This prospective, parallel-group study was conducted from 1 April 2022, to 31 July 2023, and included patients with acute exacerbation of COPD (COPDAE) and patients with stable COPD. Participants were randomly assigned in a 1:1 ratio to receive either JSHT or placebo using a computer-generated randomization sequence. Blinding was maintained throughout the study: participants, healthcare providers, and outcome assessors were all blinded to treatment allocation.

In the COPDAE group, patients in the control arm received standard treatment (intravenous corticosteroids, inhaled butanyl and ipratropium, and antibiotics if indicated) plus a placebo resembling JSHT. The JSHT group received the same standard treatment plus JSHT (one pack, three times daily for 1 week).

In the stable COPD group, both arms received standard inhaled therapy according to clinical guidelines. The JSHT group additionally received one pack of JSHT daily for 3 months, while the control group received a matched placebo.

HRQL assessments, pulmonary function tests (PFT) and laboratory tests were conducted at baseline and post-treatment. The study was approved by the Ethics Committee of Taipei Tzu Chi Hospital (IRB number 10-XD-132), and written informed consent was obtained from all participants.

JSHT is a traditional Chinese botanical drugs developed by the Buddhist Tzu Chi Medical Foundation and consists of the following botanical drugs: Houttuynia cordata Thunb. (Houttuyniae Herba, family Saururaceae, aerial parts, 14.18%), Perilla frutescens (L.) Britton (Perillae Folium, Lamiaceae, leaf, 7.09%), Glycyrrhiza glabra L. (Glycyrrhizae Radix et Rhizoma, Fabaceae, root, 7.09%), Artemisia argyi H. Lév. and Vaniot (Artemisiae Argyi Folium, Asteraceae, leaf, 21.28%), Anisomeles indica (L.) Kuntze (Anisomeles Herba, Lamiaceae, whole herb, 21.28%), Platycodon grandiflorus (Jacq.) A. DC. (Platycodonis Radix, Campanulaceae, root, 14.18%), Ophiopogon japonicus (Thunb.) Ker Gawl. (Ophiopogonis Radix, Asparagaceae, tuber, 14.18%), and Chrysanthemum morifolium Ramat. (Chrysanthemi Flos, Asteraceae, flower, 0.71%) (Hsieh et al., 2022a). The herbal formulation includes the following botanical ingredients and their used plant parts: Argyi Leaf (leaf), Kalabhangra (leaf and stem), Ophiopogonis Radix (tuberous root), Dokudami (whole plant), Balloon Flower (root), Glycyrrhizae Radix (root and rhizome), Perilla (leaf), and Chrysanthemi Flos (flower). The preparation was produced via aqueous extraction, filtration, and concentration, and packaged in GMP-certified 225 mL vacuum-sealed single-dose pouches (Hsieh et al., 2022a). The placebo used in this study was formulated with grass jelly, designed to closely resemble the JSHT preparation in taste, color, appearance, and packaging. This sensory matching was implemented to maintain effective blinding for both participants and investigators.

We appreciate the reviewer’s request for clarification. The concentrations used in this study 0.5% v/v for JSHT was selected based on both our prior experimental experience and findings reported in our previous publication (Wei et al., 2024). The concentration of 100 μg/mL LPS was chosen based on prior studies demonstrating robust inflammatory activation in epithelial cell models without compromising cell viability (Hsieh et al., 2022b). Dose-response pilot experiments confirmed this concentration was sufficient to induce inflammatory cytokinase in our model system.

Blood tests including white blood cells (WBCs), percentages of different types of WBCs (neutrophils, lymphocytes, monocytes, eosinophils, basophils), hemoglobin (Hb), hematocrit (Hct), platelets (PLT), blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA), liver enzymes (aspartate aminotransferase, alanine aminotransferase), electrolytes (sodium, potassium), C-reactive protein (CRP), and pro-brain natriuretic peptide (pPro-BNP).

