It is well known that cough in healthy individuals is a physiological defense mechanism to clear the airways of foreign particles and excessive bronchial secretions, and is a common symptom of several respiratory diseases [1]. Chronic cough can be debilitating, as the resulting distress can significantly impair the quality of life of patients [2,3]. Although most cases of cough are associated with infection, diagnostic tests for cough are made difficult by the long list of differential diagnoses. Cough variant asthma (CVA), a type of cough with asthma symptoms in addition to dry cough, is a chronic inflammatory disease characterized by airway inflammation, airway hyperresponsiveness (AHR) and airway remodeling [4,5]. Currently, cough treatment is dominated by over-the-counter (OTC) antitussive drugs, such as asmeton, but most currently available antitussive drugs have serious side effects [6]. And in many cases, cough is accompanied by severe bronchoconstriction or mucus, and there is a great need for the development of new drugs with potent cough suppressant, expectorant, and bronchodilator effects with little or no side effects.

In the field of cough suppression and asthma alleviation, traditional Chinese medicine (TCM) has been increasingly recognized for its effectiveness in recent years. An example of this is the use of San'ao decoction in combination with Scorpio and bombyx batryticatus, which has been shown to reduce airway inflammation and provide an antitussive effect in a mouse model of cough-variant asthma through the modulation of TRPA1/TRPV1/TRPV5 channels [7]. The use of Modified Dingchuan Decoction resulted in a decrease in the frequency of coughing, a longer time period between coughs, improved lung and airway health, regulated levels of inflammatory substances in the bronchoalveolar lavage fluid (BALF), and balanced the lung microbiota in the CVA model of guinea pigs [8]. Pectolinarigenin (PEC), herbal plant constituent extract isolated from Chromolaena odorata and a bioactive component of the TCM Ephedra Herb, has various biological activities. Studies have shown that PEC had anti-inflammatory effects. PEC attenuated mitochondrial dysfunction and suppressing the release of inflammatory factors to relief acute kidney injury by inhibiting JAK2/STAT3 signaling [9]. PEC has been found to be effective in reducing hyperuricemic nephropathy by blocking the TGFβ/SMAD3 and JAK2/STAT3 signaling pathways [10]. Furthermore, PEC has the ability to modulate the NF-κB/Nrf2 signaling pathway, which ultimately leads to the inhibition of macrophage inflammatory response induced by lipopolysaccharide. Additionally, PEC can also alleviate dextran sulfate sodium-induced colitis [11]. However, the function of PEC in cough suppression is less reported and the mechanism is unclear.

In the present study, we found that PEC exerted an antitussive effect by improving airway inflammation, airway remodeling and alleviating lung injury by inhibiting the Ras/ERK/c-Fos pathway.

Animals

We acquired 25 healthy females BALB/c mice from the Experimental Animal Center of Zhejiang University. The mice were between 6–8 weeks old and weighed an average of 20 g ± 2 g. They were kept at the Jiangsu University’s Animal Experiment Center. During the first week, the mice were exposed to laboratory conditions and provided access to water and full-price pellet food. The humidity level was kept at 50% to 70%. All animal procedures were approved by the Ethical Committee for the Experimental Use of Animals at Jiangsu University and met the standards outlined in the Guide for the Ethical Care and Use of Laboratory Animals (UJS-IACUC-AP-2022062116).

Model establishment and drug treatment

Twenty-five BALB/c mice, randomized into 5 groups with 5 mice each: normal group, model group, model mice treated with 25 mg/kg PEC, model mice treated with 50 mg/kg PEC and model mice treated with 25 mg/kg positive drug asmeton (Compound Methoxyphenamine Capsules, Daiichi-Sankyo). The mice in the cough model groups were constructed as reported by Wang et al. [7]. Briefly, except the normal group, all mice were injected subcutaneously with 0.1 ml of sensitizing solution, and intraperitoneally with 0.1 ml of sensitizing solution on the first day and 8th day. The sensitizing solution (0.2 ml) contains 0.02 mg of Al(OH)₃ and 0.1 mg of ovalbumin (OVA, Sigma-Aldrich) among them. Next, mice were nebulized with 20 ml of OVA (5%) for 20 min per day from day 15th to day 21st consecutively. Every other day from day 22nd to day 30th, OVA was treated in the same manner.

