Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

LV Chuan, ZHU Hui-zhi , LIU Xiang-guo, CAO Xiao-mei, XIA Yong-qi, ZHANG Qiuping, YU Zi-qi

1. The First Clinical Medical School of Anhui Univ Chinese Medicine, Hefei 230031, China

2. The First Affiliated Hospital of Anhui Univ Chinese Medicine, Hefei 230031, China

3. Anhui Univ Chinese Medicine, Hefei 230031, China

4. School of Chinese Medicine, Hefei 230032, China

Keywords:

ABSTRACT Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis.Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals.After 4 weeks, OVA atomization and gavage were used for stimulation and treatment.Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74,3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline.HE,PAS and Masson staining were used to observe the lung histopathological changes in rats.The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA.The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot.Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue.Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious.The expressions of IL-6, IL-23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01); Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased(P< 0.01).The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01).Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

1.Introduction

Bronchial asthma (BA) is one of the common chronic lung diseases, which seriously affects the quality of life of patients and brings a great burden to the field of health[1].The pathological features of asthma mainly include airway inflammation and airway remodeling[2, 3].Airway inflammation is the essence of asthma.Airway remodeling is the main cause of the occurrence,development and deterioration of the disease.It is characterized by goblet cell proliferation, collagen fiber deposition, smooth muscle cell proliferation and mucus secretion[4].It has been found that JAK2/ STAT3 signaling pathway is closely related to the occurrence of airway remodeling in asthma[5].IL-6, as its upstream factor, can participate in it by activating JAK2 / STAT3 signaling pathway.

The choice of western medicine in the treatment of asthma is relatively limited.Glucocorticoid is the first choice, which has a certain effect, but the long-term use of side effects is large, and the airway remodeling process cannot be reversed.Tradit Chin Med in the treatment of asthma has significant curative effect, distinctive characteristics, small side effects, and great potential for clinical application.Its role and related mechanism are worthy of further exploration and excavation.Yanghe Pingchuan Granule is a national famous old Chinese medicine professor Hu Guojun on the basis of‘Chuntian medical dialect’ Yanghe Decoction, and summarized in clinical diagnosis and treatment, and finally formed a prescription.It has been used in the treatment of asthma for many years, and the curative effect is obvious[6].Previous animal studies have shown that Yanghe Pingchuan Granules can reduce the secretion and proliferation of airway epithelium, goblet cells and airway mucus[7,8,9], thereby alleviating airway remodeling in asthmatic rats.However, whether it can regulate the mechanism of asthma airway remodeling by regulating the IL-6 / JAK2 / STAT3 signal axis has not been studied in depth.Therefore, the purpose of this study is to explore the mechanism of Yanghe Pingchuan Granule in preventing and treating asthma airway remodeling based on the IL-6 / JAK2 /STAT3 signal axis, and to provide further theoretical basis and data support for clinical treatment.

2.Materials and methods

2.1 Animals

Forty-two male SPF SD rats, aged 7-8 weeks, weighing 230-250 g, Zhejiang Weitong Lihua Experimental Animal Company.The feeding environment of rats : alternating light and dark for 12 h,indoor average temperature ( 20 ± 2 ) ℃, humidity 50 % ± 5 %, free eating and drinking water.All operations have been approved by the Animal Ethics Committee of Anhui Univ Tradit Chin Med ( Batch No.: AHUCM-rats-2019007 ).

2.2 Drugs and reagents

Drug composition : Mahuang 6g, Wuweizi 6 g, Shudi 15 g,Bajitian 10 g, Xuanfuhua 9 g, Tinglizi 10 g, Jiegeng 10 g, Baijiezi 6 g, Danggui 10 g, purchased from the preparation center of Anhui Provincial Hospital of Tradit Chin Med, in the form of compound decoction-free granules, 10 g / bag, 2.7 g crude drug per gram,production approval number : 21050059 ; ovalbumin ( Beijing Green Biotechnology Co., Ltd., Batch No.AP0028 ).Hematoxylin staining solution, PAS staining kit, Masson trichrome staining solution (Ebiogo, Batch No.: 05092312, 06092310, 05162314 ) ; IL-6, IL-23, IL-17A kit ( Wuhan GeneMed, Batch No.20230810 ) ;JAK2 antibody ( bioss company, batch number : AD19051026 ), P-JAK2,STAT3, P-STAT3 antibody ( abcam company, batch number :GR3215065-4, GR3105216-7, GR3380060-4 ) ; trizol ( Life technogies, Batch No.: 420810 ) ; dEPC-H2O ( Generay Biotech,lot number : JD721229 ) ; reverse Transcription Kit ( TaKaRa Company, lot number : AM62082A ).

2.3 Instruments

Ultrasonic nebulizer (Guangdong Yuehua Medical Device Co., Ltd.); microscope (OLYMPUS); centrifuge (Anhui Jiawen Instrument Co., Ltd.); electrophoresis instrument, electrophoresis tank (Shanghai Tianneng Company); pCR instrument (Thermo Scientific).

2.4 Grouping, modeling and administration of animals

Forty-two SD male rats were adaptively fed for 7 days.They were divided into normal control group, model group, Yanghe Pingchuan low, medium and high dose groups and dexamethasone group according to the random number table method, with 7 rats in each group.The rat model of asthma was replicated[10], which was divided into two stages : 2 weeks of sensitization and 4 weeks of stimulation.1 Sensitization : In addition to the normal control group,the other groups were given intraperitoneal injection of 10 % OVA +aluminum hydroxide suspension on the 0th and 7th day, 1ml per rat ;2.Excitation : 1 % OVA was atomized from the 14 th day, once a day,30 minutes each time, for 4 weeks.Administration : administered half an hour before each atomization.According to the method of human and rat body surface area[11], the low, medium and high doses of Yang and Pingchuan were calculated to be 3.87,7.74 and 15.48 g∙kg-1, respectively, and 10 mL∙ kg-1was given by gavage.From the 14 th day, the Yanghe Pingchuan low, medium and high dose groups were given intragastric administration once a day, and the dexamethasone group was given 0.0625 mg∙kg-1dexamethasone[12]once a day for 4 weeks.The rats showed obvious respiratory and systemic symptoms such as nodding breathing, shortness of breath,irritability, yellow hair, and weight loss, indicating that the asthma model was successfully replicated[13].

2.5 The lung histopathological changes were observed by HE,PAS and Masson staining

Take the right lung, fixed, made into slices, placed in a drying oven at 60 ℃ for 30 min.Slice dewaxing, descending ethanol into water.HE staining : conventional staining, dehydration, transparency,sealing, observe the degree of inflammatory cell infiltration,bronchial lumen stenosis and secretion changes and take pictures.2 PAS staining : Schiff staining solution was added dropwise,incubated, rinsed with running water, re-stained with hematoxylin,rinsed, dehydrated, and transparentized with xylene.After mounting,the proliferation of goblet cells in rat lung tissue was observed under light microscope and photographed.3 Masson staining : Bouin solution was added dropwise, incubated, rinsed with running water,stained with azure blue and hematoxylin, re-stained with aniline blue staining solution, dehydrated and mounted, and the deposition of subcutaneous collagen fibers was observed.

2.6 The levels of IL-6, IL-23 and IL-17A in serum of rats were detected by ELISA

The levels of IL-6, IL-23 and IL-17 A in the supernatant of abdominal aortic blood were detected.

2.7 The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in rat lung tissues were detected by Western blot

A total of 0.1 g of right lung tissue sample was taken and lysed with cell lysis buffer.After centrifugation for 15 min, the supernatant was collected, and the protein concentration was measured by BCA method.After the buffer was diluted, it was denatured by boiling water bath heating.Sample loading, electrophoresis ; pDVF membrane transfer ; after the completion of the closed 2 h ; primary antibody incubation ( JAK2, STAT3 dilution ratio of 1 : 1000,P-JAK2, P-STAT3 dilution ratio of 1 : 2 000 ) overnight, PBST washing 3 times ; the secondary antibody was added at 1 : 20 000,incubated for 1.2 h, and washed three times with PBST.The relative expression of the target protein was obtained by statistical analysis using Image J software.

2.8 mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissues were detected by qRT-PCR

The right lung tissue was cut and ground into powder,total RNA was extracted by Trizol method, and the RNA was reversely transcribed into cDNA according to the instructions of the kit, and then amplified by PCR.The priming sequence of β-actin (150bp) was as follows: upstream 5 ‘-CCCATCTATGAGGGTTACGC-3’,downstream5’-TTtaatGTCacGcacGATC-3’;IL-6(98bp) primer sequence:upstream 5’-GCCTTCTTGGGACTGATGTT-3’,downstream 5 ‘-ACTGGTCTGTTGTGGGTGGT-3’; JAK2(182bp) primer sequence: upstream 5 ‘-ATAGATGAGTCAACCAGGCA-3’,downstream 5’-CCATGAACAAAATCATGCCG-3’; STAT3 (115)primer sequence: upstream 5 ‘-GCAATACCATTGACCTGCCG-3’,downstream 5 ‘-AACGTGAGCGACTCAAACTG-3’.The reaction conditions of PCR amplification were as follows: predenaturation at 95 ℃ for 1min, annealing at 60 ℃ for 1min, extension at 72℃ for 30 seconds, and a total of 40 cycles were performed.The relative mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissues were calculated by 2-△△Ctmethod with β-actin as the internal reference.2-△△Ctmethod: (1) calculation of experimental group △Ct value: △Ctexperiment=Ctobjective-Ctinternalreference; (2) The control group △Ct value was calculated: △Ctcontrol=Ctobjective-CTinternalreference; (3) Calculated relative expression: △△Ct=△Ctexperimental-△Ctcontrol, relative expression =2-△△Ct.

2.9 Statistical Methods

SPSS 26.0 was used for statistical analysis of the data.The measurement data were expressed as(±s ), and one-way analysis of variance was used for comparison between groups.P < 0.05 indicated that the difference was statistically significant.

3.Result

3.1 Effect of Yang Heping Chuan Granule on lung histopathology in rats

There was no obvious abnormality in the lung tissue of the normal control group, and the alveolar cavity was clean without exudation.Compared with the normal control group, the lung tissue of the model group showed inflammatory cell infiltration, goblet cell proliferation, increased mucus secretion, and increased subepithelial collagen fiber deposition.Compared with the model group, the degree of inflammatory cell infiltration and secretion generation in each administration group were improved to varying degrees, and goblet cell proliferation, mucus secretion and subepithelial collagen fiber deposition were significantly improved.See Figure 1.

3.2 Effects of Yanghe Pingchuan Granules on serum levels of IL-6, IL-23 and IL-17A in rats

Compared with normal control group, the levels of IL-6, IL-23 and IL-17A in model group were significantly increased (P < 0.01).Compared with the model group, the levels of IL-6, IL-23 and IL-17A in the administration group were significantly decreased (P <0.01), especially in the high-dose group of Yangping Asthma.See Table 1.

2.3 Effects of Yanghe Pingchuan Granules on the expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 proteins in rat lung tissue

Compared with normal control group, the expressions of JAK-2,P-JAK2, STAT3 and P-STAT3 in model group were significantly increased (P < 0.01).Compared with model group, the expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in all drug administration groups were significantly decreased (P < 0.01).See Table 2, Figure 2.

Fig 1 Effect of Yanghe Pingchuan Granules on pathological morphology of lung tissue from rats(50×)

Tab 1 Effect of Yanghe Pingchuan Granules on levels of IL-6、IL-23、IL-17A in serum of rats(n=7, ±s)

Tab 1 Effect of Yanghe Pingchuan Granules on levels of IL-6、IL-23、IL-17A in serum of rats(n=7, ±s)

Note: Compared with normal control group **P < 0.0; Compared with model group ##P < 0.01

组别 剂量/g∙kg-1 IL-6/pg∙mL-1 IL-23/pg∙mL-1 IL-17A/pg∙mL-1正常对照组 - 102.41±6.05 92.83±10.96 153.5±11.98模型组 - 276.14±10.752** 463.70±15.932** 427.90±8.422**阳和平喘低剂量组 3.87 205.05±4.784## 404.92±4.194## 374.58±7.444##阳和平喘中剂量组 7.74 174.04±7.004## 304.94±7.894## 325.03±10.334##阳和平喘高剂量组 15.48 147.39±3.764## 250.09±5.834## 261.00±6.834##地塞米松组 6.25×10-5 158.51±3.664## 267.76±14.564## 286.55±9.434##F 498.08 867.06 640.70 P<0.01 <0.01 <0.01

Fig 2 Effect of Yanghe Pingchuan Granules on the protein expression of JAK-2、P-JAK2、STAT3 and P-STAT3 in lung tissue of rats

2.4 Effects of Yanghe Pingchuan Granules on mRNA levels of IL-6, JAK2 and STAT3 in lung tissue of rats

Compared with normal control group, mRNA levels of IL-6, JAK2 and STAT3 in model group were significantly increased (P < 0.01).Compared with the model group, the mRNA levels of IL-6, JAK2 and STAT3 in all administration groups were significantly decreased(P < 0.01), and the high dose group of Yang and asthma was the closest to the normal control group.See Table 3.

Tab 2 Effect of Yanghe Pingchuan Granules on the protein expression of JAK-2、P-JAK2、STAT3 and P-STAT3 in lung tissue of rats(n=7, ±s)

Tab 2 Effect of Yanghe Pingchuan Granules on the protein expression of JAK-2、P-JAK2、STAT3 and P-STAT3 in lung tissue of rats(n=7, ±s)

Note: Compared with normal control group **P < 0.01 ; Compared with model group ##P < 0.01.

组别 剂量/g∙kg-1 JAK2 P-JAK2 STAT3 P-STAT3正常对照组 - 0.14±0.03 0.12±0.02 0.20±0.02 0.09±0.02模型组 - 0.87±0.042** 0.82±0.062** 0.94±0.072** 0.74±0.022**阳和平喘低剂量组 3.87 0.69±0.054## 0.61±0.064## 0.73±0.054## 0.54±0.044##阳和平喘中剂量组 7.74 0.56±0.064## 0.53±0.044## 0.60±0.044## 0.40±0.084##阳和平喘高剂量组 15.48 0.46±0.044## 0.38±0.054## 0.46±0.034## 0.27±0.064##地塞米松组 6.25×10-5 0.43±0.054## 0.36±0.074## 0.43±0.054## 0.28±0.074##F 84.29 67.20 88.68 53.11 P<0.01 <0.01 <0.01 <0.01

Tab 3 Effect of Yanghe Pingchuan Granules on the mRNA expression of IL-6, JAK2 and STAT3 in lung tissue of rats(n=7,±s)

Tab 3 Effect of Yanghe Pingchuan Granules on the mRNA expression of IL-6, JAK2 and STAT3 in lung tissue of rats(n=7,±s)

Note: Compared with normal control group **P < 0.01 ; Compared with model group ##P < 0.01.

组别 剂量/g∙kg-1 IL-6/2-△△Ct JAK2/2-△△Ct STAT3/2-△△Ct正常对照组 - 1.00±0.53 1.01±0.12 1.00±0.05模型组 - 2.80±0.432** 7.24±0.852** 1.75±0.062**阳和平喘低剂量组 3.87 1.67±0.054## 3.76±0.324## 1.41±0.024##阳和平喘中剂量组 7.74 1.43±0.054## 2.96±0.164## 1.32±0.064##阳和平喘高剂量组 15.48 1.19±0.064## 1.61±0.304## 1.18±0.034##地塞米松组 6.25×10-5 1.13±0.064## 1.60±0.294## 1.15±0.054##F 77.84 181.70 193.41 P<0.01 <0.01 <0.01

3.Discussion

As a chronic respiratory disease, the mechanism of asthma mainly includes airway inflammation and airway remodeling, which are closely related.Airway remodeling is mainly caused by airway smooth muscle cell proliferation and airway wall thickening caused by changes in the cell structure, composition and function of the airway wall[14], which is considered to be an important factor in the recurrence of asthma[15].In the process of asthma,inflammatory cells in airway epithelial tissue continue to release a variety of cytokines, chemokines and other pro-inflammatory factors, prompting epithelial tissue damage-repair-re-damage-repair process [16], eventually leading to airway remodeling is an important pathological basis for the occurrence and development of asthma.

Yanghe Pingchuan Granules have the effects of tonifying kidney,activating blood and resolving phlegm.In the prescription, ephedra xuanfei pingchuan, diuresis and detumescence are the first important drugs for opening the lung and treating the sense ; inula britannica can reduce qi, reduce phlegm and asthma, Tinglizi can reduce lung qi, reduce phlegm and asthma.The two drugs are used together to help ephedra to open lung qi, reduce phlegm and asthma, and reduce lung qi to prevent lung qi from being too much.White mustard benefit qi and eliminate phlegm, platycodon grandiflorum regulate qi and eliminate phlegm, two drugs float, Xuantong lung qi, so that phlegm with qi up and down solution ; bajitian tonify kidney yang,prepared rehmannia nourishing yin and blood, two drugs tonify kidney yin and kidney yang, accompanied by schisandra astringent,tonifying kidney qi, three drugs tonify kidney qi, blood, yin and yang, enhance the kidney to absorb lung qi to achieve the function of relieving asthma ; angelica reconciles qi and blood, and blood harmony can reduce qi, so that qi and blood are not scattered and have no return.The combination of various medicines plays the role of warming the kidney and ventilating the lung, resolving phlegm and reducing qi, and activating blood and regulating the camp.In this study, a rat asthma model was established, and different doses of Yanghe Pingchuan Granules were given for intervention.The overall state of body weight, coat color and spirit of rats was significantly improved, and lung inflammation, goblet cell hyperplasia and collagen fiber deposition were significantly reduced, suggesting that Yanghe Pingchuan Granules can improve the pathological changes of lung in asthma model rats and reduce lung injury.

