Introduction: The traditional Japanese herbal medicine hochuekkito (TJ-41) has been reported to ameliorate systemic inflammation and malnutrition in patients with chronic obstructive pulmonary disease (COPD). TJ-41 has also been known to have preventive effects against influenza virus infection. However, its role in the acute exacerbation of COPD (AECOPD) remains to be elucidated. Our previous study established a murine model of viral infection-associated AECOPD that was induced by intratracheal administration of porcine pancreatic elastase (PPE) and polyinosinic-polycytidylic acid [poly(I:C)]. Here, we used this model and investigated the effects of TJ-41 in AECOPD. Methods: Specific pathogen-free C57BL/6J mice were used. A COPD model was induced by treating mice intratracheally with PPE on day 0. To generate the murine model of AECOPD, poly(I:C) was administered intratracheally following PPE treatment on days 22–24. Mice were sacrificed and analyzed on day 25. Mice were fed a diet containing 2% TJ-41 or a control diet. Results: Daily oral intake of TJ-41 significantly decreased the numbers of neutrophils and lymphocytes in the bronchoalveolar lavage fluid (BALF), which was accompanied by decreased transcripts of CXC chemokines involved in neutrophil migration, viz., Cxcl1 and Cxcl2, in whole lung homogenates and reduced Cxcl2 concentration in BALF. Conclusion: This study demonstrates the anti-inflammatory effects of TJ-41 in a mouse model of AECOPD, suggesting the effectiveness of TJ-41 for the management of COPD. Clinical investigations evaluating the therapeutic efficacy of TJ-41 in AECOPD would be meaningful.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and is an increasing economic and social burden [1]. COPD is accompanied by persistent airway inflammation and parenchymal lung tissue destruction and is primarily associated with cigarette smoke exposure. Acute exacerbation of COPD (AECOPD), which is triggered by respiratory infection or environmental insults [2], is characterized by markedly exaggerated airway inflammation. AECOPD is related to hospitalization and disease progression, and its prevention is crucial to the management of COPD [3].

Hochuekkito (TJ-41), a traditional Japanese herbal medicine, consists of 10 natural herbs and exerts beneficial effects on malnutrition and frailty [4]. Previous studies have reported that TJ-41 improved the health-related quality of life of patients with COPD [5], inhibited the entry of influenza virus into cells and influenza virus-induced autophagy [6, 7], and restored metabolic homeostasis between mitochondrial and glycolytic pathways impaired by influenza virus infection [8]. Based on these facts, we hypothesized that TJ-41 might have a favorable effect on AECOPD associated with viral respiratory infection.

In the present study, our aim was to explore the effect of TJ-41 on body weight and airway inflammation in a mouse model of AECOPD. We utilized a model established by our previous study that used porcine pancreatic elastase (PPE) to induce emphysema and polyinosinic-polycytidylic acid [poly(I:C)], a dsRNA that acts as a TLR3 agonist, to mimic viral infection [9]. Influx of neutrophils into the airway plays an important role in the pathogenesis of AECOPD [10]. Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl8, granulocyte colony stimulating factor, and leukotriene B4 are the main factors causing neutrophil recruitment [11]. We investigated the effect of TJ-41 on neutrophilic inflammation, focusing on Cxcl1 and Cxcl2, which are homologs of human Cxcl8 in mice and are key factors in poly(I:C)-induced neutrophilic inflammation [12, 13].