The Taiwan Society of Pulmonary and Critical Care Medicine provides a Chinese version of the COPD Assessment Test (CAT) (Huang et al., 2022). The CAT, consisting of eight items, assesses COPD symptoms such as cough, phlegm, chest tightness, breathlessness, limitations in daily activities, confidence in leaving the house, sleep disturbances, and energy levels. Each symptom is rated on a scale of 0–5, with a total score ranging from 0 to 40. A score of 10 or higher indicates a significant symptom burden (Huang et al., 2022). The Modified Medical Research Council (mMRC) scale, a 5-point grading system from 0 to 4, assesses dyspnea severity. A score of 0 indicates dyspnea only during intense exercise, while a score of 4 signifies breathlessness at rest (MJ Tsai et al., 2012). The 5-item Brief Symptom Rating Scale (BSRS-5) was used to assess psychological distress. The scale is a 5-point scale ranging from 0 (not at all) to 4 (extremely), with higher scores indicating more severe symptoms (Lin et al., 2023; Yang et al., 2024).

PFTs were performed using a calibrated spirometer (Medical Graphics Corp., St. Paul, MN, United States) in accordance with the guidelines of the American Thoracic Society (ATS) (Culver BH et al., 2017). The spirometric parameters measured included forced vital capacity (FVC), forced expiratory volume in one second (FEV₁), the FEV₁/FVC ratio, and maximal mid-expiratory flow (MMEF). All measurements were obtained in the pre-bronchodilator condition to evaluate baseline pulmonary function and the effects of treatment. All measurements were obtained under both pre-bronchodilator and post-bronchodilator conditions to assess baseline PFTs and treatment effects. As per the Global Initiative for Chronic Obstructive Lung Disease (GOLD) recommendations, post-bronchodilator values were used for the diagnosis and classification of airflow limitation severity (Agustí et al., 2023). Quality control procedures were rigorously followed, including daily calibration of the spirometer, regular maintenance checks, and verification of acceptable test reproducibility and performance criteria for each patient.

In the cellular study conducted on A549 cells, five groups were analyzed to evaluate the effects of JSHT. The groups were: 1) Control group: did not receive treatment; 2) lipopolysaccharide (LPS) group: treated with 100 ug/mL LPS for 16 h to induce inflammation; 3) JSHT group: treated with 0.5 volume % JSHT for 12 h; 4) Pre-JSHT + LPS group: cells were pre-treated with 0.5 volume % JSHT for 1 h, then 100 ug/mL LPS treatment for 16 h; 5) Post-JSHT + LPS group: cells were exposed to 100 ug/mL LPS for 16 h followed by 0.5 volume % JSHT for 12 h. Measurements were performed for DAMPs such as high mobility group box 1 (HMGB1), formyl peptide receptor 1 (FPR1), and extracellular adenosine triphosphate (ATP); transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB); phosphorylated mitogen-activated protein kinase (p-MAPK) and c-Jun N-terminal kinase (p-JNK); apoptotic marker cleaved Caspase 3 (cCaspase 3); and pro-inflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α).

The A549 human lung epithelial cell line used in this study was obtained from the American Type Culture Collection (ATCC, CCL-185). Cell line authentication was confirmed by short tandem repeat (STR) profiling, and cells were routinely screened for mycoplasma contamination using a PCR-based detection kit (MycoAlert™ Mycoplasma Detection Kit, Lonza). All experiments were conducted with cells within 20 passages after thawing.

A549 cells (1 × 10⁶) were seeded in 6-well plates and incubated at 37°C with 5% CO₂ for 24 h. Following exposure to five different experimental conditions, the culture medium was harvested, centrifuged at 300 × g for 10 min, and the supernatant was stored at −80°C until analysis. The levels of DAMPs, including HMGB1, FPR1, and extracellular ATP, were measured using commercially available ELISA kits. HMGB1 and ATP were quantified using kits from Abclonal (catalog numbers RK06736 and RK04252, respectively), and FPR1 was measured using a kit from Mybiosource (catalog number MBS9336637). The detection limits were 0.1 ng/mL for HMGB1, 0.35 ng/mL for ATP, and 1.0 ng/mL for FPR1.

After treatment, A549 cells were lysed in cold RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined using a BCA protein assay kit. Equal amounts of total protein were separated by electrophoresis using 4%–12% SDS-PAGE gels at 140 V for 1 h, followed by transfer onto PVDF membranes at 200 mA for 2 hours. Membranes were blocked with TOOLSpeed blocking reagent for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against cleaved caspase-3 (Cell Signaling, #9961, 1:1000 dilution, validated in PMID: 39878157), NF-κB (Abclonal, A2547, 1:1000, PMID: 26202983), phosphorylated MAPK (p-MAPK; Abclonal, AP0526, 1:1000, PMID: 31281493), and phosphorylated JNK (p-JNK; Abclonal, AP0631, 1:1000, PMID: 31614673). GAPDH (Abclonal, AC027, 1:1000, PMID: 29727616) was used as the loading control.

