Effects of compound Caoshi silkworm granules on stable COPD patients and their relationship with gut microbiota: A randomized controlled trial : Medicine

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Research Article: Clinical Trial/Experimental Study

Effects of compound Caoshi silkworm granules on stable COPD patients and their relationship with gut microbiota

A randomized controlled trial

Hu, Yibing MDa; Shi, Qinghuan MDb; Ying, Songmin MDc; Zhu, Dan MDd; Chen, Hui MDd; Yang, Xiguang MDd; Xu, Jilin MDd; Xu, Feila MDe; Tao, Feibao MDf; Xu, Bin MDf,∗

Editor(s): Aslam., Muhammad Shahzad

Author Information
doi: 10.1097/MD.0000000000020511
  • Open

Abstract

1 Introduction

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death worldwide. COPD is characterized by recurrent acute exacerbations,[1] and there has been an increase in its economic burden. The reasons why COPD is associated with an increase in the death rate globally include extended exposure to the risk factors of COPD and the age at occurrence.[2] Other factors include the lack of knowledge regarding COPD among patients, patient refusal of standard treatments, and non-compliance during the period of stable COPD. Therefore, effective treatment of acute exacerbations of COPD, particularly the management of stable COPD, is important.

Compound Caoshi silkworm granules (CCSGs) are widely used as a dietary supplement in Jinhua, Zhejiang, China. Some COPD patients may take CCSGs (mainly comprising Caoshi silkworm and Astragalus) as a treatment option during the period of stable COPD. However, no evidence-based study has been published so far, and the effect of CCSGs on stable COPD patients is unclear.

The gut-lung axis has been widely recognized in recent years.[3] Over 100 trillion microbial cells are harbored in the human gut, and they play a role in immune, metabolic, and physiological functions. Not surprisingly, gut microbial homeostasis is involved in the development of lung diseases, including COPD. This axis may be a new treatment target for COPD. Thus, there is an urgent need to investigate the relationship between changes in gut microbiota and the progression of COPD.

Therefore, the aim of this prospective randomized controlled trial was to determine the effects of CCSGs in patients with stable COPD grades II–III and to assess their relationship with gut microbial homeostasis.

2 Methods

The study was performed at the Jinhua Hospital of Zhejiang University. The protocol was designed by the principal investigators in accordance with the latest clinical practice guidelines. The study was approved by the institutional ethics committee of Jinhua Hospital and was registered at http://www.chictr.org.cn/ (ChiCTR1800020441). All participants received training before the start of the trial to ensure the accuracy of the data. All patients provided informed consent. All research work was conducted strictly in accordance with the Declaration of Helsinki (1964).

2.1 Patients

Forty outpatients with an accurate diagnosis of stable COPD were enrolled from January 2019 to April 2019 at the Jinhua Hospital of Zhejiang University in China. The inclusion criteria were as follows: confirmed COPD grades II and III based on the 2013 Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines, age ranging from 40 to 80 years, and history of 2 or more exacerbations at least 2 years earlier. Exclusion criteria were as follows: confirmed heart, kidney, or liver disease or history of major lung diseases (eg, asthma, lung transplantation, cancer, or pneumonectomy) before study entry, history of alcohol or drug abuse, acquired or congenital immune deficiency, severe COPD (GOLD VI grade), any type of mental disease, pregnant or breast-feeding women, and participation in any drug clinical trial during the past 6 months.

2.2 Study design

We conducted a 3-month randomized controlled trial. Eligible participants were randomly assigned to one of the following 2 groups at a 1:1 ratio: Group A: the routine treatment (RT) group; Group B: the routine treatment plus CCSGs (RT plus CCSGs) group. Patients in the RT group were only given Western medicine treatment recommended by the GOLD guidelines. Patients in the RT plus CCSGs group, who were given Western medicine treatment, were additionally given CCSGs. Each bag of CCSGs contained Caoshi silkworm 30 g and Astragalus 5 g. These granules were given orally, one bag at a time and once a day. The treatment duration was 3 months and clinical visits were scheduled at the end of 3 months. During the treatment period, patients with any of the following conditions were removed from the study: poor compliance, serious adverse events, and any one of the above-mentioned exclusion criteria. Serious adverse events included anaphylactic shock, hospitalization due to exacerbation, leukopenia, and thrombocytopenia. Anticipated adverse events included gingivitis, toothache, dizziness, insomnia, acute exacerbation of COPD, and pharyngitis.