Mouse Models

Specific pathogen-free female C57BL/6J mice (6 wks) were purchased from The Jackson Laboratory Japan (Kanagawa, Japan). The mice were provided with free access to diet and water. We used 4–5 mice per group per experiment. All animal experiments were conducted with the approval of the Animal Ethics Committee of the University of Tokyo (P19-120) and in accordance with institutional guidelines. Mice were anesthetised with an intraperitoneal injection of medetomidine (0.3 mg/kg; Zenoaq, Fukushima, Japan), midazolam (4 mg/kg; Sandoz, Tokyo, Japan), and butorphanol (5 mg/kg; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) before intratracheal administration, and double doses were used for euthanasia. PBS (Fujifilm Wako Pure Chemical Corporation), PPE (Merck KGaA; Darmstadt, Germany), or poly(I:C) (Tocris Bioscience; Bristol, UK) were intratracheally administered. Specifically, we administered 0.25 U of PPE on day 0 and/or 100 μg of poly(I:C) on days 22–24. The detailed methods of histopathological evaluation and bronchoalveolar lavage fluid (BALF) analysis are described in our previous study [9]. Briefly, the left lungs were inflated to achieve a constant pressure of 25 cmH2O for 15 min using a 20-gauge intratracheal cannula with 10% normal buffered formalin for fixation. The tissues were embedded in paraffin 24 h later and then cut in 5-μm mid-sagittal sections for evaluation. Histopathological preparations were performed by Genostaff Co., Ltd. (Tokyo, Japan). To obtain BALF, the animals were tracheotomised, cannulated with a 20-gauge catheter, and their lungs were washed three times with 1 mL of PBS. The resultant BALF was centrifuged at 350×g for 5 min, the supernatants were stored at −80°C for ELISA analysis, pellets were dissolved in 1 mL of PBS, and the cells were counted using the Acridine Orange/Propidium Iodide cell viability kit (Logos Biosystems; Gyeonggi-do, South Korea). Differential cell counts were performed on cytospin preparations stained with May-Grünwald-Giemsa (Muto Pure Chemicals; Tokyo, Japan) according to the manufacturer’s instructions. A minimum of 400 cells were visually assessed to determine cell fractionation.

TJ-41

TJ-41 supplied by Tsumura & Co. (Tokyo, Japan) was added to the standard rodent diet at a concentration of 2% (MF; Oriental Yeast Co., Tokyo, Japan). Because we mixed TJ-41 with chow and administered it orally, the mice ingested different amounts. TJ-41 consisted of Astragali radix (16.7%), Atractylodis lanceae rhizoma (16.7%), Ginseng radix (16.7%), Angelica radix (12.5%), Bupleuri radix (8.3%), Zizyphi fructus (8.3%), Aurantii nobilis pericarpium (8.3%), Glycyrrhizae radix (6.3%), Cimicifugae rhizoma (4.2%), and Zingiberis rhizoma (2.0%).

Quantitative RT-PCR

We extracted RNA from whole lung homogenates using the RNeasy Mini Kit (Qiagen; Hilden, Germany). Reverse transcription was achieved using the iScript cDNA Synthesis Kit (Bio-Rad; CA, USA) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the Thermal Cycler Dice Real Time System III (Takara Bio; Shiga, Japan) and TB Green Fast qPCR Mix (Takara Bio), as described in our previous study [9]. Gapdh was used as an internal control. All primer sequences are presented in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000536348).

ELISA

The concentrations of Cxcl1 and Cxcl2 were measured in duplicate by ELISA according to the manufacturer’s instructions (R&D Systems; MN, USA). The isolated serum was diluted 2-fold before use, whereas BALF was prepared as described above, thawed, and then used undiluted.

Statistics

Data are expressed as mean ± SEM. Statistical significance was set at p < 0.05 for all comparisons (JMP Pro 17.2.0; SAS Institute, NC, USA). Two-tailed Student’s t test was used for comparisons between two groups, whereas two-way repeated measures ANOVA was used to examine the effect of two within-subject factors.

Mouse Models of COPD and AECOPD

PPE alone or both PPE and poly(I:C) were intratracheally administered as models for COPD or AECOPD, respectively. We also evaluated control group mice that were only administered PBS or poly(I:C). After 7 days, mice were fed a standard rodent diet with or without TJ-41 for 18 days (Fig. 1a). TJ-41 treatment exerted no effect on body weight in PBS-treated, poly(I:C)-treated, PPE-treated, and PPE/poly(I:C)-treated groups (Fig. 1b). The H&E-stained lung sections revealed PPE-induced emphysema and poly(I:C)-induced inflammatory cell infiltration (Fig. 1c), which was consistent with our previous observations [9].

Fig. 1.

Mouse models of COPD and AECOPD. a Experimental protocol. Poly(I:C): polyinosinic-polycytidylic acid. PBS (50 μL) only, poly(I:C) 100 μg only, PBS with PPE 0.25 U alone, or PPE 0.25 U and poly(I:C) 100 μg was intratracheally administered. b Changes in body weight. n = 5 per group. n.s.: not significant with two-way repeated measures ANOVA. c H&E-stained lung tissue. Bar: 100 μm. Blue arrows: neutrophils. Orange arrows: macrophages. PPE, porcine pancreatic elastase.