After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 hour at room temperature. Signals were detected using enhanced chemiluminescence (ECL) reagents and visualized with the Bio-Rad ChemiDoc XRS+ imaging system. Band intensities were quantified using ImageJ software. The expression level of each target protein was normalized to GAPDH and presented as the ratio of target to GAPDH to account for loading variability.

A549 cells were cultured in a 6-well plate at 37°C for 24 h. After treatment under five experimental conditions, the culture medium was collected and stored at −80°C. Inflammatory cytokines including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α) were quantified using ELISA kits from Abclonal (catalog numbers RK00001, RK00004, RK00011, and RK00030, respectively). The detection limits for these cytokines were 3.9 pg/mL for IL-1β, 0.7 pg/mL for IL-6, 1.7 pg/mL for IL-8, and 6.9 pg/mL for TNF-α. All ELISA procedures were conducted according to the manufacturers’ instructions. Each sample was assayed in triplicate to ensure accuracy and reproducibility. Absorbance readings were obtained using an Infinite 200 PRO microplate reader (Tecan, Männedorf, Switzerland).

Data are presented as mean ± standard deviation (SD). Statistical analyses were conducted using SPSS (version 24.0). For the clinical study, paired t-tests compared baseline and post-treatment outcomes within each group. In the cellular study, ANOVA assessed differences among the five groups (control, JSHT, LPS, pre-JSHT, post-JSHT), with post hoc comparisons using the least significant difference method. Statistical significance was set at p < 0.05.

There were 4 and 5 patients with COPDAE in the control and JSHT groups, respectively. The baseline characteristics of the patients with COPDAE are shown in Table 1. Age, sex, smoking status, BH, BW, BMI, and lung function test results were not significantly different between the two groups (all p > 0.05).

The HRQL values are listed in Table 2. In the control group, improvements were observed in the CAT total score, chest tightness, breathlessness, limitation of activities, dyspnea, mMRC score, and sleep difficulties (p < 0.05). In the JSHT group, significant improvements were observed in more aspects, including the CAT total score, cough, phlegm, chest tightness, breathlessness, limitation of activities, confidence in leaving home, mMRC dyspnea, sleep difficulties, and anxiety (p < 0.05).

Laboratory data of patients with COPDAE are shown in Table 3. In both groups, most parameters, including blood cell count, hemoglobin level, renal function, and liver function, showed no significant differences after treatment (p > 0.05). However, a significant decrease in potassium levels was observed in the control group (p = 0.017), which was not evident in the JSHT group (p = 0.098). The JSHT group showed a reduction in CRP levels (from 5.7 ± 8.5 to 1.1 ± 1.6), but this change did not reach statistical significance (p > 0.05).

There were 8 and 9 patients with stable COPD in the control and JSHT groups, respectively. The baseline characteristics of the patients with stable COPD are shown in Table 4. Age, sex, smoking status, BH, BW, BMI, and lung function were not significantly different between the two groups (all p > 0.05).

The HRQL scores of patients with stable COPD are shown in Table 5. Significant reductions in breathlessness and mMRC scores were observed in the control group (p < 0.05). The total CAT score and cough, phlegm, chest tightness, and other symptoms also decreased; however, these changes were not statistically significant (p > 0.05). In the JSHT group, there were significant decreases in the total CAT score, cough, phlegm, breathlessness, and mMRC score (all p < 0.05). However, the BSRS scores were not significantly different post-treatment in either the control or JSHT groups (all p > 0.05).

The laboratory data of the patients with stable COPD are shown in Table 6. In both groups, most parameters, including blood cell count, Hb, electrolytes, renal function, and liver function, showed no significant differences after treatment (p > 0.05). In the JSHT group, percentage of basophils showed a significant decrease from 0.5% ± 0.3% to 0.3% ± 2.0% (p = 0.036).