The dosing regimen was as follows, from day 19th to day 30th, mice in the dosing group were given mouse PEC (25 mg/kg/day or 50 mg/kg/day) or positive drug asmeton (25 mg/kg/day) by gavage daily. Mice in the normal and model groups were given equal volumes of distilled water.

Cough response test

Mice were tested for cough response on day 30th. In this study, capsaicin was used to induce cough in mice as reported by Wang et al. [7]. First, each mouse was carefully placed into a transparent sealed box (measuring 270 mm × 180 mm × 160 mm) with a nebulizer attached. A solution of capsaicin, with a concentration of 100 μM, was then sprayed into the box using the nebulizer at a rate of 1 ml/min. This was done to stimulate the mice for a period of 1 min. For the next 4 min, the mice were continuously observed and the number of coughs, as well as the latency of cough, were recorded. The coughs were analyzed by two trained personnel who were blind to the treatment and cough response criteria were based on previously reported standards [12]. The results were finally analyzed.

Histological analysis of lung tissues

Lung tissues from various groups of mice were collected and fixed with 4% paraformaldehyde for 24 h, paraffin-embedded, and cut into 4 μm sections. Sections were stained with hematoxylin-eosin (H&E), periodic acid-Schiff (PAS) and Masson. Collagenous areas on the airway basement membrane were analyzed by a Leica-Qwin image processing system (Leica Imaging Systems). The results were expressed as the area of collagen staining per micrometer length of the fine bronchial basement membrane. Finally, tissues were observed under a light microscope (Eclipse Ni-U; Nikon) at 400× magnification for at least 5 fields of view.

Enzyme-linked immunosorbent assay (ELISA)

The experiment involved intubating mice and injecting 0.5 ml of phosphate buffered saline (PBS) into their left lung. This was followed by collecting BALF three times to obtain samples, which were then centrifuged for 10 min at 1,000 r/min to obtain the supernatant. Using ELISA kits from Beyotime, the inflammation marker tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were detected according to the manufacturer's instructions. Finally, the absorbance value was measured at 450 nm.

Airway reactivity testing

The response of the airways to various physical or chemical stimuli or allergens is known as airway reactivity [13]. On day 31st of the experiment, we performed airway reactivity tests on all experimental mice. A noninvasive spirometer (gyd-003; EMKA) was used to measure the level of expiratory pause (Penh) after nebulizing different concentrations of acetylmethacholine (0, 6.25, 12.5, 25, and 50 mg/ml) in mice.

Inflammatory cell populations counting

A portion of the obtained BALF centrifuged cell sediment was collected and resuspended in PBS, and the rest was made into a cell smear. The total number of white blood cells in the 6 observation fields was counted using a blood cell counter (YA0811; Solarbio). Different types of inflammatory cells were observed by Diff-Quik staining (D030-1-1; Nanjing Built) as previous reported by Wang et al. [14]. The cell smears were air-dried and then fixed with RI reagent at room temperature for 10 sec. After removing the excess liquid, the sections were immediately transferred to R2 reagent for 8 sec and then promptly removed. The liquid was then drained from the sections, and the sections were rinsed in R3 reagent for 8 sec. The sections were then dried in R3 reagent for 10 sec and rinsed again in R3 reagent for 8 sec. Finally, the dye solution was carefully removed from the cell smears by running water. The cell smears were then dehydrated twice with a 100% ethanol solution and sealed. Lastly, under a microscope (TS100; Nikon), the types of inflammatory cells were identified and counted.

Immunofluorescence (IF)

Lung tissue from different groups were collected and 1 ml of 4% paraformaldehyde was added and fixed at room temperature for 20 min. After washing with PBS for 3 times, the tissues were blocked with 2% albumin from bovine serum for 1 h at room temperature and incubated with primary antibodies against COL1A1 (Abcam) overnight at 4°C. Then, immunoglobulin G-Alexa Fluor 488 (Abcam) were treated for 1 h. The stained coverslips were fixed and examined under a fluorescence microscope (Zeiss) after being treated with DAPI (1:1,000, Sigma). The fluorescence intensity was assessed using Image J software.