2.3 Stool sample collection

Fresh stool samples were obtained from all patients before the treatment. Stool samples were frozen and stored at –80°C.

2.4 DNA isolation, PCR amplification, and sequencing

Microbial total genomic DNA was extracted from stool samples using the cetyltrimethylammonium bromide/ sodium dodecyl sulfate method. DNA concentration and purity were monitored with 1% agarose gels. DNA was diluted to 1 ng/μl according to the concentration. The V4 region of the 16S rRNA gene was amplified using 515F and 806R. PCR reactions were conducted using the following parameters: 1 minute of denaturation at 98°C, 30 cycles for 10 seconds at 98°C, 30 seconds of annealing at 50°C, 30 seconds of elongation at 72°C, and a final extension at 72°C for 5 minutes. PCR products were mixed with the same volume of loading buffer, and electrophoresis was performed on 2% agarose gels. Samples with a length between 400 and 450 bp were chosen for further detection. Sequencing libraries were generated using the NEB Next Ultra DNA Library Prep Kit for Illumina (NEB, USA, Ipswich, MA, USA) following the manufacturer's recommendations. The library quality was assessed on the [email protected] 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq platform and 250 bp paired-end reads were generated.

2.5 St. George's Respiratory Questionnaire (SGRQ)

The SGRQ consists of 50 items that yield a total score based on the sum of the total number of items, as well as the following 3 scores: Symptoms, Activity, and Impact. SGRQ assessments were performed at the baseline (month 0) and at month 3.

2.6 Functional prediction

To normalize the operational taxonomic unit abundance table, we conducted 16S rRNA functional prediction. This was performed to eliminate the effect of the 16S marker gene copy number. Functional Annotation of Prokaryotic Taxa (FAPROTAX) was used for functional prediction.

3 Statistical analysis

Statistical analysis was performed by using SPSS 20.0 (IBM, Chicago, IL, USA). The data with normal distributions were presented as means and standard deviations. The independent sample t test was used. For non-normal data, the independent samples Mann–Whitney U test was used. Categorical variables were expressed as number (percentage) and compared using the chi-squared test or Fisher exact test. A P value <.05 was considered statistically significant.

4 Results

4.1 Baseline characteristics of stable COPD patients and healthy population, and the difference in gut microbiota

A total of 80 subjects were enrolled, comprising 40 stable COPD patients and 40 healthy subjects. The baseline characteristics were similar in the stable COPD patient and healthy population groups (Table 1). The Venn diagram showed that the 2 groups had different types of gut microbiota. However, some component parts of the gut microbiota were the same (Fig. 1).

T1
Table 1:
Baseline characteristics of stable COPD patients and healthy population.
F1
Figure 1:
Venn diagram of gut microbiota operational taxonomic units. Blue: healthy population; Green: stable chronic obstructive pulmonary disease patients.

4.2 Baseline characteristics of the RT and RT plus CCSGs groups

The 40 stable COPD patients were further divided into the following 2 groups: 20 patients were included in the RT group and 20 patients were included in the RT plus CCSGs group. All patients received a β2 receptor agonist and inhaled glucocorticoids as the basic treatment. The demographic data, including age, body mass index (BMI), and sex, were not significantly different between the 2 groups. Table 2 shows the baseline levels of alanine aminotransferase, aspartate transaminase, blood urea nitrogen, serum creatinine, erythrocyte sedimentation rate, and C-reactive protein (all P > .05).

T2
Table 2:
Baseline characteristics of patients in the RT and RT plus CCSGs groups.

4.3 St. George's Respiratory Questionnaire scores in the RT and RT plus CCSGs groups

Before and after 3 months of treatment, we assessed the SGRQ scores in both of these groups. At baseline, there was no significant difference in the SGRQ scores. However, the scores in the 3rd month changed in the RT plus CCSGs group and were statistically significantly different from those in the RT group (Table 3).

