HONG Mei, Wang Guang-yao, PEI Kai, PENG Si-min, WANG Lin, ZHANG Yanyan, XU Guang-lan?

1.Guangxi University of Traditional Chinese Medicine, Nanning 530200, China

2.The First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine, Nanning 530023, China

ABSTRACT Objective: To explore the mechanism of Qingjin Huatan Decoction in regulating iron metabolism by regulating inflammatory factors in the inflammatory environment of chronic obstructive pulmonary disease.Methods: The COPD model was prepared by tracheal drops of lipopolysaccharide (LPS) combined with smoke.40 rats were randomly divided into normal group, COPD group, roxithromycin western medicine group and Qingjin Huatan Decoction traditional Chinese medicine group (n=10).Post-molding drug administration intervention,Qingjin Huatan Decoction Chinese medicine group was given 14.62 g/kg (Qingjin Huatan Decoction), roxithromycin western medicine group was given 0.015 75 g/kg (roxithromycin),COPD group and normal group were given normal saline 10 mL/kg, twice a day.The expressions of Hepcidin, IL-1β, IL-10 and TGF-β1 in serum of rats in each group were detected by ELISA.WesternBlot and qRT-PCR were used to detect the expression of TF and IREB2 protein and mRNA in lung tissues of each group.Results: Compared with the normal group, the contents of serum Hepcidin and IL-10 in COPD group were significantly decreased(P＜0.01), while the contents of serum IL-1β and TGF-β1 were significantly increased(P＜0.01, P＜0.05).Compared with COPD group, the contents of serum Hepcidin and IL-10 were significantly increased in the two drug groups (P＜0.01), while the contents of serum IL-1β and TGF-β1 were significantly decreased in the two drug groups (P＜0.05).Compared with normal group, TF mRNA and protein expression in COPD group were increased (P＜0.01),while IREB2 mRNA and protein expression were decreased (P＜0.01).Compared with COPD group, TF mRNA and protein expressions in lung tissues of the two-drug group were decreased(P＜0.01, P＜0.05), while IREB2 mRNA and protein expressions were increased (P＜0.01).Conclusion: Iron metabolism is related to inflammation.Qingjin Huatan Decoction can regulate iron metabolism in inflammatory environment to treat COPD.

Keywords:

Qingjin Huatan Decoction

Chronic obstructive pulmonary

disease

Iron metabolism

Inflammation

Traditional Chinese medicine

1.Introduction

Chronic obstructive pulmonary disease (COPD) is recognized as a serious debilitating inflammatory lung diseases associated with smoking, its current cannot be cured and limited life.Chronic airway inflammation and airway epithelial cell injury are the main pathological features and important pathological basis.In the course of disease development, a large number of inflammatory cells gather, infiltrate lung tissue and airway, release inflammatory mediators, form an inflammatory environment, and slowly destroy lung tissue structure, causing a series of clinical manifestations.

Iron is one of the essential trace elements in the body.It is mainly produced by the liver, but a small amount of iron is expressed in the lung.Iron is closely related to the normal division, proliferation,growth and death of cells in the body.When the iron supply in the body is maintained at the required level, it can support the functional stability of a single cell or even the whole body.The transport and utilization of iron in the body has a special and complex mechanism, which depends on maintaining iron homeostasis in the whole body[1].Iron, as one of the most common catalysts for the production of reactive oxygen species[2], often leads to oxidative stress toxicity through reactive oxygen species in the process of reaction, and then participates in the development of inflammation,degenerative diseases, cancer and other diseases[3].A large number of studies have shown that during the acute episode of COPD, the body releases a large number of inflammatory mediators that can destroy lung iron homeostasis.It leads to compensatory changes in the expression of iron-related proteins such as Transferrin (TF) and Ironregulatoryprotein2 (IREB2)[4].

At present, TCM treatment of COPD has been extensively studied[5,6].Qingjin Huatan decoction from "miscellaneous disease Guangyao", by catharsis lung heat of scutellaria, gardenia and mulberry white bark;Fritillaria, fructus trichosanthes and platycodon for removing heat and promoting phlegm;Nourishing lung Yin,cough moistening lung ophiopogon, bosom mother; Transport spleen dampness of poria cocos; Shun qi and eliminate phlegm of orange red; The glycyrrhiza, a total of 11 herbal composition, has the effect of clearing heat and eliminating phlegm, suppressing cough.Combined with the previous studies of our research group and those of other domestic researchers, it is clear that Qingjin Huatan Decoction has an obvious effect on alleviating the inflammatory reaction of COPD[7-11].In this study, phlegm-heat stagnation lung model of COPD rats was prepared, and the expression of Hepcidin,TF and IREB2 regulated by Qingjin Huatan Decoction through inflammatory factors IL-1β, IL-10 and TGF-β1 was observed, and the mechanism of its treatment of COPD was summarized, so as to broaden the thoughts and direction for clinical treatment of COPD.

2.Materials

2.1 Animal

There were 40 SD (Sprague-Dawley) rats, weighing 180-200 g, Animal Qualification certificate (SCXK (Hunan) 2019-0004),Animal Ethics (DW20220511-099).

2.2 Drugs

Qingjin Huatan Decoction Qingjin Huatan Decoction (no decoction granules) composition:Radix Skullcap 15 g (batch number,2108021C), Radix Platycodon platycodon 15 g (batch number,2109007S), Ophiopogonis japonicus (batch number, 2110021C)10g(batch number, 21100212s), Fructus jasminoides 15 g (batch number, 2109002S), Fritillaria Fritillaria 10g (batch number,2110004C), Poria cocos 10 g (batch number, 2110006S),Mulberry white bark 12g (batch number, 2103002S), orange red 15g (batch number, 2108005S), Zhimu 12g (batch number, 2104001C), Fructus Trichosanthes kernel (fried)15 g (batch number, 2110005C),Licorice 10 g (batch number, 2104001C)2109006C) Pharmaceutical granules were purchased from the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine.The particles were thoroughly mixed according to the above dosage and dissolved in ultra-pure water.The final concentration of the liquid was 2 g/mL.The dissolved Chinese medicine liquid is stored in the refrigerator (-20℃) and heated to room temperature by microwave oven when necessary.Western medicine roxithromycin Dispersive Tablets (Harbin Pharmaceutical Group, lot number H19990031).

2.3 Main reagents

Cigarettes (Zhenlong Brand, China Tobacco Guangxi Industry Co., LTD.);Lipopolysaccharide (LPS, Sigma10 mg);Murine monomolecular antibodies against TF, IREB2 and β-actin (AbVang-A1448, AbVang-A6382 and AbVang-AC004);ABclonal AS014 for Goat against rabbit secondary antibody;ECL Chemiluminescent Substrate (Ultra Sensitive) (biosharp,BL520A);qPCR Master MIX(promega,A6001);EastepTM Super Total RNA Extraction Kit(promega,LS1040);Immunosorbent assay (ELISA) detection kit(Interleukin-10, Interleukin -1β, transforming growth factor -β1)(provided by Hangzhou Like), Hepcidin ELISA kit (provided by Rising Biotics).

2.4 Main instrument

Micropipette (Eppendrof, model 3120000.216);Centrifuge(Tianmei, model NU-C200R-E);Full-wavelength enzyme label instrument (Biotek Eooch2);Chemiluminescence Imager (Bio-Rad);Fluorescence quantitative PCR instrument (Roche SW-CJ-2D model).