Western blot analysis

As previously reported by Lu et al. [15], we isolated the total protein from the lung tissues of each group of mice. Cellular protein lysates were put onto a 10% SDS-polyacrylamide gel and separated by electrophoresis. Next, the proteins were then transferred to polyvinylidene difluoride membranes (Millipore) with 5% nonfat milk and left to sit at room temperature for an hour. Specific antibodies were then incubated for an overnight period at 4°C and membranes were washed 3 times in TBST following day, and incubated with HRP-conjugated secondary IgG antibody (ab205718, 1:2,000, Abcam) for 1 h at room temperature. Finally, the membranes were washed with enhanced chemiluminescence reagent (Beyotime) and image J was used for quantification. The specific antibodies which used in the study included anti-Ras (ab206969, 1:5,000), anti-p-ERK (ab201015, 1:5,000), anti-ERK (ab184699, 1:5,000), anti-c-Fos (ab208942, 1:1,000) and anti-β-actin (ab8227, 1:1,000). All the antibodies were acquired from Abcam.

Statistical analysis

GraphPad Prism software (7.04) was used for statistical analysis. One-way ANOVA followed Dunnett’s multiple comparisons test as used for comparisons between multiple groups. All data are described as mean ± standard deviation and p < 0.05 indicated a statistically significant difference.

PEC alleviated lung tissue injury in mice model with cough

We used an injection of sensitizing solution and OVA to construct a mouse cough model to investigate the pharmacological effects of PEC, a monomer of Chinese medicine. To begin, we obtained lung tissues from each group of mice and analyzed them using H&E staining. The results of the H&E staining were shown in Fig. 1A. In the lung tissues of OVA-induced airway inflammation model with cough, there was a noticeable infiltration of inflammatory cells in the perivascular and peribronchial connective tissues. In contrast, in the PEC and positive drug asmeton treatment groups, inflammatory cells were suppressed and gradually repaired the OVA-induced lung tissue damage with the increase of the administered dose. In the meantime, PAS staining was also shown in Fig. 1B and found that there was significant mucus production in the epithelial layer of the mice in the OVA-induced airway inflammation model with cough group, while mucus production decreased after administration of the drug. Then, ELISA was used to reveal that the levels of inflammatory factors TNF-α and IL-6 were considerably higher in the BALF of mice in the OVA-induced airway inflammation model with cough group. However, the administration of 50 mg/ml of PEC greatly reduced these levels (p < 0.001, compared with the OVA-induced airway inflammation model with cough group). Interestingly, the high dose of PEC had a similar effect on inflammatory factors as the positive drug asmeton (Fig. 1C). These results hinted that PEC could alleviate OVA induced lung tissue injury and reduce the release of inflammatory factors.

Figure 1. Pectolinarigenin (PEC) alleviated lung tissue injury in mice model with cough. (A) Pathological sections of the lung tissue were performed by H&E in the OVA-induced airway inflammation model with cough group treated with the PEC and positive drug asmeton. Mice were executed on day 30th (Scale bar: 200 μm or 100 μm). (B) Pathological sections of the lung tissue were performed by PAS in the OVA-induced airway inflammation model with cough group treated with the PEC and positive drug asmeton (Scale bar: 200 μm or 100 μm). (C) The inflammatory factor levels (TNF-α and IL-6) in BALF were assessed using ELISA. Values are presented as mean ± SD. OVA, ovalbumin; PAS, periodic acid-Schiff; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; BALF, bronchoalveolar lavage fluid. ^^^p < 0.001 compared with the sham group; ^#p < 0.05, ^###p < 0.001 compared with the OVA-induced airway inflammation model with cough group.

PEC inhibited airway obstruction and the number of inflammatory cells in cough model mice

Next, lung resistance was measured to quantitatively evaluate AHR. As shown in Fig. 2A, AHR was dramatically increased in the model group of mice compared to healthy control animals (p < 0.001), whereas PEC and positive drug administration reduced AHR in the OVA-induced airway inflammation model with cough group. In addition, inflammatory cell populations in the BALF of mice was also measured. It was found that leukocytes, eosinophil, neutrophils, lymphocytes and monocytes were significantly higher in the OVA-induced airway inflammation model with cough group than which in the control group (p < 0.01). However, the upregulation of inflammatory cell populations in the OVA-induced airway inflammation model with cough group was reversed by treatment with PEC or positive drug asmeton (Fig. 2B). These results suggested that PEC played an important role on improving AHR and suppressing inflammatory cell populations in OVA-induced airway inflammation model with cough.