T3
Table 3:
SGRQ scores in the RT and RT plus CCSGs groups.

4.4 Pulmonary function in the RT and RT plus CCSGs groups

There was no significant difference between the RT and RT plus CCSGs groups in terms of pulmonary function, including forced expiratory volume-one second (FEV1), forced vital capacity (FVC), and forced expiratory volume-one second/forced vital capacity (FEV1/FVC), both before and after 3 months of treatment (Table 4).

T4
Table 4:
Comparison of pulmonary function indices between the RT and RT plus CCSGs groups.

4.5 Safety of patients in the RT and RT plus CCSGs groups

No serious adverse events occurred in the RT and RT plus CCSGs groups. The incidence of adverse events showed no significant difference between these 2 groups (P > .05). In the RT group, five patients showed the following drug-related adverse effects: gingivitis, toothache, dizziness, insomnia, and pharyngitis. In the RT plus CCSGs group, 4 patients showed the following drug-related adverse effects: gingivitis, toothache, dizziness, and insomnia (Table 5).

T5
Table 5:
Comparison of adverse events between the RT and RT plus CCSGs groups.

4.6 Difference in the gut microbiota in the routine treatment plus Compound Caoshi silkworm granules group

To investigate the potential reasons why CCSGs could improve the SGRQ scores, we further divided the RT plus CCSGs group into 2 groups. Patients with the top 10 SGRQ scores were classified as Group N and those with the lowest 10 SGRQ scores were classified as Group T. We compared the gut microbiota components between Groups N and T using the fecal samples collected before treatment. The Venn diagram, observed-species value, Shannon value, top 10 levels of phylum, and heatmap of functional predictions between Groups N and T are shown in Figure 2 A to E. The networks of the gut microbiota in Groups N and T are presented in Figure 2 F to G.

F2
Figure 2:
Differences in gut microbiota components between Groups N and T. (A) The Venn diagram of the gut microbiota between Groups N and T. (B, C) Comparison of the observed-species and Shannon values between Groups N and T. (D) The top 10 levels of phylum between Groups N and T. (E) Comparison of the heatmap of functional predictions between Groups N and T using FAPROTAX. (F) The network of gut microbiota in Group N. (G) The network of gut microbiota in Group T.

5 Discussion

In this study, we found that there was a difference in the composition of fecal microbiota between stable COPD patients and the healthy population. Recently, a growing number of studies have demonstrated that the gut microbiota may have a relationship with a range of gastrointestinal (GI) diseases and non-GI conditions, such as lung diseases. This relationship was termed the “gut-lung axis”. Zhang et al[4] showed that there was a close correlation between inflammatory factors, such as C-reactive protein and interleukin-6, in the peripheral serum of children with bronchial asthma and the intestinal flora and GI function. A greater level of inflammatory factors in the serum indicated a higher probability of disturbance in the intestinal flora in asthma.

In the field of COPD, Ottiger et al[5] suggested that the plasma levels of the metabolite trimethylamine-N-oxide, which is dependent on the gut microflora, was a predictive factor for COPD exacerbation and had an association with long-term all-cause mortality in COPD patients. Using the administrative health databases in Canada, Vutcovici et al[6] concluded that inflammatory bowel disease (IBD) had an impact on the mortality of COPD. Among 273,208 COPD patients, 687 eventually developed IBD. Based on the digestive condition, IBD increased the risk of death in COPD patients (hazard ratio 4.45, 95% CI 2.39–8.30). Moreover, IBD could increase the risk of all-cause mortality in COPD patients ((hazard ratio 1.23, 95% CI 1.09–1.4). Zheng et al[7] used Sprague-Dawley rats to establish a chronic bronchitis model and they found that the respiratory and intestinal microflora had a dynamic relationship with the disease process. All of the above-mentioned studies revealed that there is a complex relationship between the gut and lung.

