Study on the effect of modified Liu-an decoction on Th1/Th2 function in Guinea pigs with cough variant asthma through the Notch signal pathway

Fangwei Xu; Jian Deng; Ningning Zhang; Kimberly XinTing Leow; Panpan Li; Chen Lu; Yuhang Chen; Ye Zhang; Liqun Wu

doi:10.1371/journal.pone.0327356

. 2025 Sep 15;20(9):e0327356. doi: 10.1371/journal.pone.0327356

Study on the effect of modified Liu-an decoction on Th1/Th2 function in Guinea pigs with cough variant asthma through the Notch signal pathway

Fangwei Xu ¹, Jian Deng ¹, Ningning Zhang ², Kimberly XinTing Leow ³, Panpan Li ⁴, Chen Lu ⁵, Yuhang Chen ², Ye Zhang ², Liqun Wu ^6,^*

Editor: Misbahuddin Rafeeq⁷

PMCID: PMC12435660 PMID: 40953067

Abstract

Objective

To explore the effects and mechanism of Modified Liu-an Decoction (MLAD) on Th1/Th2 function and the Notch signaling pathway in guinea pigs with cough variant asthma (CVA).

Methods

60 SPF Hartley guinea pigs were randomized into six groups (n = 10): the blank control group, model group, montelukast sodium group (MS group), as well as MLAD group of low-dose, mid-dose and high-dose. Intraperitoneal injection of ovalbumin, aluminum hydroxide sensitization, and nebulized inhalation of ovalbumin were performed in all guinea pigs of non-blank control groupto induce CVA. Cough frequency and airway resistance were recorded. The chronic inflammatory infiltration in the airways was detected by using HE and Masson staining smears. The IL-2, IL-4, IL-12, and IL-13 levels were measured by enzyme-linked immunoassay (ELISA). The expression of Delta, Jagged1, Notch1, and NICD proteins in the lung tissues were analyzed by Western blot.

Results

In terms of airway sensitivity, coughing time and airway resistance were reduced in the MLAD and MS groups compared to the model group (P < 0.05). MLAD reduced the inflammatory infiltration in the lungs of CVA guinea pigs compared with the model group (P < 0.05).For cytokines in BALF, the contents of IL-2 and IL-12 were increased in the MS group and each dose group of MLAD compared with the model group, and the contents of IL-4 and IL-13 were decreased compared with them.The contents of IL-12 were significantly different in each dose group of MLAD compared with the model group; the contents of IL-2 and IL-4 differed with increasing concentrations of MLAD. 4 content with increasing MLAD concentration, the more significant difference. IL-13 content decreased significantly in the MLAD low dose group compared with the model group (P < 0.05). In the serum cytokine model group IL-2, IL-12, IL-13 content were significantly higher than the blank group, IL-4 content was lower than the blank group, and the difference between each intervention group and the model group was not regular.

The expression levels of Jagged1, Notch, and NICD proteins in the CVA guinea pigs in the MS group and the low-dose MLAD group were decreased compared with those in the model group, and the expression of Delta1 protein was increased compared with that in the model group (P < 0.05).

Conclusion

The addition of Liuan Decoction to the lungs can clear lungs and remove heat and strengthen the spleen to resolve phlegm, congestion, edema degree, reduce airway inflammation, and at the same time can reduce peri-tracheal collagen deposition, effectively improve airway remodeling, by regulating the dynamic balance of Th1/Th2. The specific downstream regulatory mechanisms of Notch signaling pathway need to be explored in further studies.

1. Introduction

Cough variant asthma (CVA) is a unique type of asthma with cough as the main clinical manifestation, which often occurs in the morning and at night, and triggered by exposure to cold air and irritating odors, and aggravated after exercise. CVA typically lasts for more than a month, meanwhile, recurrent attacks can adversely affect the children’s quality of life. It is reported that CVA is the leading cause of chronic cough in Chinese children, accounting for 41.95% [1,2].The optimal treatment window is often missed due to the absence of wheezing, limited clinician awareness, and diagnostic experience. Approximately 30% of untreated CVA cases in children may progress to typical asthma [3–5].The etiology and mechanisms underlying CVA are complex, and antibiotic treatments are ineffective. Currently, standard treatments include glucocorticoids, bronchodilators, and leukotriene receptor antagonists. However, due to the psychiatric side effects, parents of Chinese children are very cautious about long-term oral administration of montelukast sodium, which affects compliance and overall efficacy [6,7].Traditional Chinese medicine can play an alternative and complementary role to some extent. In recent years, traditional Chinese medicine (TCM) has shown promising results in treating CVA, with better therapeutic efficacy, fewer side effects, and higher acceptance rates among patients. Modified Liu-an Decoction (MLAD) has been effective in treating children with phlegm-heat and phlegm-dampness syndromes, and it has been found to stimulate appetite more effectively than Montelukast sodium [8,9]. MLAD is a well-established prescription for chronic cough in children, composed of Pinellia Ternata, Citri Reticulatae, Poria Cocos, Sinapis Alba, pumice, Lepidium Apetalum, Semen Trichosanthis, Rhizoma Arisaema Cum Bile, and Raphanus Sativus. Previous animal studies have demonstrated that MLAD can reduce serum levels of tumor necrosis factor-α (TNF-α) and interleukin-5 (IL-5), decrease eosinophils (EOS) in bronchoalveolar lavage fluid (BALF), and reduce airway inflammation and collagen deposition. These effects contribute to improved airway remodeling and reduced cough frequency [10–12]. Building on these findings, this study aims to analyze the relationship between CVA airway inflammation and Th1/Th2 cell balance and explore the role of cytokines and the Notch signaling pathway in the therapeutic mechanisms of MLAD in treating CVA.

2 Materials and methods

2.1 Experimental animal

60 healthy 3-week-old pure male guinea pigs, clean SPF grade and weighing 230 to 280g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd. (License Number: SYXK (Beijing) 2019−0013). As guinea pigs are social animals and should not be raised in a single cage, thus 5 guinea pigs were placed per cage according to their size. The guinea pigs were raised under standard conditions in the clean SPF-grade animal room of Dongfang Hospital of Beijing University of Chinese Medicine, with 12h light and night cycle, temperature (23 ± 2)℃, humidity 50%−60%. The experimental protocol was approved by the Ethics Committee for Animal Experiments of Dongfang Hospital of Beijing University of Chinese Medicine, Ethics Approval No. 201918. The guinea pigs were euthanized by releasing carbon dioxide into the box. This experiment follows national and institutional guidelines on the care and use of laboratory animals.

2.2 Experimental drug

The MLAD was composed of Pinellia Ternata 6g, Citrus maxima 6g, Poria Cocos 6g, Prunus Armeniaca 6g, Sinapis Alba 6g, Glycyrrhiza uralensis 3g, pumice 20g, Lepidium Apetalum 6g, Semen Trichosanthis 10g, Rhizoma Arisaema Cum Bile 4g, Raphanus Sativus 10g. The full scientific species (Latin binomial nomenclature) names of all ingredients of MLAD are shown in Table 1. The low-dose group, mid-dose group, and high-dose group were prepared into solutions with concentrations of 0.74g/mL, 1.48g/mL, and 2.22g/mL respectively. MLAD was provided by Beijing Kangrentang Pharmaceuticals and identified by the Department of Pharmacy, Dongfang Hospital, Beijing University of Traditional Chinese Medicine. Montelukast sodium (Hangzhou Merck Pharmaceutical Co., Ltd., specification: 4 mg/ tablet) was made into suspension with normal saline and stored in a refrigerator at 4℃. The drugs were provided by the traditional Chinese Medicine Pharmacy of Dongfang Hospital of Beijing University of Chinese Medicine and Kang Ren Tang Pharmaceutical Co., Ltd. Ovalbumin (OVA) (batch number: 421C033, Solarbio Company) and Aluminum hydroxide (batch number: 20131125, Sinopharm Chemical Reagent Co., Ltd and capsaicin (purity > 95%, batch number: P12D9S77366, Yuan Ye Biology Co., Ltd.).