Figure 2. Pectolinarigenin (PEC) inhibited airway obstruction and the number of inflammatory cells in cough model mice. (A) Airway hyperresponsiveness in the OVA-induced airway inflammation model with cough group treated with the PEC and positive drug asmeton as measured by the response to different concentrations of acetylmethacholine (0, 6.25, 12.5, 25, 50 mg/ml). (B) Inflammatory cell populations (leukocytes, eosinophil, neutrophils, lymphocytes, and monocytes) in the BALF of mice was measured. Values are presented as mean ± SD. OVA, ovalbumin; BALF, bronchoalveolar lavage fluid. ^^^p < 0.001 compared with the sham group; ^###p < 0.001 compared with the OVA-induced airway inflammation model with cough group.

Inhibition of cough frequency and airway wall collagen deposition by PEC in OVA-induced airway inflammation model with cough

In order to investigate the effect of PEC on coughing in mice, we induced coughing with capsaicin in each group of mice, and then observed and recorded the number of coughs and the latency period of coughing in mice. It was found that after treatment with capsaicin, the frequency of cough increased and the latency of cough shortened in the OVA-induced airway inflammation model with cough group compared with the control group. PEC and positive drug asmeton treatment decreased cough frequency and prolonged cough latency (Fig. 3).

Figure 3. Pectolinarigenin (PEC) inhibited cough frequency. (A) Frequency of cough in 4 min. (B) Latent period of cough was measured in the model group treated with the PEC and positive drug asmeton. Values are presented as mean ± SD. ^^p < 0.01, ^^^p < 0.001 compared with the sham group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with the ovalbumin-induced airway inflammation model with cough group.

Several studies have shown that collagen deposition in the airways leading to airway remodeling, which could exacerbate airway pathologies such as asthma and cough [16,17]. To investigate airway remodeling, we used IF and Masson trichrome staining to detect collagen deposition. Fig. 4A, B showed that COL1A1 expression was higher in OVA-induced airway inflammation model with cough, while treatment with PEC or the positive drug asmeton was decreased COL1A1 expression. Fig. 4C, D showed that normal mice had only a small amount of collagen deposition in the airway wall and around blood vessels, while the OVA-induced airway inflammation model with cough groups had significantly increased levels (p < 0.01). However, treatment with either PEC or the positive drug asmeton was effective in reducing OVA-induced collagen deposition. To summarize, these results suggested that administration of PEC to model mice inhibited coughing frequency and collagen deposition in the airway wall.

Figure 4. Pectolinarigenin (PEC) inhibited airway wall collagen deposition. (A, B) COL1A1 expression in the lung tissue was performed IF and the staining results were also quantified. Scale bar: 200 μm. (C, D) Histological analysis of the lung tissue was performed Masson trichrome staining and the staining results were also quantified. Scale bar: 200 μm. Values are presented as mean ± SD. IF, immunofluorescence; OVA, ovalbumin. ^^^p < 0.001 compared with the sham group; ^###p < 0.001 compared with the OVA-induced airway inflammation model with cough group.

PEC inhibited the Ras/ERK/c-Fos pathway

To further investigate the specific mechanism of PEC in cough suppressant and anti-inflammatory, we performed Western blotting experiments. It has been shown that OVA induced the activation of Ras/ERK/c-Fos pathway [18]. As reported, in this experiment, the protein expression of Ras, p-ERK and c-Fos was increased in the OVA-induced airway inflammation model with cough group, whereas treatment with PEC or positive drug asmeton inhibited the protein expression of Ras, p-ERK and c-Fos (Fig. 5). These results hinted that the role of PEC on model mice might be related to the inhibition of the Ras/ERK/c-Fos pathway.

Figure 5. Pectolinarigenin (PEC) inhibited the Ras/ERK/c-Fos pathway. The protein level of Ras, p-ERK, ERK and c-Fos in the model group treated with the PEC and positive drug asmeton was measured by Western blotting. Values are presented as mean ± SD. ^^^p < 0.001 compared with the sham group; ^###p < 0.001 compared with the ovalbumin-induced airway inflammation model with cough group.