Further, we showed that patients with COPD who took CCSGs as a dietary supplement experienced an improvement in symptom scores. The clinical symptoms of COPD patients were also alleviated. Moreover, our present study showed that the incidence of drug-related adverse effects was similar in the RT and RT plus CCSGs groups. The main adverse effects included gingivitis, toothache, dizziness, insomnia, and pharyngitis. A longer period of time and closer follow-up are needed to study the effects of CCSGs. However, there was no significant change in pulmonary function based on the FEV1, FVC, and FEV1/FVC measurement. We speculated that there was a minimal effect of CCSGs on bronchodilation. CCSGs are extracted from traditional Chinese medicine but are considered a dietary supplement in Jinhua, China. Recently, traditional Chinese medicine has provided novel insights into the treatment of COPD. Wang et al[8] performed a multicenter, double-blind randomized controlled trial to evaluate the efficacy of Bushen Yiqi granule and Bushen Fangchuan tablet in the treatment of stable COPD patients. They reported that both treatment groups showed decreased SGRQ scores and increased FVC, FEV1, and FEV1/FVC values. The levels of inflammatory cytokines, such as IL-17 (P = .0219), were also reduced in the 2 treatment groups. Li et al[9] enrolled 352 patients in their four-center, open-label, randomized controlled trial and demonstrated that Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shen granules, and Yi-Qi Zi-Shen granules could reduce the frequency of acute exacerbations of COPD (FAS: P = .000; PPS: P = .000). Ma et al[10] evaluated the role of YuPingFeng granules in preventing COPD exacerbation and improving the symptom score. After treatment for 1 year, the YuPingFeng granule group had a lower exacerbation rate than the placebo group (P = .002) and a lower COPD assessment test (CAT) score, P = .001). However, the pulmonary function of patients in this group showed no significant changes.

There was an interesting difference in fecal microbiota between the top 10 SGRQ score and lowest 10 SGRQ score groups, indicating that fecal microbiota may be involved in the effect of CCSGs on COPD patients. Barcik et al[11] reported that histamine secretion from gut bacteria could have an effect on lung mucosal sites. They utilized a recombinant bacterial strain, which was able to secrete histamine, in wild type mice and they found that pulmonary eosinophilia was ameliorated and inflammatory cytokine secretion was suppressed in mouse lung cells. Some scholars have reported that gut microbes may shape the response to cancer immunotherapy.[12,13] Routy et al[14] reported negative results for overall survival (OS) among 239 advanced non-small cell lung cancer (NSCLC) patients who took an antibiotic during treatment with programmed cell death protein 1/programmed cell death protein ligand 1 inhibition. In combination with previous studies suggesting that an antibiotic can transiently change the composition of the gut microbiome, Blaser MJ suggested that gut microbiota dysbiosis may influence the therapeutic efficacy of.[15] Therefore, fecal microbiota may have an important regulatory role during the administration of CCSGs.

The limitations of this research work need to be described. Firstly, a 3-month treatment period is not long enough and a longer duration of follow-up is necessary. Secondly, different parts of the gut have different gut microbiota components. Thus, fecal samples may not be representative of the total gut microbiota. Thirdly, mucosa-related microbiota is more associated with the host, and fecal samples only show some components of the luminal microbiota, which should be taken into consideration.

6 Conclusion

We believe that our research work will contribute to the development of the concept of control for stable COPD patients. The use of CCSGs for 3 months, in combination with regular treatment, may improve the patients’ symptom scores. The effects may have an association with differences in gut microbiota. CCSGs can be considered an effective intervention for patients with stable COPD, and gut microbiota may be a new target. However, more multicenter clinical trials with a large sample size are warranted to obtain consolidated evidence.

Author contributions

Data curation: Yibing Hu, Qinghuan Shi, Dan Zhu, Jilin Xu.

Formal analysis: Yibing Hu.

Funding acquisition: Bin Xu, Hui Chen.

Investigation: Yibing Hu, Hui Chen, Xiguang Yang, Feila Xu.

Methodology: Yibing Hu, Qinghuan Shi, Songmin Ying, Hui Chen.

Project administration: Songmin Ying.

Resources: Songmin Ying, Hui Chen.

Software: Qinghuan Shi, Songmin Ying.

Validation: Feibao Tao.

Visualization: Feibao Tao.

Writing – original draft: Yibing Hu.

Writing – review & editing: Bin Xu.

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Keywords:

Compound Caoshi silkworm granules; chronic obstructive pulmonary disease; gut microbiota

Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc.