.003 7±0.001 0△0.003 8±0.000 7△Model0.003 8±0.000 20.003 9±0.000 4Theophylline0.004 5±0.000 1△0.004 5±0.000 8△Low-dose Shenge Yifei capsule0.003 9±0.000 3△◇0.003 7±0.000 8△◇High-dose Shenge Yifei capsule 0.003 9±0.000 3△◇○0.004 2±0.000 5△◇○

Note: Compared with model group,△P>0.05; compared with theophylline group,◇P>0.05; and compared with low-dose Shenge Yifei capsule group,○P>0.05.

3.4ComparisonsofserumTGF-β1levelamongdifferentgroupsofrats12and18weeksafterdrugadministrationThe specific results are shown in Table 2.

GroupAt week 12At week 18Control163.40±19.92▲160.37±18.65▲Model233.60±13.33234.93±7.15Theophylline170.80±7.35▲161.15±9.36▲Low-dose Shenge Yifei capsule195.43±6.72▲◆176.00±9.43▲High-dose Shenge Yifei capsule 170.23±7.58▲●165.05±16.56▲

Note: Compared with model group,▲P<0.05; compared with theophylline group,◆P<0.05; and compared with low-dose Shenge Yifei capsule group,●P<0.05.

3.5ExpressionofTGF-β1,Smad2,Smad3andSmad7inairwaytissuesofratsineachgroup12and18weeksafterdrugadministrationThe specific results are shown in Table 3.

GroupTGF-β1∥ρ/ng/LAt week 12At week 18Smad2At week 12At week 18Smad3At week 12At week 18Smad7At week 12At week 18Control1.03±0.30▲0.027±0.01▲1.00±0.13▲1.04±0.38▲1.06±0.45▲1.02±0.28▲1.00±0.03▲1.07±0.49▲Model4.06±0.740.23±0.117.43±2.164.40±0.433.54±0.874.56±1.420.29±0.040.27±0.11Theophylline1.34±0.42▲0.033±0.08▲1.33±0.63▲1.87±0.41▲1.17±0.39▲1.16±0.18▲0.59±0.08▲0.85±0.19▲Low-dose Shenge Yifei capsule2.30±0.09▲◆0.034±0.01▲3.44±1.75▲2.47±0.48▲2.16±0.52▲◆2.33±0.46▲0.48±0.04▲◆0.72±0.03▲High-dose Shenge Yifei capsule1.48±0.32▲●0.021±0.001▲2.07±0.24▲1.53±0.60▲●1.08±0.26▲●1.48±0.33▲0.65±0.07▲●0.99±0.10▲

Note: Compared with model group,▲P<0.05; compared with theophylline group,◆P<0.05; and compared with low-dose Shenge Yifei capsule group,●P<0.05.

3.6ExpressionofTGF-β1,Smad3andSmad7inairwaytissuesofratsineachgroup12and18weeksafterdrugadministrationThe specific results are shown in Table 4.

GroupTGF-β1∥ρ/ng/LAt week 12At week 18Smad3At week 12At week 18Smad7At week 12At week 18Control0.74±0.10▲0.66±0.19▲0.75±0.12▲0.45±0.22▲1.35±0.14▲1.06±0.18▲Model2.01±0.171.26±0.141.66±0.191.41±0.140.48±0.110.60±0.171Theophylline1.00±0.09▲1.10±0.21▲1.12±0.13▲1.11±0.13▲0.75±0.18▲1.00±0.12▲Low-dose Shenge Yifei capsule1.00±0.13▲1.03±0.10▲1.23±0.12▲1.21±0.09▲0.78±0.10▲0.92±0.12▲High-dose Shenge Yifei capsule0.75±0.08▲◆●0.97±0.15▲0.76±0.15▲◆●0.89±0.13▲●1.08±0.15▲◆●1.01±0.15▲

Note: Compared with model group,▲P<0.05; compared with theophylline group,◆P<0.05; and compared with low-dose Shenge Yifei capsule group,●P<0.05.

3.7Western-blotresultsofTGF-β1,Smad3andSmad7inratsofeachgroupThe results are shown in Fig.4.

Note: 1. control group; 2. model group; 3. theophylline group; 4. low-dose Shenge Yifei capsule group; 5. high-dose Shenge Yifei capsule group.

Fig.4Western-blotresultsofTGF-β1,Smad3andSmad7inratsofdifferentgroup

4 Discussion

So far, the research on the pathogenesis of COPD has been more and more detailed, but the exact mechanism has not yet been elucidated. A lot of research has confirmed that TGF-β1 plays the regulatory role through the TGF-β/Smad pathway, and it mainly regulates proliferation, differentiation, growth, migration and apoptosis of cells. Inflamed by smoke or smoke particles, TGF-β is synthesized and released by various inflammatory cells, and it activates the TGF-β/Smads signalling pathway and promotes the release of inflammatory mediators such as TNF-α and IL-8[4-5]. Studies have also shown that in the airway remodeling of COPD, Smad2 and Smad3 play a regulatory role in the TGF-β1/Smads signal transduction pathway, while Smad7 plays an inhibitory role[6]. Chinese medicine has received the attention of most researchers in the prevention and treatment of COPD. Many experimental and clinical studies have proven that traditional Chinese medicine has a good effect in treating COPD[7].

COPD belongs to the category of lung distention, and misdiagnosis is the root cause of lung distention. The main pathogenesis during the COPD remission period is asthenia in origin and asthenia in superficiality, with deficiency of lung, spleen and kidney as fundamental symptom and intermingled phlegm and blood stasis as incidental symptom, and qi deficiency, blood stasis and phlegm resistance run though the disease. Based on the Kaiyu Tongxuan method in Xuanfu theory, Shenge Yifei capsule developed from Shenge San inPrescriptionsforUniversalReliefof Ming Dynasty was used for the treatment of COPD in this experiment. It is composed of Radix Ginseng Rubra, gecko, pseudo-ginseng, Szechuan Lovage Rhizome and Radix Liquiritiae, with functions of tonifying lung and kidney, removing blood stasis, expelling phlegm and invigorating stagnation. This experiment shows that Shenge Yifei capsule can down-regulate the expression ofTGF-β1,Smad2 andSmad3 and up-regulate the expression ofSmad7, and reduce the degree of airway remodeling in COPD rats at remission stage. High-dose Shenge Yifei capsule is more effective in inhibitingSmad2 expression, indicating that Shenge Yifei capsule can regulate the TGF-β1/Smads signaling pathway, prevent the remodeling of COPD during the remission period and delay the development of COPD.