Table 1. The full scientific species (Latin binomial nomenclature) names of all ingredients of Modified Liu-an Decoction.

Chinese name	English name	Latin name
BAN XIA	Ternate Pinellia	Pinellia Ternata
JU HONG	--	Citrus maxima
FU LING	Indian Bread	Poria Cocos
XING REN	Apricot Seed	Prunus Armeniaca
BAI JIE ZI	White Mustard Seed	Sinapis Alba
GAN CAO	Licorice	Glycyrrhiza uralensis
LAI FU ZI	Garden Radish Seed	Raphanus Sativus
HAI FU SHI	Pumice	Pumex
TING LI ZI	Pepperweed Seed Equivalent	Lepidium Apetalum
GUA LOU ZI	Snakegourd Seed	Semen Trichosanthis
DAN NAN XING	Bile Arisaema	Rhizoma Arisaema Cum Bile

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2.3 Experimental grouping

The experimental animals were numbered according to body weight in ascending order. In the initial grouping, 10 animals were randomly selected as the blank control group(Group A) by using the random number table method, and the remaining 50 animals were treated by unified modeling. After successful modeling, the animals were randomly divided into 5 groups: Group B was the blank control group, Group B was the CVA model group (hereinafter referred to as the model group), Group C was the Montelukast sodium group, Group D, E and F were the low-dose group, mid-dose group and high-dose group of MLAD, respectively.

2.4 Model replication

Based on previous studies and the exploration of the CVA model by the previous research group [13], we adopted the following modeling methods: intraperitoneal injection of immunosuppressant cyclophosphamide (30 mg/kg) on the 1^st day, 1 mL of 2 mg OVA and 200 mg aluminum hydroxide suspension on the 3^rd day, and intraperitoneal injection of 0.1 mg ovalbumin and 100 mg aluminum hydroxide mixture on the 22^nd day to enhance the immune response. From the 23^rd day, 1% OVA solution atomization was performed (the atomization rate was 3 mL/min for 20 seconds and gradually extended to 90s), once every other day, for 7 times. The normal control group was given the same dose of normal saline. Evaluation of the animal model: after modeling, it was mainly assessed by the behavioral performance, the key manifestations are nodding and shrugging, coughing without obvious wheezing after stimulation, cough times observed more than 5 times within 3 minutes, mental irritability or sluggishness and so on. If the above conditions are met, it is determined that the modeling is successful.

2.5 Administration method and dosage

The dosage for each group was calculated according to the conversion formula of body surface area between humans and guinea pigs. From the 23rd day of the experiment, 30 minutes before the stimulation with a nebulizer, each group was given intragastric administration intervention for 14 days. The blank control group and model group were fed with drinking water 1mL/100g/d once a day. The C group was administered with Montelukast sodium suspension 1 mg/kg (0.1 mg/mL, 1mL/100g/day) orally once a day. D group: oral administration of 0.74g/mL of MLAD solution, 1mL/100g/d, once a day. E group: oral administration of 1.48g/mL MLAD solution, 1mL/100g/d, once a day. F group: oral administration of 2.22g/mL MLAD solution, 1mL/100g/d, once a day. The three concentration groups of MLAD are equivalent to 1:2:3 of the adult dosage.

2.6 Observation index

2.6.1 The general condition of CVA Guinea pigs.

The appearance (coat color, hair quality, mental state, etc.), abnormal behavior (cough, sneezing, accelerated breathing, abdominal twitching, etc.), appetite, and body weight were the primary observation indices for each group. Body weight was used as quantitative statistical data.

2.6.2 Airway sensitivity of CVA Guinea pigs.

Except for the blank control group, all the other guinea pigs were placed in a confined space to inhale the 10^-4mol/L capsaicin solution (capsaicin was added to Tween-80 solution, absolute ethanol, and normal saline to prepare a 10^-4mol/L capsaicin solution, ready to mix and use [14]) for 60 seconds after being subjected to a 1% atomization challenge on the 36th day. Thereafter, the atomizer was switched off and the box lid was lifted after 60 seconds. The guinea pigs were then retrieved from the box, and the number of coughs for each guinea pig in 3 minutes was recorded.

2.6.3 Airway resistance of CVA Guinea pigs.

The animal pulmonary function test (PFT) system was used to observe the changes in airway resistance (RI). The guinea pigs of each group were anesthetized by an intraperitoneal injection of 20% urethane solution at 1g/kg. The volumetric box was later sealed. After determining the base value, the stimulating reagent and sequence were as follows: 0.5mL of normal saline and 0.5mL of methacholine with concentrations of 0.02, 0.04, and 0.08 mg/mL for excitation. After each injection, the airway resistance at each concentration level was recorded. The subsequent injection was performed after the total RI returned to normal levels.

2.6.4 Detection of IL-2, IL-4, IL-12 and IL-13 levels in serum and BALF by ELISA.

After completing PFT, CO2 was euthanized. Immediately, blood was drawn from the abdominal aorta and balf was extracted by alveolar perfusion from the right lung. serum and balf supernatant were extracted by centrifugation after standing at room temperature for 2h. Elisa assay for the detection of IL2, IL4, IL12 and IL13 levels in serum and balf, according to the instructions of the relevant kits.

2.6.5 Lung histopathology scoring.

After collecting BALF, the right lung hilar was quickly ligated, and the middle and lower lobes of the left lung were cut and preserved in formalin (Solarbio) for fixation 48h. The fixed lung tissue was routinely paraffin-embedded, sectioned at 4μm, deparaffinized, subjected to various levels of ethanol to water washing, water-dried and hematoxylin-stained for 5 min, rinsed with water and blow-dried and then differentiated with hydrochloric acid-ethanol for 30s. It was soaked with water for 15 min and then blow-dried, and then placed in eosin solution for 2 min, and then dehydrated, clarified, and sealed with neutral resin, and photographed using an orthogonal fluorescence microscope (Leica DM3000).The quantitative grading criteria of the pathological sections [15] are shown in Table 2.

Table 2. Histopathological Scoring System for Evaluating Changes in Pulmonary Inflammation in Guinea Pigs with Antigen Challenge.

Histopatholog-ical score	Eosinophilic infiltration in blood vessels and bronchi	Edema	Epithelial cell injury
0	Normal	Normal	Normal
1	Small amount of cell infiltration, no obvious histopathological changes	Mild diffuse edema	Mild cell injury
2	Small to medium amount of cell infiltration, mild tissue damage	Moderate alveolar and bronchiolar edema	Mild cell injury
3	Moderate cell infiltration and mild tissue injury	Regional and focal edema	Moderate cell injury
4	Moderate to large amount of cell infiltration, obvious tissue damage	Significant edema	Moderate cell injury
5	Massive cell infiltration, severe tissue damage	Pneumatosis edema	Epithelial metaplasia, mucinous cell hyperplasia

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2.6.6 Detection of Delta1, Jagged1, Notch1, and NICD proteins in lung tissue by Western blot.

After the collection of BALF, followed by rapid ligation of the left hilar, the tissue of the left upper lobe of the lung was taken and preserved in liquid nitrogen, and then the proteins were separated by gel electrophoresis, and the electrophoretically separated bands were transferred to NC/PVDF membranes, and the membranes were treated with antibodies against Delta1, Jagged1, Notch1, and NICD proteins, and finally the proteins were detected, imaged, and analyzed as a result. The optical density values of the target bands were analyzed by Quantity One image analysis software.

2.7 Statistical methods

The SPSS 26.0 statistical analysis software was used for statistical analysis. The experimental data were all measurement data, expressed as mean±standard deviation, in line with normal distribution, homogenous variance. The one-way ANOVA was selected, and LSD was used for the pairwise comparison method test. If it did not conform to a normal distribution, the Kruskal-Wallis test was used. p < 0.05 was regarded as a statistically significant difference.

3. Experimental results

3.1 General condition of guinea pigs in each group

Throughout the whole process, the guinea pigs in the blank control group were sensitive and alert, had moist and smooth fur, loud calls, stable vital signs, a normal diet, and normal weight gain (body weight was measured every 3 days). In the model group, steady breathing was observed before OVA atomization. The guinea pigs gradually experienced varying degrees of rapid breathing, abdominal convulsions, coughing, and sneezing during the atomization process. There was also occasional nodding, shrugging, and face wiping observed in the guinea pigs. In terms of mental state, it was manifested as restlessness or sluggishness, and some guinea pigs would chew on the hair of other guinea pigs. With the increase in OVA atomization, the aforementioned symptoms, were progressively aggravated. The symptoms of each intervention group were similar to those of the model group at the initial stage of OVA atomization. After drug intervention, the mental state symptoms of the MLAD groups improved compared to those in the model group and the MS group.

On the 7^th day after modeling, one guinea pig in the high-dose MLAD group died by accident. No abnormal findings were found after the autopsy.During the OVA atomization challenge, due to the large individual differences and different sensitivities to ovalbumin, 7 guinea pigs (2 each in the model group and the high dose group, and 1 each in the other groups) died during the atomization process. It was found that the lungs of died guinea pigs were hyperemic and the rest were normal. Therefore, the deaths could be due to high bronchial spasms and respiratory distress.

In terms of diet and weight, the guinea pigs in the high-dose MLAD group gained the most weight. The fur of the model group was slightly loose, and there was no significant increase in body weight in the later stage of the experiment, and some of the guinea pigs even lost weight. The body weight of the guinea pigs in each group was significantly lower than that of the blank control group, and the weight of the model group was significantly lower than that of the blank control group (p < 0.05). The body weight of the guinea pigs in the high-dose MLDA group was significantly higher than that of the other groups. There was no statistically significant difference amongst the other groups (p < 0.05). See Table 3.

Table 3. Changes in Body Weight (g) of Guinea Pigs in each group before and after modeling.

Group	n	Before OVA induction	n	Before execution
Blank control group	10	262.66 ± 1.73	10	436.20 ± 37.44
Model group	10	262.54 ± 6.52	8	384.86 ± 20.25*
MS group	10	267.71 ± 3.68	9	408.38 ± 21.01
Low-dose Modified Liu-an decoction group	10	257.00 ± 4.87	9	404.00 ± 12.82
Mid-dose Modified Liu-an decoction group	10	261.80 ± 3.50	9	408.71 ± 19.70
High-dose Modified Liu-an decoction group	10	264.28 ± 7.16	7	457.80 ± 9.47^#

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Note: Compared with the blank control group, *p < 0.05, Compared with the model group, ^# p < 0.05, Compared with the MS group, ^△p < 0.05.

3.2 MLAD reduces airway sensitivity and airway resistance in cva guinea pigs

The number of the number of coughs showed normal distribution and the variance was homogenous. Using the one-way analysis of variance, the cough frequency among the six groups was not the same. Compared with the blank control group [(1.80 ± 0.58) times] (normal saline atomization stimulation), the number of coughs in the model group [(11.00 ± 1.48) times], MS group [(6.40 ± 1.29) times] and low-dose MLAD group [(4.40 ± 0.93) times], mid-dose MLAD group [(5.00 ± 1.38) times] and high-dose MLAD group [(5.00 ± 1.14) times] were significantly increased, and the difference was statistically significant (p < 0.05). Compared with the model group, the number of coughs in each MLAD dose group and the MS group was significant, with a statistically significant difference (p < 0.05). The number of coughs in the low-dose MLAD group was the lowest. Compared with the MS group, the cough frequency in each MLAD dose group was lower, but there was no significant difference between the groups (p > 0.05). (Table 4)

Table 4. Number of coughs of Guinea Pigs in each group after modeling.

Group	n	Cough frequency	*P	#P	△P
Blank control group	10	1.80 ± 0.58	–	0	0.011
Model group	8	11.00 ± 1.48	0	–	0.011
MS group	9	6.40 ± 1.29	0.011	0.011	–
Low-dose Modified Liu-an decoction group	9	4.40 ± 0.93	0.13	0.001	0.24
Mid-dose Modified Liu-an decoction group	9	5.00 ± 1.38	0.066	0.001	0.407
High-dose Modified Liu-an decoction group	7	5.00 ± 1.14	0.066	0.001	0.407

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Note: Compared with the normal group, *p < 0.05, Compared with the model group, ^# p < 0.05, Compared with the MS group, ^△p < 0.05.

Compared with the blank control group, there were significant differences in airway resistance in normal saline, MeCH 0.02 mg/mL, and MeCh 0.04 mg/mL dose of the other five groups (p < 0.05). There was a statistically significant difference in airway resistance between the low and high-dose MLAD groups and the model group at the MeCh 0.02 mg/mL dose. The airway resistance of the blank control group and the low and mid-dose MLAD groups gradually elevated with the increased stimulant concentration. On the other hand, the airway resistance of the model group, MS group, and high-dose MLAD group peaked at the MeCh0.04mg/mL dose.(Table 5 and Figs 1 and 2)

Table 5. Comparison of the Area under the Total Airway Resistance Curve (RL-area) under Different Doses of Methacholine Challenge Test (cmH₂O/mL).

Group	Saline injection before stimulation	Acetylcholine content (mg/mL)
Group	Saline injection before stimulation	0.02	0.04	0.08
Blank control group	48.6 ± 7.46^#△	72.54 ± 5.69^#△	90.35 ± 12.11^#△	110.99 ± 19.48^#△
Model group	171.34 ± 11.69^*	206.02 ± 11.29^*	299.14 ± 23.55^*	246.92 ± 76.05
MS group	174.86 ± 50.93^*	188.19 ± 54.98^*	240.18 ± 66.41^*	138.03 ± 35.91
Low-dose Modified Liu-an decoction group	113.61 ± 23.02^#	122.13 ± 30.77^*#	238.14 ± 67.55^*	240.90 ± 85.45
Mid-dose Modified Liu-an decoction group	162.02 ± 21.31^*	175.64 ± 22.03^*	224.37 ± 42.90^*	328.87 ± 112.60^*△
High-dose Modified Liu-an decoction group	89.75 ± 7.82^#△	112.11 ± 15.17^#△	131.58 ± 4.69^#△	171.2 ± 27.41

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Note: Compared with the normal group, *p < 0.05, Compared with the model group, ^# p < 0.05, Compared with the MS group, ^△p < 0.05.

3.3 MLAD regulates Th1/2 cytokine expression levels

The IL-2, IL-12, and IL-13 levels in the model group were significantly higher, while the IL-4 levels were lower than those in the normal group (p < 0.05). The IL-12 levels in the MS group and the MLAD groups were lower than those in the model group. The IL-2 levels in the MS group and the low-dose MLAD groups were significantly different from the model group (p < 0.05). The IL-4 levels in each drug intervention group were higher than that in the model group, but there was no significant difference between the low and medium-dose MLAD groups and the model group. There was no statistically significant difference in IL-13 levels between each group and the model group (Fig 3).

3.4 Comparison of IL-2, IL-12, IL-4 and IL-13 levels in BALF of guinea pigs

The IL-2 and IL-12 levels in the model group were lower, and the IL-4 and IL-13 levels in the model group were higher than those in the blank control group (p < 0.05). Compared with the model group, the IL-2 and IL-12 levels in the MS group and the various MLAD dose groups were higher, whereas the IL-4 and IL-13 levels were lower. Among them, the various cytokine levels in the MS group were significantly different from those in the model group (p < 0.05). The IL-12 levels in each MLAD dose group were significantly higher than that of the model group (p < 0.05). Among them, the IL-2 and IL-4 levels were significantly different between the high-dose MLAD group and the model group (p < 0.01). There were significant differences between the MS group, the middle-dose MLAD group and the model group (p < 0.05). The IL-13 levels decreased after treatment, with the low-dose MLAD group experiencing a significant decrease compared with the model group (p < 0.05). However, there was no statistically significant difference between the middle-high-dose MLAD group and the model group. (Fig 4)

3.5 MLAD attenuates airway structural damage in cva guinea pig lung tissue

Regarding the histopathologic inflammation grading scores of guinea pig lungs in each group, the difference between the blank control group(2.4 ± 0.25) and the other 5 groups was statistically significant, and the difference between the low-dose MLAD group(6.3 ± 0.44) and the model group(8.1 ± 0.43) was statistically significant (p < 0.05), there was no statistically significant difference in the middle-dose MLAD group(7.6 ± 0.91), MS group(7.5 ± 0.50) and the high-dose MLAD group (7.1 ± 0.91). As shown in Figs 5 and 6 Histopathologic inflammation grading scores of guinea pig lungs in each group, the airway structure of the guinea pigs in the blank control group retained its normal integrity, with a clear and complete alveolar structure, without any evident inflammatory cell infiltration observed. The rest of the groups showed varying degrees of alveolar collapse, septal widening, partial alveolar dilatation, low columnar bronchial mucosal epithelium, an irregular proliferation of some airway mucosal epithelial cells, significant reduction of folds, as well as severe airway epithelial cell necrosis and shedding. There was also massive infiltration of monocytes, plasma cells, and eosinophils in the submucosal and alveolar septa, and hyperplasia of the airway smooth muscle. Furthermore, there was loosening, edema, and fibrosis around the bronchi and blood vessels.

3.6 MLAD inhibits NOTCH pathway expression

Compared with the normal group, the expression of the Notch ligand protein Delta1 in the remaining 5 groups was significantly decreased, while the expression of ligand Jagged1, receptor Notch1, and nuclear-localized Notch protein fragment NICD was significantly increased (p < 0.05). Compared with the model group, the expression of Jagged1, Notch1, and NICD proteins in the guinea pigs treated with MS and low-dose MLAD group decreased, while the expression of Delta1 protein increased (p < 0.05). However, there was no statistically significant difference between the 2 groups, and there was no statistically significant difference in protein levels between the middle-dose and high-dose MLAD group and the model group. (Figs 7 and 8)

4 Discussion

It is widely recognized that the pathophysiological process of CVA is not fundamentally different from that of typical asthma, and it is a subtype of bronchial asthma, with the same pathogenesis, which mainly includes airway immune-inflammatory mechanisms and neuromodulatory mechanisms and their interactions [16,17]. In this study, we investigated the efficacy of the traditional Chinese medicine Modified Liu-an Decoction in the treatment of CVA from the perspective of airway immune-inflammatory mechanisms, including airway inflammatory response, airway hyperresponsiveness, airway remodeling,and further explored specific mechanisms of action from the perspective of airway immunoinflammation.

4.1 Airway inflammatory

The inflammatory cells involved in the pathogenesis of CVA mainly include T lymphocytes, eosinophils (EOS), and mast cells. T lymphocytes play an essential role in the pathogenesis of CVA. By affecting the differentiation of Th1/Th2 cells, the release of Th1 cytokines decreases and Th2 cytokines increases, resulting in chronic airway inflammation [18,19].The decrease in the Th1/Th2 cell ratio is the initiation and maintenance factor of airway inflammation and airway hyperresponsiveness, and Th2 dominance is the central mechanism for the formation and progression of CVA [20]. Some studies have shown that children with CVA have higher levels of IL-4 and INF-γ/IL-4, and there is an imbalance of Th1/Th2 cells, which manifests as Th2 cell hyperactivity [21]. Therefore, by regulating the balance of Th1/Th2 cells and affecting the release of relevant cytokines, chronic airway inflammation in CVA children can be alleviated. The lung tissue contains the most Notch ligands and mRNA receptors. The Notch signaling pathway plays a significant role in lung tissue conduction, especially on the differentiation of Th1 and Th2 cells in T lymphocytes in the lung tissue. The Notch ligand Jagged is a dendritic-derived signal that plays a role in the differentiation of IL-4-independent Th2 cells, while the Notch ligand Delta is also a dendritic-derived signal that promotes the differentiation of Th1 cells [22]. Therefore, the Notch signaling pathway regulates T lymphocytes in the lung tissue through different ligand-receptor binding, so that the Th1/Th2 cells are in a state of relatively balanced state to keep the body healthy.

Among the observed indices in this study, IL-2 is mainly a 15kD glycoprotein secreted by Th1 cells, stimulated by IL antigens or mitogens, and induced by IL-1, which can stimulate the expression of MHC class II antigens in T cells and produce cytokines such as IFN-γ and TNF-α, which can promote the differentiation of Th0 cells into MHC and selectively inhibit the secretion of Th2 cytokines [23,24]. IL-12 appears earlier in the process and it can promote the differentiation of CD4 + T cells into Th1 cells, and promote Th1 cells to secrete IL-2 and IFN- γ, thereby regulating the ratio of Th1 and Th2 to develop into Th1 [25,26]. Xu Hui et al [27] found that IL-12 could reduce the expression of IL-4 mRNA in the lung tissue of asthmatic mice, thereby reducing the chronic inflammatory infiltration of the airway. IL-4 is mainly secreted by Th2 cells with a variety of biological promoters, with the induction of the IgE-mediated immune response being the most important mechanism [28]. Matsukura et al [29] showed that IL-4 activates the inflammatory cells in the respiratory tract by upregulating the expression of eotaxin-1 and eotaxin-3 mRNA, which increases the number of acute attacks in asthmatic patients. IL-13 is produced by activated airway hyperresponsiveness (AHR)-related cells including Th2 cells, which can thicken the airway wall by promoting goblet cell proliferation [30] and mucus secretion [31]. When AHR-inducing factors are produced, IL-13 increases abnormally, which not only promotes the synthesis of IgE but also down-regulates the secretion of cytokines produced by Th1, such as IL-12 and IFN- γ, thus leading to the occurrence of disease [32,33]. IL-2 and IL-4 are key cytokines in the differentiation of Th0 cells into Th1 cells and Th2 cells. When the specific antigen-presenting cells are sensitized, they can differentiate into Th0A and Th0B. Th0A can secrete IL-2 and IL-4, while Th0B can secrete IL-2, IL-4, and IFN- γ. Under the action of high levels of IL-4, most Th0A and part of Th0B can differentiate into Th2. If the effect of IL-4 is blocked, Th0A will lose the ability to differentiate into Th2 and can express IFN- γ and differentiate into Th0B, and Th0B will further differentiate into Th1 [34]. IL-2 and IL-12 are also called NK cells and T cell growth factors respectively. The phenotypic difference between their inducing lymphocyte growth is that the main function of IL-2 is to promote the growth of T cells. On the one hand, it can stimulate T cells to produce cytokines such as IFN-γ and TNF-α after binding to the receptor and exerting its effector function. On the other hand, the IL-2 levels in the peripheral blood or plasma can also affect the balance shift of Th1/Th2 [35]. IL-12 mainly promotes the expression of NK cells and also maintains the growth of B cells. IL-12 can stimulate the production or apoptosis of various cells, promote the differentiation of Th0 into Th1, and produce a variety of cytokines such as IFN-γ, IL-2, GM-CSF, etc.[36]. IL-12 can reduce the number of eosinophils and the IgE levels in the lung tissue or blood by inducing eosinophil apoptosis and controlling the activation of mast cells [37]. IL-4 is the IgE regulator with the highest biological effect known to date, and IL-12 inhibits the activation of mast cells by reducing the production of IL-4, thereby reducing chronic airway inflammation [38]. IL-4 and IL-13 are Th2 cytokines that are closely linked in function and gene locus and have multiple overlapping functions. These functions play an important role in allergic airway inflammation and airway hyperresponsiveness. Their functional overlap is due to the α-chain of IL-4, which forms an important functional signaling component in the IL-4 and IL-13 receptors. The role of IL-4 and IL-13 in asthma is different. IL-4 may use its α- chain in the early and later stages when B cells are isotype-switched to secrete IgE, while IL-13 uses this receptor to promote the production and development of airway hyperresponsiveness and inflammation in the late stage of the allergic reaction. In experimental studies, it was found that the IL-4 and IL-13 functions were positively correlated at the molecular level and in asthma [39–41]. The results of this study suggest that MLAD can reduce chronic airway inflammation by up-regulating the IL-2 and IL-12 levels in BALF, down-regulating the IL-4 and IL-13 levels, and regulating the dynamic balance of Th1/Th2.A number of domestic animal experimental studies have found [42] that traditional Chinese medicine compound can promote the expression of signal transducer and activator of ranscription4 (STAT4) in lung tissues by up-regulating the expression of helper T cell (Th)1 cytokine IL-12 and down-regulating the secretion of Th2 cells, thus controlling the development of the disease. of ranscription4 (Stat4) in lung tissues, reducing airway inflammation and thus controlling the development of the disease. The results of the study suggest that MLAD can regulate the dynamic balance of Th1/Th2 by up-regulating the content of IL-2 and IL-12 and down-regulating the content of IL-4 and IL-13 in BALF, thus reducing the chronic inflammation of airways, which is similar to the results of the above study.

The Notch signaling pathway plays an important role in lung tissue conduction, especially the differentiation of Th1 and Th2 cells in T lymphocytes in the lung tissue. The overexpression of the Notch1 and Jagged1 proteins can promote the expression of Th2 cell-specific transcriptional factor GATA3 and pro-inflammatory factor IL-4, thereby intensifying the polarization of Th2 cells in the body [43,44]. Experimental studies [45] have suggested that the expression of Jagged1, Notch1 proteins and their downstream transcriptional target Hes1 protein were significantly increased in OVA-induced chronic bronchial asthma mice. Therefore, the Notch signaling pathway regulates T lymphocytes in the lung tissue through different ligand-receptor binding, so that the Th1/Th2 cells are in a state of relatively balanced state to keep the body healthy. From the results of the Notch ligand/receptor protein levels in the lung tissue of the guinea pigs in each group, it was found that both MS and MLAD could increase the expression of Delta1 protein while inhibiting the expression of Jagged1, Notch1, and NICD proteins. The effect of the low-dose MLAD group was similar to that of the MS group. However, no salvage experiments were performed in this experiment, the specific downstream regulatory mechanisms need to be explored in further studies.

4.2 Airway hyperresponsiveness

In the results of this experiment, compared with the blank control group, the number of coughs of the guinea pigs in the other groups increased significantly after the last OVA protein atomization stimulation. The number of coughs in all groups was more than 5 times, suggesting that the model was successfully established. The number of coughs in the low-dose group was the least. The response of the guinea pigs to most protein antigens is a delayed hypersensitivity reaction mediated by specific sensitized effector T cells, which can be passively transferred by CD4 + T cells [46]. During the course of the experiment, it was also found that the cough was not very obvious on the day of atomization, and the guinea pigs in each group experienced more frequent coughing after drug administration the next day. After the intervention, the airway sensitivity of the guinea pigs in each group was stimulated with capsaicin atomization. The airway sensitivity of the guinea pigs in each treatment group was lower than that of the model group, suggesting that the intervention treatment was effective. The MLAD and MS sodium groups have similar efficacy in reducing airway sensitivity in CVA guinea pigs. In the pulmonary function challenge test, the airway resistance of the model group and MS group both peaked at the dose of MeCh0.04mg/m, considering that the guinea pig airway spasm has reached the limit at this concentration, and the airway spams have reached the limit when the concentration was increased. The smooth muscle of the airway failed to respond to increased concentrations of re-stimulation. However, the airway resistance of the normal group and each MLAD dose group gradually increased with the increasing concentration of the stimulant, suggesting that the airway hyperresponsiveness of the guinea pigs decreased after treatment with MLAD. The resistance value of the high-dose MLAD group was higher than that of the blank control group, but the trend was very consistent. It is considered that MLAD may increase the airway sensitivity threshold of CVA guinea pigs, thereby controlling neurogenic inflammation and reducing the number of coughs. Nerve-related airway factors can increase tracheal vascular permeability, promote airway glandular secretion, and stimulate neurogenic inflammation such as airway smooth muscle spasm and contraction, while neuropeptide antagonists can effectively treat cough and relieve cough caused by capsaicin stimulation [47,48]. MLAD may also play a similar role in the treatment of CVA, but its specific mechanism requires further theoretical and experimental research.

4.3 Airway remodeling

Long-term chronic non-specific inflammatory stimulation of the airway can lead to varying degrees of pathological changes such as airway mucosal epithelial injury, cilia exfoliation and inflammatory cell infiltration, airway mucosal edema and hypertrophy, smooth muscle spasm, and airway inflammatory secretion obstruction in the airway. In this study, except for the blank control group, all the other groups had varying degrees of the aforementioned pathological changes. After the treatment with montelukast and MLAD, the pathological inflammation scores of the lung tissue of the guinea pigs in each treatment group decreased, indicating that the treatment was effective, and the curative effect of the low-dose MLAD group was better than that of the MS group. The hyperresponsiveness of the airway can restrict the airflow to different degrees. In mild cases, the pathological changes are reversible. However, if left unchecked, it will lead to airway remodeling [49]. Through Masson staining of the lung tissues of the guinea pigs in each group, we could see a thin layer of collagen deposition around the trachea of the guinea pigs’ lung tissue in the blank control group, and a thicker layer of collagen deposition around the trachea of the guinea pigs’ lung tissue in the model group. The collagen area and collagen volume fraction were significantly increased in the model group compared with the blank control group and significantly reduced in each treatment group. According to the Chinese medicine theory of “strange diseases have more phlegm” and “all diseases are caused by phlegm”, it is believed that the key to the pathogenesis of cough variant asthma lies in the internalization of phlegm, which is caused by the deficiency of the lungs, spleen and qi, and the external cause is caused by wind evils that attack the lungs, and the combination of internal and external causes leads to the disease. From a microscopic point of view, this invisible phlegm in the lungs coincides with chronic inflammation of the airways, inflammatory cell infiltration, release of inflammatory factors, congestion and edema of the airway mucosa, increased mucus secretion, mucus plugs, collagen deposition and other modern pathologies, and the addition of Liuan Decoction to the lungs can clear lungs and remove heat and strengthen the spleen to resolve phlegm, congestion, edema degree, reduce airway inflammation, and at the same time can reduce peri-tracheal collagen deposition, effectively improve airway remodeling, which is consistent with the results of the previous rat experiments [11].

5 Conclusion

In this study, we explored the effectiveness and possible mechanisms of MLAD in the treatment of CVA. First we found that MLAD reduced the number of coughs and airway resistance in CVA guinea pigs. Secondly, MLAD effectively regulated the levels of inflammatory factors IL12,IL2,IL4,IL13 in serum and balf, inhibited airway inflammation, and at the same time attenuated inflammatory infiltration in lung tissue. MLAD could increase the expression of Delta1 protein while inhibiting the expression of Jagged1, Notch1, and NICD proteins, the specific downstream regulatory mechanisms need to be explored in further studies.In summary, we verified that mlad prevented CVA by ameliorating airway inflammatory infiltration and airway remodeling induced by th1/2 differentiation imbalance, and its mechanism may be related to the inhibition of Notch pathway expression.

Data Availability

The data from this studyis available from the Dryad database under the registration number DOI: https://doi.org/10.5061/dryad.x95x69pxg.

Funding Statement

This study was supported by [the National Natural Science Foundation of China (No. 81874487), and the Guangzhou Science and Technology Bureau Research Project, No. 2023A04J1255] in the form of a grant awarded to [WLQ] and [XFW]. The specific roles of this author are articulated in the ‘author contributions’ section. The two authors are the leads of these two research projects, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Reviewer #1: Dear authors,

The statistical methods presented in a transparent way help readers understand how the data was analyzed, increasing the reproducibility and credibility of the study. Sensitivity analyzes are responsible for testing the robustness of the results of different assumptions or analytical choices in the study. Defining the limitations and biases of the study are important to clarify to the reader the possible restrictions present in the research design, collection or analysis of the data obtained, providing an adequate interpretation of the results, in addition to helping to understand the potential impact of these factors, promoting transparency to readers. The statistical analyzes of the research were well executed, but I suggest presenting improvements in methodological transparency and sensitivity analyses, which could further increase the robustness and credibility of the results obtained. Providing a detailed description of the statistical methods used would also be interesting. Furthermore, researchers could define the limitations surrounding the study and define the biases present in the research, if any.

Reviewer #2: I acknowledge the efforts of the authors in an attempt to provide a therapeutic solution for asthmatic conditions. While the research addresses a clinically relevant question with potentially significant implications for treatment planning, several areas are noted with flaws and clinical concerns. Some of these are summarized below;

MAJOR CONCERNS:

1. CLINICAL CONCERN: In this study, the use of numerous herbs (more than 10) appears somewhat arbitrary, suggesting a trial-and-error approach by combining multiple ingredients to achieve results. No identifiable compounds other than concocting too many herbs without analyzing their constituents. This method raises health concerns due to the potential toxicity of unknown constituents. Employing such a mixture without a thorough analysis of each component’s active and potentially toxic compounds is neither scientifically rigorous nor acceptable for ensuring safety. Therefore, it is highly important to provide a detailed analysis of the constituents of the herbs used in a study like this, especially for a few key reasons:

a) Pinpoint specific compounds responsible for therapeutic effects to identify the active agents understanding that this knowledge allows researchers to attribute biological activity (such as anti-inflammatory, bronchodilatory, or immune-modulatory effects) to particular compounds. Without this, attributing effects solely to the herbal mixture may limit the scientific rigor of the study and make it difficult to determine which components are beneficial.

b) Detect and manage potentially harmful compounds. Herbs can contain toxic compounds that may pose health risks, particularly if they are administered in higher doses or for prolonged periods. Detailed analysis helps identify any potential toxins, ensuring safer use in clinical or experimental settings.

c) Facilitate consistent replication of treatment effects in future studies. If the herb mixture (like MLAD in this case) is to be recommended for future therapeutic use, having a clear understanding of its components is essential for reproducibility. It ensures that other researchers or clinicians can recreate the mixture with similar composition and dosage, leading to consistent results and therapeutic efficacy.

d) Understand how individual components affect biological pathways. If one herb contains a compound that modulates the Notch signaling pathway (central to the study’s hypothesis), identifying it could enhance understanding of how the herbal mixture exerts its effects on Th1/Th2 function.

2. POOR METHODOLOGY:

a) Experimental model: Using 3-week-old guinea pigs in a study involving respiratory function, immune response, and airway inflammation seem unethical and thus should be approached with caution. At this age, guinea pigs are quite young, and their respiratory and immune systems are still maturing, which could influence the results and interpretation of the study.

I. At 3 weeks, guinea pigs are weaned but still undergoing significant physiological development, including in the lungs. Immature lung structure and function could lead to different responses to asthma or inflammation compared to fully mature animals.

II. The immune response in young animals may not fully mimic that in adults, especially in conditions involving Th1/Th2 balance and chronic inflammation. Young guinea pigs may have an underdeveloped adaptive immune response, which could affect how they respond to allergens and treatments.

III. For studies aimed at understanding asthma-like conditions in humans, using older guinea pigs (e.g., 6–8 weeks or older) would generally provide a more stable model, as their respiratory and immune systems would more closely recapitulating those of adult humans. Young animals might not fully replicate the chronic and complex immune dynamics of asthma, especially cough variant asthma (CVA).

IV. Young animals may experience more stress and potential adverse effects from experimental procedures, particularly if they involve repeated allergen exposure and invasive testing and may therefore violate ethical considerations and animal welfare.

3. Regarding the statement, "MLAD was composed of Pinellia Ternata 6g, Citrus maxima 6g, Poria Cocos 6g, Prunus Armeniaca 6g, Sinapis Alba 6g, Glycyrrhiza uralensis 3g, pumice 20g, Lepidium Apetalum 6g, Semen Trichosanthis 10g, Rhizoma Arisaema Cum Bile 4g, Raphanus Sativus 10g ", some concerns are raised;

I. The statement does not provide any rationale for the specific quantities of each ingredient. While traditional formulations may prescribe certain amounts, discussing why these specific doses are used could enhance understanding, particularly for readers unfamiliar with TCM.

II. The study failed to show where each of the MLAD composition was sourced; root. fruits, stem or so. Different parts can contain varying concentrations of active compounds and may have distinct therapeutic effects. Including this detail would enhance the study’s reproducibility and scientific rigor.

III. Though minor, while scientific names are provided, they are inconsistent in capitalization and formatting. Scientific names should always be italicized, with the genus capitalized and the species name in lowercase (e.g., Pinellia ternata).

4. Under “1.3 Experimental grouping”, the grouping method used in this study is flawed due to a lack of consideration for body weight balance across the groups. Ensuring that each group has a relatively similar mean body weight is essential for minimizing variability that could affect treatment response and overall outcomes. However, this crucial step was overlooked. In Table 3, both the administration of ovalbumin and the treatments appear to be based on body weight when the animals were euthanized for analysis, and for the endpoint outcomes. This oversight introduces a significant bias, as variations in body weight on group level could influence the animals' responses to both the sensitization and treatment, potentially skewing the results. This lack of initial weight-based grouping represents a fundamental methodological flaw that weakens the study’s reliability and validity.

5. The behavioral assessment criteria used in this study are flawed and inadequate. The criteria for assessing successful model induction—such as nodding, shrugging, and coughing—are subjective and may result in inconsistent evaluations. Implementing a validated scoring model for behavioral assessment, such as the Asthma Severity Score or Pulmonary Symptom Scoring System, would improve objectivity. Such models assess symptoms based on standardized metrics like cough frequency, respiration rate, and activity level, providing consistent criteria that reduce observer bias and enhance the reproducibility of results.

6. Cyclophosphamide, a powerful immunosuppressant, is administered on Day 1 without clarification of its specific role. Given that CVA is an immune-driven condition, the inclusion of an immunosuppressant may affect the immune response’s development in ways not typical of CVA, potentially influencing the model's relevance.

7. The cytokine analysis in this study is limited by its focus on Th2 responses, limited from Th1.

a) It omits critical data on Th1 cytokines, such as IFN-γ and other pro-inflammatory markers, which are crucial for understanding the balance between Th1 and Th2 pathways in asthma. The study would benefit from investigating how Th1 cytokines might be downregulated in an asthmatic context, along with potential pathway crosstalk between Notch signaling and Th1/Th2 regulation.

b) Additionally, the role of effector cells such as macrophages and Tcells (both) in this model remains unexplored. Effector cell activity and differentiation are key to understanding the immune response and would add depth to the study’s conclusions. A simple flow analysis could have improved the study rigor.

c) For robust conclusions on cytokine expression, it’s essential to account for possible confounding factors such as underlying health conditions or infections (e.g., intestinal or blood pathogens) that can influence cytokine levels. Cytokine regulation is heavily influenced by the JAK-STAT pathway; yet, the study lacks details on how, or if, JAK-STAT interacts with Notch signaling. Clarifying these pathway interactions would strengthen the study’s insights into the mechanisms at play in asthma.

8. The authors claimed that they stained Masson Trichrome for fibrosis analysis in the lungs, only to be seen on the section slide. In fact, I had expected IHC stain to establish the findings here but to no avail.

MINOR CONCERNS:

1. The “Conclusion” section is placed before the “Discussion.” This is unconventional; the authors should justify this arrangement or consider reordering the sections.

2. There are numerous capitalization errors throughout the manuscript that need to be addressed.

3. In the dosing description, there’s an error in consistency and clarity: “Low-dose MLAD decoction group: oral administration of 0.74g/mL MLAD solution, 1mL/100g/d, once a day. Mid-dose MLAD group: oral administration of 1.48g/mL MLAD solution, 1mL/100g/d, once a day. Low-dose MLAD group: oral administration of 2.22g/mL MLAD solution, 1mL/100g/d, once a day. The three concentration groups of MLAD are equivalent to 1:2:3 of the adult dosage.” Something is wrong here. This should be clarified and corrected to accurately describe each dosage group.

4. The statement “From the day of stimulation, that is, from the 23rd day of the experiment, 30 minutes before the stimulation with a nebulizer, each group was given intragastric administration intervention for 14 days” lacks specificity. What was the purpose of this intervention?

5. The sentence, “The dosage for each group was calculated according to the conversion formula of body surface area between humans and guinea pigs,” is vague and should be clarified, perhaps with the formula provided for transparency.

6. Avoid starting sentences with numerals, as in “60 healthy 3-week-old pure male guinea pigs...” Instead, write, “Sixty (60) healthy 3-week-old male guinea pigs...” This applies throughout the manuscript.

7. Some sentences are overly lengthy and difficult to follow, such as: “Through our previous animal experimental studies, we discovered that MLAD could reduce the levels of serum tumor necrosis factor-α (TNF-α) and interleukin-5 (IL-5) in CVA rats, reduce the number of eosinophils (EOS) in the bronchoalveolar lavage fluid (BALF), reduce airway inflammation and airway collagen deposition, thereby improving the pathological state of airway remodeling and reducing cough frequency.” This should be broken down for clarity.

8. The authors mention a previous clinical trial: “Our previous clinical trials have shown that treatment of CVA using Modified Liu-an Decoction (MLAD) and Montelukast sodium was effective in children with phlegm-heat and phlegm-dampness syndromes.” Specific details, including location and timing, should be provided to give context to this claim.

9. Many statements appear to be derived from the literature without proper citation. For instance, “The etiology and mechanism of CVA are complicated, and antibiotic treatment is ineffective,” lacks a reference.

10. The abbreviation “CVA” is used without being defined at first use. It should be spelled out initially for clarity.

11. There is no “Introduction” section, which is unusual for a scientific manuscript.

12. The introduction should provide a rationale, emphasizing how this new agent could potentially offer advantages over current treatments and why it is important to pursue this line of research.

13. The poor design and readability of figures and graphs diminish the accessibility and interpretability of the data. Enhanced visuals with clear labeling would allow readers to better understand the results.

In summary, the study would benefit from a stronger structural foundation, improved clarity and consistency, more rigorous methodology, comprehensive data analysis, detailed justification for MLAD's use, and enhanced figures. Addressing these points would lead to a more reliable and scientifically robust study.

**********

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Reviewer #1: No

Reviewer #2: Yes: Kamoru Adedokun

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Attachment

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PLoS One. 2025 Sep 15;20(9):e0327356. doi: 10.1371/journal.pone.0327356.r002

Author response to Decision Letter 1

27 Dec 2024

The editors may not be particularly familiar with TCM medications; the medications used in this study have been clinically tested over a long period of time and there are clinical trials to prove their safety�HU Jiao Jiao. Study on the effect of slowing croup six-an decoction on small airway function in children with asthma in remission [D]. Beijing University of Traditional Chinese Medicine,2019.�. The guinea pigs have already entered puberty at 13 weeks, so we chose 3-week-old guinea pigs for the pediatric study, including the effects of the drug on their growth and development.

PLoS One. doi: 10.1371/journal.pone.0327356.r003

Decision Letter 1

Misbahuddin Rafeeq

24 Mar 2025

Dear Dr. XU,

Please submit your revised manuscript by May 08 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #3: No

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #3: The authors have done great work, but there are still some questions and suggestions for them.

Q1: There are some format errors. For example, in the first line of the second paragraph of 2.1 General condition of guinea pigs in each group, the 7th should be 7th ("th" superscripted). The spacing before and after punctuation marks (such as “,”, “<” and “±”) is inconsistent. Also, the "n"s indicating sample size and the "P"s indicating significance should be italicized. The capitalization of 3.3 is not consistent with that of 3.1 and 3.2. I hope the authors can improve the format of this manuscript.

Q2: Figure 1, 3, 4, 6, 7, 8 should each include a title, not just a legend.

Q3: In Figure 7, the protein weights should be written as kDa and the font should be standardized (I suggest that they be standardized to Arial).

Q4: I suggest that the bars in Figure 8 should be grouped by proteins, rather than experimental groups. This could help with the clarity of the results. Also, I think the * and # showing significance are misplaced, as they are not positioned directly above the bars.

Q5: Is it possible to add some indicators that can directly demonstrate Th1/Th2 conversion, such as ① flow cytometry or ② the detection of Th1 marker T-box transcription factor (T-bet) and Th2 marker GATA-binding protein 3 (GATA-3) or ③ Th1-associated proinflammatory cytokines interferon (IFN)-γ and tumor necrosis factor (TNF)-α by CD4+ T cells?

Q6: I suggest that the original Western Blot data should be provided in supplementary materials.

Q7: There are some Chinese characters in the 3 Discussion part of Revised Manuscript with Track Changes.

Reviewer #4: Comments to the Authors�

First, I would like to declare that I have reviewed the Revised Manuscript with Track Changes. The manuscript by Fangwei Xu et al., titled "Study on the Effect of Modified Liu-an Decoction on Th1/Th2 Function in Guinea Pigs with Cough Variant Asthma through the Notch Signal Pathway", concludes that the potential mechanism of Modified Liu-an Decoction (MLAD) in treating CVA involves regulating the dynamic balance of Th1/Th2 by modulating the expression of Notch pathway-related proteins, thereby alleviating chronic airway inflammation and improving airway remodeling. I find this study highly significant, but there are issues with its design and manuscript presentation that I hope the authors can address and improve.

Comments:

1.The study design includes six groups labeled A-F, where Group A is the blank control group, Group B is the model group, and the remaining groups (C-F) can be referred to as model drug groups (MS group and MLAD groups). I believe the primary role of Group A is to validate the successful modeling of Groups B-F. The authors also indicate that the animal model is mainly evaluated based on behavioral performance, with the effective observation indicators being 1.6.1-1.6.2. When analyzing the experimental data, considering the influence of a single factor, comparisons between the blank control group and the model group, as well as between the model group and the model drug groups, are statistically meaningful. However, in the description of the experimental results, there are comparisons between the blank control group data and the model drug group data, such as in 2.1 regarding diet and weight, 2.2 regarding airway sensitivity, 2.3 regarding airway hyperresponsiveness, and 2.6 regarding lung tissue of guinea pigs in each group.

2.The manuscript exhibits a lack of precision in its presentation. For instance, in the Introduction, the phrase "[7]" is used; in Section 1.2, the notation "AI(OH)3" appears; in Section 1.3, the text states "divided into 5 groups"; in Section 1.5, there is a duplication of "E group" and an omission of "F group"; in Section 1.7, the term "mean±standard" is employed; and in Sections 2.4 and 2.7, the expression "the normal group" is utilized. These inaccuracies and inconsistencies require correction.

3.Certain paragraphs are overly lengthy and would benefit from appropriate segmentation. For example, in the Discussion, the second paragraph regarding Airway Inflammatory, as well as the sections on Airway Hyperresponsiveness and airway remodeling, could be restructured for improved clarity and readability.

4.In the Introduction, the authors state, "Through our previous animal experimental studies, we discovered that MLAD could reduce the levels of serum tumor necrosis factor-α (TNF-α) and interleukin-5 (IL-5) in CVA rats, reduce the number of eosinophils (EOS) in the bronchoalveolar lavage fluid (BALF), reduce airway inflammation and airway collagen deposition, thereby improving the pathological state of airway remodeling and reducing cough frequency." However, in the Results section of the Abstract, the authors mention, "Compared with the model group, MLAD reduced the inflammatory infiltration and collagen deposition in the lungs of CVA guinea pigs (P<0.05)." This raises the question of whether the same data has been reported in multiple publications.

5.Due to the current font used, certain details in the figures are difficult to discern, particularly in Fig. 2, Fig. 3, and Fig. 4. It is recommended to enhance the clarity and resolution of these figures to ensure accurate interpretation of the data.

**********

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Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2025 Sep 15;20(9):e0327356. doi: 10.1371/journal.pone.0327356.r004

Author response to Decision Letter 2

29 May 2025

Thanks for the valuable suggestions for changes, not required at this time

Attachment

Submitted filename: The Reply to reviewers and the Editor.docx

pone.0327356.s004.docx^{(16.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0327356.r005

Decision Letter 2

Misbahuddin Rafeeq

15 Jun 2025

Study on the Effect of Modified Liu-an Decoction on Th1/Th2 Function in Guinea Pigs with Cough Variant Asthma through the Notch Signal Pathway

PONE-D-24-12851R2

Dear Dr. Fangwei XU,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0327356.r006

Acceptance letter

Misbahuddin Rafeeq

PONE-D-24-12851R2

PLOS ONE

Dear Dr. XU,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: REVIEWER REPORT.pdf

pone.0327356.s001.pdf^{(185.8KB, pdf)}

Attachment

Submitted filename: The Reply to reviewers and the Editor.docx

pone.0327356.s004.docx^{(16.2KB, docx)}

Data Availability Statement

The data from this studyis available from the Dryad database under the registration number DOI: https://doi.org/10.5061/dryad.x95x69pxg.

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PERMALINK

Study on the effect of modified Liu-an decoction on Th1/Th2 function in Guinea pigs with cough variant asthma through the Notch signal pathway

Fangwei Xu

Jian Deng

Ningning Zhang

Kimberly XinTing Leow

Panpan Li

Chen Lu

Yuhang Chen

Ye Zhang

Liqun Wu

Roles

Abstract

Objective

Methods

Results

Conclusion

1. Introduction

2 Materials and methods

2.1 Experimental animal

2.2 Experimental drug

Table 1. The full scientific species (Latin binomial nomenclature) names of all ingredients of Modified Liu-an Decoction.

2.3 Experimental grouping

2.4 Model replication

2.5 Administration method and dosage

2.6 Observation index

2.6.1 The general condition of CVA Guinea pigs.

2.6.2 Airway sensitivity of CVA Guinea pigs.

2.6.3 Airway resistance of CVA Guinea pigs.

2.6.4 Detection of IL-2, IL-4, IL-12 and IL-13 levels in serum and BALF by ELISA.

2.6.5 Lung histopathology scoring.

Table 2. Histopathological Scoring System for Evaluating Changes in Pulmonary Inflammation in Guinea Pigs with Antigen Challenge.

2.6.6 Detection of Delta1, Jagged1, Notch1, and NICD proteins in lung tissue by Western blot.

2.7 Statistical methods

3. Experimental results

3.1 General condition of guinea pigs in each group

Table 3. Changes in Body Weight (g) of Guinea Pigs in each group before and after modeling.

3.2 MLAD reduces airway sensitivity and airway resistance in cva guinea pigs

Table 4. Number of coughs of Guinea Pigs in each group after modeling.

Table 5. Comparison of the Area under the Total Airway Resistance Curve (RL-area) under Different Doses of Methacholine Challenge Test (cmH2O/mL).

Fig 1. Changes in airway resistance after excitation with different concentrations of acetylmethacholine.

Fig 2. Airway resistance curve during airway excitation in guinea pigsNote.

3.3 MLAD regulates Th1/2 cytokine expression levels

Fig 3. Concentration of cytokines in the serum of guinea pigs in each group.

3.4 Comparison of IL-2, IL-12, IL-4 and IL-13 levels in BALF of guinea pigs

Fig 4. Cytokine concentration in BALF of guinea pigs in each group.

3.5 MLAD attenuates airway structural damage in cva guinea pig lung tissue

Fig 5. HE staining results of lung tissue of guinea pigs in each group (×200,scale = 50μm).

Fig 6. Histopathologic inflammation grading scores of guinea pig lungs in each group.

3.6 MLAD inhibits NOTCH pathway expression

Fig 7. Protein expression of Delta1, Jagged1, Notch1, and NICD in lung tissues by Western blot assay.

Fig 8. Semi-quantitative analysis of Delta1, Jagged1, Notch1, and NICD protein levels in lung tissues.

4 Discussion

4.1 Airway inflammatory

4.2 Airway hyperresponsiveness

4.3 Airway remodeling

5 Conclusion

Data Availability

Funding Statement

References

Decision Letter 0

Misbahuddin Rafeeq

Roles

Author response to Decision Letter 1

Decision Letter 1

Misbahuddin Rafeeq

Roles

Author response to Decision Letter 2

Decision Letter 2

Misbahuddin Rafeeq

Roles

Acceptance letter

Misbahuddin Rafeeq

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Table 5. Comparison of the Area under the Total Airway Resistance Curve (RL-area) under Different Doses of Methacholine Challenge Test (cmH₂O/mL).