The PFT results of patients with stable COPD are shown in Table 7. None of the PFT parameters showed significant differences after treatment in both groups (p > 0.05).

The effects of JSHT on the attenuation of the LPS-induced release of DAMPs such as HMGB1, FPR1, and extracellular ATP in A549 cells are shown in Figure 1. Compared with the control group, LPS significantly increased HMGB1 (Figure 1A), FPR1 (Figure 1B), and ATP levels (Figure 1C) (all p < 0.05). Both the pre-JSHT and post-JSHT groups demonstrated significantly reduced levels of HMGB1, FPR1, and ATP compared with the LPS group (all p < 0.05). There were no significant differences between the pre-JSHT and post-JSHT groups (p > 0.05).

Figure 2 represents the Western blot results showing (Figure 2A) representative blots and quantified levels of NF-kB (Figure 2B), pMAPK (Figure 2C), pJNK (Figure 2D) and cCaspase 3 (Figure 2E) in A549 cells after LPS exposure with or without JSHT treatment. LPS significantly increased the expression of NF-κB, pMAPK, pJNK, and cleaved caspase-3 compared with the control group (all p < 0.05). The pre-JSHT group showed reduced levels of pMAPK, pJNK, and cleaved caspase-3 compared with the LPS group (all p < 0.05), but no significant difference in NF-κB (p > 0.05). The post-JSHT group showed reduced levels of NF-κB, pMAPK, pJNK, and cleaved caspase-3 compared with the LPS group (all p < 0.05). No significant differences were observed between the pre-JSHT and post-JSHT groups across all markers (p > 0.05).

LPS significantly increased levels of IL-1 (Figure 3A), IL-6 (Figure 3B), IL-8 (Figure 3C) and TNF-α (Figure 3D) compared with the control group (all p < 0.05). The pre-JSHT group significantly reduced IL-1β, IL-6, IL-8, and TNF-α levels compared with the LPS group (all p < 0.05). The post-JSHT group reduced IL-8 and TNF-α levels (p < 0.05), but showed no significant differences in IL-1β and IL-6 compared with the LPS group (p > 0.05). IL-1β levels were higher in the post-JSHT group than in the pre-JSHT group (p < 0.05), while no significant differences were observed between the pre-JSHT and post-JSHT groups for IL-6, IL-8, and TNF-α levels (p > 0.05).

This study had several limitations. First, the limited sample size in both the exacerbated and stable COPD groups may have reduced the statistical power of our analyses, potentially increasing the risk of Type II errors. As this study was designed as a preliminary investigation, the findings should be interpreted with caution. Larger, well-powered randomized controlled trials are warranted to confirm the therapeutic benefits of JSHT and to ensure the reproducibility and generalizability of the results. Despite the sample size limitations, this study offers meaningful preliminary insights into the potential benefits of JSHT as an adjunctive treatment for COPD. Second, the relatively short duration of treatment and follow-up limits our ability to assess the sustained effects and long-term safety of JSHT in patients with COPD. While the observed improvements in symptoms and inflammatory markers are encouraging, it remains unclear whether these benefits persist beyond the study period. An extended study duration with longer follow-up is essential to evaluate the durability of JSHT’s therapeutic effects, its impact on disease progression, exacerbation frequency, and long-term clinical outcomes. Third, this study is the lack of detailed chemical profiling of the JSHT formulation. As our primary focus was to evaluate the overall clinical efficacy and anti-inflammatory effects of JSHT in COPD, we did not conduct component-level analysis in this phase. To address this, future studies will incorporate high-performance liquid chromatography, mass spectrometry, and related phytochemical techniques to establish a chemical fingerprint of JSHT. These efforts will help elucidate the roles of individual constituents and support mechanistic validation at the molecular level. Fourth, this study lacks a direct assessment of oxidative stress, a key contributor to COPD pathogenesis. Cigarette smoke and related irritants induce excessive production of reactive oxygen species (ROS), contributing to epithelial injury, chronic inflammation, and disease progression. While our study focused on evaluating the anti-inflammatory effects of JSHT through the measurement of DAMPs and cytokine expression, we did not include assays to quantify ROS levels or antioxidant responses. As oxidative stress and inflammation are closely interconnected in COPD, future studies assessing the potential antioxidant properties of JSHT would provide a more comprehensive understanding of its therapeutic effects.