Fig. 1.

Mouse models of COPD and AECOPD. a Experimental protocol. Poly(I:C): polyinosinic-polycytidylic acid. PBS (50 μL) only, poly(I:C) 100 μg only, PBS with PPE 0.25 U alone, or PPE 0.25 U and poly(I:C) 100 μg was intratracheally administered. b Changes in body weight. n = 5 per group. n.s.: not significant with two-way repeated measures ANOVA. c H&E-stained lung tissue. Bar: 100 μm. Blue arrows: neutrophils. Orange arrows: macrophages. PPE, porcine pancreatic elastase.

Close modal

BALF Cell Counts and Expression Levels of Cxcl1 and Cxcl2

We next compared the degree of lung inflammation in each group with and without TJ-41 treatment. First, we collected BALF and counted the numbers of total cells, macrophages, neutrophils, and lymphocytes (Fig. 2a). PPE treatment did not influence the total cell numbers or cell fractions, whereas the administration of both PPE and poly(I:C) markedly increased the numbers of total cells, macrophages, neutrophils, and lymphocytes. Under this condition, TJ-41 treatment significantly decreased the numbers of neutrophils and lymphocytes in the PPE/poly(I:C)-treated group. There was also a decrease in the number of macrophages, although not statistically significant.

Fig. 2.

BALF cell counts and expression levels of Cxcl1 and Cxcl2. a Differential BALF cell counts. b Expression levels of Cxcl1 and Cxcl2 in whole lung homogenates. c Concentrations of Cxcl1 and Cxcl2 in the serum and BALF measured by ELISA. n = 4–5 per group, per experiment. *p < 0.05. **p < 0.01. ***p < 0.001. ND: not detected. n.s.: not significant with two-tailed Student’s t test between two groups.

Fig. 2.

BALF cell counts and expression levels of Cxcl1 and Cxcl2. a Differential BALF cell counts. b Expression levels of Cxcl1 and Cxcl2 in whole lung homogenates. c Concentrations of Cxcl1 and Cxcl2 in the serum and BALF measured by ELISA. n = 4–5 per group, per experiment. *p < 0.05. **p < 0.01. ***p < 0.001. ND: not detected. n.s.: not significant with two-tailed Student’s t test between two groups.

Close modal

The CXC chemokines Cxcl1 and Cxcl2 are critical for the dsRNA-induced influx of neutrophils in the lung [12]. We next evaluated their transcript levels in whole lung homogenates and found that the administration of both PPE and poly(I:C) induced the expression of Cxcl1 and Cxcl2 (Fig. 2b). TJ-41 treatment attenuated the induction of Cxcl1 and Cxcl2 by PPE and poly(I:C). Finally, we measured the serum and BALF concentrations of Cxcl1 and Cxcl2 (Fig. 2c). Serum levels of Cxcl1 and Cxcl2 showed no apparent differences, whereas the administration of both PPE and poly(I:C) increased the concentration of Cxcl2 in BALF, and this effect was significantly inhibited by TJ-41 treatment. These findings were consistent with the results of whole lung homogenates.

In this study, we demonstrated that TJ-41 attenuated the infiltration of inflammatory cells in the lung in a murine model of AECOPD that was induced by PPE and poly(I:C). This result was accompanied by the downregulation of Cxcl1 and Cxcl2, the chemokines involved in neutrophil recruitment. Altogether, TJ-41 exerted inhibitory effects on inflammatory responses in an experimental model of AECOPD.

The results of this study were consistent with our previous observation that TJ-41 attenuated lipopolysaccharide (LPS)-induced lung inflammation in a mouse model of lung emphysema [14]. LPS is a major component of the outer membrane of gram-negative bacteria that is recognized by TLR4, and its administration mimics bacterial infection. On the other hand, poly(I:C), used in the present study, is a dsRNA that is recognized by TLR3 and its administration mimics viral infection. Overall, TJ-41 might exert protective effects against acute inflammation caused by both bacterial and viral infection in the setting of pulmonary emphysema. Given that most patients with AECOPD have either viral or bacterial infection or a combination of both [15], these observations support the potential benefit of using TJ-41 for patients with COPD in the clinical setting. In a preliminary experiment, we investigated a model in which the administration of TJ-41 commenced 1 week prior to the administration of poly(I:C) (data not presented). This experiment did not reveal any significant anti-inflammatory effects associated with TJ-41. Consequently, from a clinical perspective, it may be imperative to initiate TJ-41 therapy well before an impending acute exacerbation.