3.Methods

3.1 Preparation and administration of COPD model rats

Combined with the previous modeling summary of the research group, LPS+ smoke was combined to prepare COPD rat models[9].Among the total 40 rats, 30 rats were randomly assigned for modeling, and the remaining 10 rats were normal group.LPS was injected at d1 (the first day of modeling, same as below), d14 and 26 d.LPS was mixed with sterile normal saline into 2 g/mL.After anesthesia, the limbs were fixed with medical glue, the incisor teeth were fixed with thin rubber bands, and the rats were kept in supine position.The neck skin of the rat was wiped with iodine to expose the required parts of the operation.A small incision of about 1.5 cm was made in the middle of the neck of the rat with a sterile knife, and the subcutaneous tissue was bluntly removed to expose the white annular trachea.The rat plate was padded, LPS solution was pushed into 200 μL through the trachea with a syringe, and then shaken for 1-2 min.The wound was quickly and gently sutured, wiped and disinfected with iodine, and placed in a clean rat cage.The whole operation should ensure aseptic conditions.On the second day after each operation, the rats were placed in a transparent smoking chamber (50 cm×50 cm×50 cm), in which 20 cigarettes were put.After lighting the chamber, the lid was closed for timing and smoking, 45min each time, twice a day, with a total period of 30 d.

3.2 Group administration

Thirty rats were randomly divided into COPD group, roxithromycin western medicine group and Qingjin Huatan Decoction Chinese medicine group, with 10 rats in each group.Experimental rats were given daily dose and intragastric administration according to relevant literature[12].Qingjin Huatan Decoction group was given Qingjin Huatan Decoction 15 g/kg, roxithromycin group was given roxithromycin 0.015 75 g/kg, COPD group and normal group were given 20 mL/kg/d normal saline.The time of each intragastric administration was 9 a.m.and 21 p.m., twice a day, often for 2 weeks.

3.3 Indicator Detection

3.3.1 Detection of HE staining

After the modeling, appropriate amount of lung tissue (n=3) was taken from rats in each group and fixed with 4% paraformaldehyde tissue was given.Then 3-5 pathological sections were prepared by steps such as dehydration, transparent treatment, paraffin embedding section and batik decolorization, and the results were observed under a microscope.

3.3.1.1 Inflammation scores of pathological sections of rat lung tissue

Pathological pictures of rats in each group were observed, and scores were performed to judge the degree of inflammatory response in rats according to the method of Liu Junbo, Kong Yanhua, Cao Shiying et al.[13-15].The total pathological score was performed based on the degree of inflammatory cell infiltration, degree of emphysema (alveolar wall thinning, lung dilation and fusion), and degree of mucus secretion, and the score was shown in Table 1.

Tab 1 Evaluation of inflammatory response in rats (points)

3.3.2 The contents of Hepcidin, IL-1β, IL-10 and TGF-β1 in serum of rats were determined by ELISA

Serum of treated rats in each group was centrifuged at 3000 r/min for 10 min, and supernatant was absorbed, according to the instructions of each ELISA kit.OD values were measured, the standard curve was calculated, and the contents of Hepcidin, IL-1β,IL-10 and TGF-β1 were analyzed and calculated.

3.3.3 mRNA expression levels of TF and FTH1 in lung tissues were detected by qRT-PCR

Proper amount of lung tissues of rats in each group were collected into EP tubes without nucleation and enzyme, total RNA of each group was extracted according to RNA kit, RNA concentration was determined, denatured and reverse transcription was performed to obtain a 20 microliter system, which was diluted to 120 microliters with nucleation and enzyme free water, and then added into qPCRMasterMIX for machine reaction.The internal parameter was β-actin, and finally the expression of the target gene was calculated by 2－ΔΔCt method.The primer sequence was shown in Table 2.

Tab 2 Primer Sequence

3.3.4 The expression of TF and IREB2 proteins in lung tissues was detected by Western Blot

About 20 μg of lung tissue was collected from each group into a centrifuge tube.After grinding, the lung tissue was placed on ice for 30min (cracking).After centrifugation for 5min, the supernatant was obtained, the protein was measured, the concentration was adjusted, and the sample was denatured.Separation of total protein,transmembrane, sealing, 1×TBST washing, primary antibody incubation (TF, IREB21:1 000;β-actin1:100 000), 1×TBST washing, secondary antibody incubation (1:5 000), 1×TBST washing again, the strip was placed in a dark box, and the luminescence solution was soaked on both sides for development in the developer.The gray values of the developed bands were calculated by ImageJ, and the relative proteins were normalized by β-actin.

3.4 Statistical Methods

All the results of the experimental data are derived from SPSS26.0 analysis, article results expressed in(±s), P ＜ 0.05 results are different.

4.Results

4.1 Inflammatory response scores of rats in each group

Compared with the normal group, the inflammatory reaction and pathological changes of rats in COPD group were obvious (P＜0.01),and the inflammatory reaction and pathological reaction of rats in Qingjin Huatan decoction group were reduced (P＜0.01).(See Table 3)

Tab 3 groups of rat lung tissue inflammation pathological total score (points,n=3,±s)

Tab 3 groups of rat lung tissue inflammation pathological total score (points,n=3,±s)

Note: Compared with the normal group, *P＜0.01;Compared with COPD group, ▲P＜0.01;Compared with roxithromycin group, #P＞0.05

group n Total score of lung tissue inflammation level Normal group 3 0.33±0.29 COPD group 3 8.67±0.58*Roxithromycin group 3 3.67±0.76▲Qingjin Huatan Decoction group 3 3.17±0.58▲#F-108.062 P-0.386

4.2 HE staining results of rat lung tissue

In the normal group, relatively complete lung tissue and trachea structure were observed, no damage was observed in alveolar rules and lung tissues, no abnormal cells (inflammatory cells) were infiltrated, and the mucosal structure was normal.In COPD group,the shape of alveoli was not clear, the air duct wall was thickened,the alveolar wall was thinner, the lung tissue was destroyed, and the inflammatory cells were everywhere.Compared with the normal group, a small amount of inflammatory cells could be seen between the alveolar tissues in the roxithromycin western medicine group and the Qingjin Phlegm decoction group, and the airway wall was slightly thickened.Compared with the COPD group,the inflammatory cells were significantly reduced and the tissue structure was clearly visible.(See Figure 1)

Note: a is the normal group;b is COPD group;c is roxithromycin group;d was for Qingjin Huatan Decoction group Fig 1 Results of rat lung tissue under HE microscope (HE×200)

4.3 Contents of Hepcidin, IL-1β, IL-10 and TGF-β1 in serum of rats in each group

Hepcidin and IL-10: Compared with normal group, serum content in COPD group was significantly decreased (P＜0.01);Compared with COPD group, the serum expression level of rats in the western medicine group of roxithromycin after drug intervention and the Qingjin Huatan Decoction group was increased at different levels(P＜0.01);Compared with roxithromycin group and Hepcidin group,there was no significant difference in serum content (P＞0.05), and the effect was comparable.The serum content of Qingjin Huatan Decoction group treated with IL-10 had statistical difference(P＜0.01), and the effect of Qingjin Huatan decoction was more obvious.

IL-1β, TGF-β1: compared with normal group, serum expression levels of COPD group were increased (P＜0.01;P＜0.05);Compared with COPD group, levels of roxithromycin in western medicine group and gold clearing phlegm decoction group were decreased in different ways (P＜0.05);Compared with roxithromycin group,the serum content of Qingjin Huatan decoction group was not significantly different (P＞0.05), and the effect was similar.(See Table4)

Tab 4 Contents of Hepcidin, IL-1β, IL-10 and TGF-β1 in serum of rats in each group(n=3, ±s)

Tab 4 Contents of Hepcidin, IL-1β, IL-10 and TGF-β1 in serum of rats in each group(n=3, ±s)

Note: Compared with the normal group, *P＜0.01;Compared with COPD group, ▲P＜0.05;Compared with roxithromycin group, #P＞0.05

Group Doseg/kg Hepcidin(ng/ml) IL-1β(pg/ml) IL-10(pg/ml) TGF-β1(pg/ml)Normal group - 33.94±1.54 209.32±5.08 576.01±6.31 115.36±0.67 COPD group - 17.72±1.51* 408.65±18.5* 292.52±5.54* 262.26±14.38*Roxithromycin group 0.015 75 35.79±2.12▲ 301.49±21.33*▲ 486.07±17.92*▲ 151.29±2.77*▲Qingjin Huatan Decoction group 15 33.28±2.51▲# 312.99±12.01*▲# 560.23±15.05▲ 144.8±1.54*▲#F-54.518 82.433 328.881 229.505 P-0.471 0.084 0.156 0.006

4.4 Expression levels of TF and IREB2 mRNA in lung tissues of rats in each group

Compared with normal group, TFmRNA expression in COPD group was increased (P＜0.01), while IREB2mRNA expression was decreased (P＜0.01).Compared with COPD group, TFmRNA expression level in lung tissues of roxithromycin group and Qingjin Huatan Decoction was significantly decreased (P＜0.01), while IREB2mRNA expression was significantly increased (P＜0.01).Compared with roxithromycin group, the TFmRNA expression level of Qingjin Huatan Decoction group was statistically significant(P＜0.01), while the IREB2mRNA expression level of Qingjin Huatan Decoction group was not significantly different (P＞0.05)(see Table 5).

Tab 5 Comparison of TF and IREB2mRNA expression levels in lung tissues of rats in each group(n=9,±s )

Tab 5 Comparison of TF and IREB2mRNA expression levels in lung tissues of rats in each group(n=9,±s )

Note: Compared with the normal group, *P＜0.01;Compared with COPD group, ▲P＜0.01;Compared with roxithromycin group, #P＜0.05

group Doseg/kg TF IREB2 Normal group - 1.00±0.08 1.00±0.08 COPD group - 5.34±0.25* 0.26±0.02*Roxithromycin group 0.015 75 0.41±0.05*▲ 0.90±0.10▲Qingjin Huatan Decoction group 15 0.62±0.11*▲# 0.99±0.13▲F-2774.464 137.153 P-0.002 0.002

4.5 Expression levels of TF and IREB2 in lung tissues of each group

Compared with normal group, TF protein content in lung tissue of COPD group was significantly increased (P＜0.01), while IREB2 protein content was significantly decreased (P＜0.01).Compared with COPD group, TF expression level in lung tissues of roxithromycin western medicine group and gold clearing phlegm decoction group was significantly decreased (P＜0.05), while IREB2 expression level was significantly increased (P＜0.01).Compared with roxithromycin group, there was no significant difference in the expression level of TF in Qingjin Huatan Decoction group (P＞0.05), but the expression level of IREB2 in Qingjin Huatan Decoction group was statisticallysignificant (P＜0.01) (see Table 6, Figure 2).

Tab 6 Comparison of TF and IREB2 protein expression levels in lung tissues of rats in each group(n=9,±s)

Tab 6 Comparison of TF and IREB2 protein expression levels in lung tissues of rats in each group(n=9,±s)

Note: Compared with the normal group, *P＜0.01;Compared with COPD group, ▲P＜0.05;Compared with roxithromycin group, #P＞0.05

group Dose g/kg TF/β-actin IREB2/β-actin Normal group - 0.63±0.24 0.80±0.20 COPD group - 1.38±0.01* 0.15±0.05*Roxithromycin group 0.015 75 0.84±0.22▲ 0.78±0.13▲Qingjin Huatan Decoction group 15 0.96±0.25▲# 1.15±0.03*▲F-7.096 34.2 P-0.172 0.166

Fig 2 Electrophoresis of TF and IREB2 protein expression in lung tissue of rats in each group

5.Discussion

Traditional Chinese medicine, according to the manifestations of "cough", "phlegm" and "asthma", puts COPD into the scope of"cough", "asthmatic syndrome" and "lung distension", and considers its pathogenesis to be mainly the deficiency of lung, spleen and kidney, and the stagnation of phlegm and heat as the standard solid.Studies suggest that phlegm-heat stagnation of lung is the most common syndrome type in COPD[16].Qingjin Huatan Decoction is a classic prescription left by the long-term practice of traditional Chinese medicine that can stand the test.It is a representative prescription of clearing heat and eliminating phlegm, and has a remarkable effect on the treatment of COPD in clinical application.According to the research group's preliminary clinical research results on COPD, Qingjin Huatan decoction can effectively improve the symptoms of patients with chronic obstructive pulmonary disease and reduce the occurrence of inflammation[7].Cigarette smoke and harmful gases/particles can lead to iron deposition in the lungs, loss of structural integrity or dysfunction of epithelial cells, resulting in COPD, weakened defense ability, inability to eliminate pathogens, long-term development of chronic airway inflammation, which interferes with iron balance[17].Iron deficiency in the body can lead to anemia and impaired oxygen transport,which impairs cell function.Excessive iron in the body will promote the formation of oxygen free radicals and lipid peroxidation, leading to cytotoxicity;The increase of iron will also promote the growth of various pathogenic microorganisms, increase their toxicity, and promote the occurrence of infection[18].Many studies have also shown that TCM affects the development of diseases by regulating iron balance[19].