Studies have shown that the activation of IL-6/JAK2/STAT3 signaling axis is involved in many aspects of the occurrence and development of asthma[17,18].Inhibition of its activation can alleviate airway inflammation, oxidative stress and airway remodeling in asthma.In the process of asthma, IL-6 can bind to the receptor IL-6R on the cell surface and exist in the form of a complex.The complex then binds to the membrane glycoprotein gp130 to form a new complex and induces phosphorylation of JAK2 kinase, which in turn promotes the expression of its downstream STAT3[19].Activated STAT3 further activates the key nuclear factor RORγt of Th17 cell differentiation and promotes Th17 to secrete inflammatory factors such as IL-17A[20].STAT is a signal transducer of various cytokines and growth factors in mammals.It plays a key role in regulating the homeostasis, differentiation and cell function of immune cells, and is involved in the occurrence of asthma[21,22].STAT is a downstream target of JAK, and its key regulation is mediated by phosphorylation, usually completed by JAK2[23].Airway smooth muscle cell proliferation is one of the typical characteristics of airway remodeling.Studies have found that JAK2 can promote the proliferation and migration of airway smooth muscle cells in asthmatic patients by up-regulating the expression of miR-375[24].Relevant studies have shown that STAT3 can promote the proliferation of airway smooth muscle, and inhibiting its expression can significantly improve the quality of life of asthmatic patients[25,26].The results of this study showed that the expression levels of IL-6, JAK2 and STAT3 in the model group increased significantly, indicating that the airway remodeling of the model group was aggravated under the persistent airway inflammation stimulation.Compared with the model group, the expression of JAK2 and STAT3 in each administration group decreased, and the expression of IL-6 also decreased, suggesting that Yanghe Pingchuan Granules may inhibit the conduction of JAK2 / STAT3 signaling pathway by inhibiting IL-6, thereby reducing airway smooth muscle proliferation and alleviating airway remodeling.

IL-6, IL-23 and IL-17 A are common inflammatory factors in various inflammatory reactions and are closely related to airway remodeling in asthma.As an important pro-inflammatory factor in the body, IL-6 can determine the type of adaptive immunity,regulate CD4 + T cells and reduce the differentiation of Th1 cells.At the same time, IL-6 can also inhibit Treg cells and promote Th17 differentiation, thereby inducing airway remodeling in asthma[27].In addition, analysis of human bronchial tissue samples showed that IL-6 was involved in airway remodeling during asthma[28].IL-23 is an inflammatory mediator produced by activated dendritic cells, macrophages and monocytes, which can promote the growth and proliferation of activated Th17 cells, and then secrete inflammatory mediators such as IL-17A[29,30].IL-17A is a major member of the IL-17 family, which can promote the aggregation of a variety of inflammatory cells, thereby promoting the occurrence of inflammation[31].When Th17 / Treg immune imbalance occurs,Th17 cells, as the goalkeeper of mucosal dynamic balance, can promote fibroblast deposition by secreting IL-17A, which in turn leads to airway remodeling[32].The results of this study showed that the expression levels of IL-6, IL-23, and IL-17A in the model group were significantly increased, indicating that the inflammatory response was an important part of the process of asthma.After administration, the pathological damage of lung tissue in each group was reduced, and the expression levels of IL-6, IL-23, and IL-17A were reduced to varying degrees, with the most significant reduction in the high-dose group of Yanghe Pingchuan Granule, indicating that Yanghe Pingchuan Granule alleviated airway inflammation and improved airway remodeling in asthmatic model rats by inhibiting the release of IL-6, IL-23, and IL-17A inflammatory factors.

In summary, Yanghe Pingchuan Granules may inhibit the activation of IL-6 / JAK2 / STAT3 signaling axis, reduce the expression of inflammatory factors in the lungs, reduce goblet cell formation and collagen fiber proliferation, inhibit airway smooth muscle proliferation, and ultimately achieve the purpose of improving airway remodeling in asthma.In the future, our research group will apply relevant pathway inhibitors to further explain the specific mechanism of this pathway in the improvement of asthma by Yanghe Pingchuan Granules.

This article has no conflict of interest.

Author ‘s contribution : Lv Chuan is responsible for the design and writing of the article ; lv Chuan, Xia Yongqi and Yu Ziqi were responsible for the implementation of animal experiments and data collection.Cao Xiaomei and Zhang Qiuping were responsible for experimental management and data analysis.Zhu Huizhi and Liu Xiangguo are responsible for the quality control and revision of the article.