Cough is a common respiratory symptom. However, frequent coughing from acute to chronic often causes great pain to patients, such as chest tightness, throat tickling, wheezing, etc. In severe cases, it may turn into CVA, which seriously affects people's quality of life. For cough treatment, it is dominated by OTC antitussive drugs, such as diastolic bronchial smooth muscle antitussive drugs (asmeton, ephedrine), opioid antitussive drugs (codeine, dihydrocodeine, and loperamide) and so on, but most currently available antitussive drugs have serious side effects [6]. If someone takes too much opioid antitussive medication, they may have three common symptoms: decreased consciousness or even go into a coma, breathing that is abnormally slow or stops completely, and pinpoint pupils. Respiratory depression can cause cyanosis and result in severe neurological damage due to lack of oxygen to the brain, which can even be fatal [19]. While asmeton also has drowsiness, vertigo, tachycardia and gastrointestinal adverse effects [20]. Therefore there is an urgent need to explore effective and potent antitussive drugs with fewer adverse reactions [21].

PEC isolated from C. odorata. It is also a bioactive component of the TCM Ephedra Herb. Among them, an aqueous decoction of the herb ephedra has been widely used in the treatment of cough and asthma [22,23]. It has not been researched whether PEC, which is one of the active components in ephedra, can be utilized for treating cough.

A number of studies had found that PEC had anti-inflammatory effects. PEC could reduce the release of inflammatory factors [9-11]. As reported in the literature, this study also found that PEC inhibited the release of inflammatory factors (TNF-α and IL-6) and reduced the number of inflammatory cell populations in cough model mice. In addition to airway inflammation, cough is triggered by AHR and airway remodeling. In this study, we assessed AHR and found that PEC reduced respiratory resistance in OVA-induced airway inflammation model with cough to attenuate AHR. In addition to this, we used Masson trichrome staining to observe the collagen deposition in the airway wall. We found that PEC could reduce collagen deposition in the airway wall and around blood vessels in model mice, indicating that PEC could attenuate airway remodeling. Meanwhile, PEC administration reduced the cough frequency and shortened the cough latency in the OVA-induced airway inflammation model with cough.

We conducted further research on the antitussive effect of PEC and discovered that it hindered the Ras/ERK/c-Fos pathway. This pathway is a significant signaling cascade in the MAPK signaling pathways and contributes significantly to the progression of several ailments. It has been reported that Ras/ERK/c-Fos activation increased airway hypersensitivity and cough frequency in guinea pig models [24]. Furthermore, Ras/ERK/c-Fos activation increased airway inflammatory responses and promoted OVA-induced asthma [18]. Thus, the Ras/ERK/c-Fos pathway is a possible target for the treatment of respiratory diseases. Besides, positive drug asmeton, whose main ingredient is orthoxine. Among them, orthoxine has methoxy and anti-inflammatory effects, and is used in the treatment of asthma. It has been found that orthoxine inhibited histamine-induced bronchoconstriction in anesthetized guinea pigs [25]. Interestingly, PEC also contains methoxy, which may have similar results, so PEC also exerts an anti-inflammatory and antitussive effect.

However, this study still had limitations. On the one hand, we have validated PEC only in animals, yet not in clinical samples; on the other hand, other possible molecular mechanisms of PEC remained to be elaborated. Therefore, follow-up studies should focus on developing new dosage forms suitable for clinical administration and experimental validation; secondly, other potential molecular mechanisms of PEC should be investigated to improve this study.

In conclusion, the present study demonstrated that PEC had an antitussive effect. PEC could reduce the release of airway inflammatory factors, improving AHR and airway remodeling to suppress the number of cough and the latency to cough in mice by inhibiting the Ras/ERK/c-Fos pathway.

None.

This work was supported by the Jiangsu Senile Health Research Project (Grant No. LK2021057), Zhenjiang Key Research and Development Project (Grant No. SH2022081), TCM Science and Technology Development Program of Jiangsu Province (Grant No. MS2022125), and Zhenjiang Key Research and Development Project (Grant No. SH2023034), Jiangsu Province Postgraduate Research and Practice Innovation Program (Grant No. JX10214039).

The authors declare no conflicts of interest.