Cxcl1 and Cxcl2 bind to their cognate receptor Cxcr2 and elicit neutrophil recruitment, and Cxcr2 is critical for the poly(I:C)-induced influx of neutrophils in the lung [12]. Together with our observation that TJ-41 significantly decreased the transcript levels of Cxcl1 and Cxcl2 in whole lung homogenates, the downregulation of Cxcl1 and Cxcl2 by TJ-41 might substantially contribute to the attenuation of lung inflammation. The protein levels of Cxcl1 and Cxcl2 in the serum may not exhibit a direct correlation with their production in the lungs because they could be subject to the influence of systemic inflammatory conditions. Chemokine levels detected in BALF more accurately correspond to their release within the airway and alveolar compartments. Consequently, they may not necessarily reflect the overall expression levels within the entire lung tissue. In this study, we found concordant downregulation of Cxcl2 both in the lung tissue and BALF, suggesting its significant role in the anti-inflammatory effect of TJ-41 in the lungs. Although not examined in this study, it may be worthwhile to measure the levels of other factors involved in neutrophil recruitment, such as Cxcl3, Cxcl5, and leukotriene B4, in this model. In this study, inflammation induced by PPE or poly(I:C) alone was not suppressed by TJ-41, whereas inflammation induced by PPE+poly(I:C) was. The mechanism for this was not elucidated in this study, but there was a trend for TJ-41 to suppress inflammation in the poly(I:C) alone group, although this was not statistically significant. The anti-inflammatory effect of TJ-41 may be more pronounced with PPE+poly(I:C), as inflammation was more pronounced with PPE+poly(I:C).

Assuming an average weight of 20 g with a dietary intake of 3 g/day, the mice consumed 3 g/kg/day of TJ-41; in contrast, the recommended daily intake for humans is 7.5 g/day. Although our experimental dose of TJ-41 according to body weight was considerably higher than that in humans, dividing the dose for mice by 12.3 gives the human equivalent dose [16]. Thus, the dose used in this study can be extrapolated to humans. Herbal medicines contain multiple constituents and are presumably metabolized in a complicated manner. Hence, it is difficult to characterize their pharmacokinetic properties and elucidate the detailed mechanism by which TJ-41 metabolites exerted anti-inflammatory effects. Despite this limitation, the present study is the first to our knowledge to demonstrate the therapeutic efficacy of TJ-41 in an experimental model of viral infection-associated AECOPD. Clinical investigations evaluating the therapeutic efficacy of TJ-41 in AECOPD would be meaningful.

We thank our laboratory members for their technical support and useful discussion. We also would like to thank Enago (www.enago.jp) for the English language review.

All animal experiments in this study were conducted with the approval of the Animal Ethics Committee of the University of Tokyo (P19-120) and in accordance with institutional guidelines.

T.J. belongs to an endowed department by Tsumura & Co. Other authors have no conflicts of interest to declare.

This study was supported by Tsumura & Co. The funding source provided TJ-41 and pharmacological data on TJ-41 but was not involved in the study design; data collection, analysis, and interpretation; manuscript writing; and decision to submit the article for publication.

Kensuke Fukuda, Hirotaka Matsuzaki, and Yoshihisa Hiraishi conceived of and designed the study. Kensuke Fukuda and Hirotaka Matsuzaki collected and analyzed the data. Naoya Miyashita, Takashi Ishii, Masaaki Yuki, Hideaki Isago, Hiroyuki Tamiya, and Akihisa Mitani aided in interpreting the study results and conducting literature reviews. Akira Saito, Taisuke Jo, and Takahide Nagase supervised the study. Study design and data interpretation were discussed among all authors. All authors provided critical revisions and approved the final draft of the manuscript for submission.

All data generated or analyzed during this study are included in this article and its online supplementary material files. Further inquiries can be directed to the corresponding author.

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