Hepcidin is the main iron regulatory hormone in the body, and many evidence shows that hepcidin is also related to IL-1β, IL-10, TGF-β and other inflammatory factors.When iron depletion occurs in macrophages, it can simulate the hypoxia state to compensate and promote the synthesis of IL-1β, and it can induce ferrimodulin in hepatocytes by secreting IL-1β.The effects of IL-10 on iron metabolism in LPs-induced inflammatory mice by regulating the expression of ferrimodulin have been shown to inhibit inflammatory factors, inhibit the STAT3 signaling pathway, reduce the expression of ferrimodulin, inhibit the expression of transferrin receptor to improve iron metabolism and relieve anemia.TGF-β is involved in inflammatory induced ferrimodulin levels through SMAD1/5/8 signaling[20].TF is an indispensable component in body fluid and an indispensable "carrier" protein for iron transport and metabolism, which can regulate the homeostasis of iron ion levels inside and outside cells.Transferrin (TF, also known as TRF)is an indispensable component in body fluid and an indispensable"carrier" protein for iron transport and metabolism.It can regulate iron level homeostasis in and out of cells.IREB2 plays a key role in maintaining iron balance and is strongly linked to nicotine receptor genes[21].It is this that has led to IREB2 being studied in relation to respiratory diseases such as chronic obstructive pulmonary disease.When iron deficiency occurs in cells, IRPs bind to specific stem rings in IREs to stabilize transferrin receptors and promote the separation of iron in transferrin, preventing the biosynthesis process of ferritin and iron ions, so that iron cannot be stored and metabolized[22].When the cells are iron-rich, the reverse is true.

It is believed that in COPD disease model prepared by cigarette plus lipopolysaccharide, under the influence of smoke, the amount of iron deposited in the lung increases, TF level rises, iron is transported into cells, binds to ferritin, intracellular ferritin concentration increases,IREB2 begins to degrade, reduces the storage of iron, increases excretion, and regulates membrane ferritin to transport intracellular iron to the extracellular.Create an iron metabolism cycle.When the level of Hepcidin in the blood is reduced, it leads to a compensatory increase of membrane iron transporter, which is involved in systemic circulating iron metabolism.

Qingjin Huatan Decoction can reduce lung inflammation, improve the pathological changes of lung tissue in COPD rats, and relieve COPD.Its mechanism may be related to increasing the content of IL-10 inflammatory factor in serum and decreasing the content of IL-1β and TGF-β1, so as to reduce the pulmonary inflammatory reaction.Pulmonary iron metabolism disorders exist in COPD, and inflammation is closely related to iron metabolism.Qingjin Huatan Decoction can promote the balance of lung iron metabolism,improve the disorder of lung iron metabolism during COPD, and delay the development of COPD.It may play a role by regulating inflammatory factors to increase the expression of Hepcidin in serum, down-regulate TFmRNA and protein levels in lung tissue,and up-regulate IREB2mRNA and protein levels.

6.Author contribution description

Hong Mei: Animal modeling, complete the experiment, analyze the data, write the paper;Wang Guangyao, Xu Guanglan: Experimental thought guidance correction;Pei Kai: Collaborative experiment to guide data analysis;Peng Simin, Zhang Yanyan, Wang Lin: Related literature collection and analysis.

There is no conflict of interest among the authors and all of them have contributed to this article.