Intervention effect of Danbei Yifei formula on pulmonary fibrosis based on urine metabolomics by UHPLC-Q-Exactive

Xiao-Jun Cai,Bai-Hua Jiang,Bi-Hai Zhang,Zhen-Hua Lu,Tao Wang,Qiang Li,Guan-Nan Jin

Heilongjiang Academy of Chinese Medicine Sciences,Harbin 150036,China

Keywords:Danbei Yifei formula Pulmonary fibrosis LC-MS technology Biomarker Metabolic pathway

ABSTRACT Objective:Determine the urinary biomarkers and pathogenesis of pulmonary fibrosis rats,and elaborate the intervention mechanism of DanBei YiFei formula.Methods:Bleomycin was injected into the trachea to induce pulmonary fibrosis in rats after anesthesia,and the diagnostic indexes of clinical pulmonary fibrosis,including superoxide dismutase,glutathione and malondialdehyde,were measured.High-throughput metabolic data of rats with pulmonary fibrosis were obtained by the latest high-resolution liquid-mass spectrometry technology,the multidimensional data were processed by Chemometrics algorithm to screen biomarkers related to pulmonary fibrosis.While,metabolic function indexes of rats after administration was observed,and the effective mechanism of DanBei YiFei formula on pulmonary fibrosis was expounded.Results:The clinical biochemical indexes showed that there were significant differences in metabolism in the model group,which confirmed the success of the preparation of the model of pulmonary fibrosis.Metabolisms research showed that the metabolic contour of the rats with pulmonary fibrosis was found to be significantly deviated,and the metabolism in vivo was abnormal.After the DanBei YiFei formula was given,the overall metabolic contour of the rats showed a trend of back modulation,and developed in the direction of healthy rats.With database matching and data processing 12 biomarkers,including Fumaric acid,Arginine and Spermidine,were obtained which were radically different from those of healthy rats and pulmonary fibrosis rats.Conclusion:DanBei YiFei formula has definite therapeutic effect on pulmonary fibrosis rats.Regulation of Tricarboxylic acid cycle and Arginine metabolic pathway may be the mechanism of its treatment of pulmonary fibrosis.

1.Introduction

Pulmonary fibrosis is an insidious progressive disease characterized by abnormal deposition of extracellular matrix,resulting in irreversible damage to lung structure and dysfunction of gas exchange[1].Recent studies have shown an increase in both the incidence and incidence of pulmonary fibrosis[2-3].It is a serious result of the repair process of abnormal lung tissue damage.This pathophysiological disorder involves a complex interaction between epithelial injury,oxidative stress,coagulation disorders,and inflammation that ultimately results in the conversion of multiple cell types to myofibroblasts and extracellular matrix deposition[4-6].Alveolar e7pithelial cells secrete TNF as a mediator of inflammatory signaling pathways in the presence of invaders[7].Inflammation is enhanced by activating nuclear transcription factors.Among them,inflammatory response is the cause of leukocyte and lymphocyte aggregation and oxidative stress injury[8].At present,pirfenidone is a representative therapeutic drug for the clinical treatment of pulmonary fibrosis,but it is often accompanied by a certain degree of side effects and a heavy financial burden on the patient's family[4-5].Therefore,there is an urgent need for new therapeutic drugs to effectively treat pulmonary fibrosis.

For thousands of years,TCM has provided effective treatments for chronic lung diseases,including reducing clinical symptoms,improving lung function and exercising.At present,the study of TCM formula and monomer has been proved to be an effective method to treat pulmonary fibrosis[9-10].Dan Bei Yi lung recipe is from Heilongjiang Academy of Chinese Medicine Sciences and is developed by Professor Jiang Baihua from his own experience.This recipe consists of danshen 30g,Astragalus 30g,Pingbi 20g,Radix Codonopsis 20g,Ophiopogon japonicus 20g,radix platycodonis 20g,radix platycodonis 15g,Ligusticum chuanxiong 15g,peach kernel 15g,Fructus schisandrae 15g,Fructus psoraleae 15g and earthworm 15g respectively.Its curative effect on pulmonary fibrosis is definite[11-26].Therefore,in this study,metabolomics technology was used to study the intervention effect of Danbeit on rats with pulmonary fibrosis,so as to provide experimental basis for the clinical pathogenesis of pulmonary fibrosis and the pharmacodynamic material basis and mechanism of Danbeit.

2.Materials and methods

2.1 Instruments and reagents

UHPLC-Q-Exactive system(American Thermo Fisher Scientific Company),TOMS Analytical Balances (Grainger);Zjhk-40l Ultra pure water machine (Tianjin Zhuojinghongke Instrument Equipment Technology Co.,LTD.);Danshen,Pingbei,Taoren,Earthworm,Chuanxiong,Radix platycodonis,Astragalus,Codonopsis,Psoraleae,Ophiopogon,Schisandrae were purchased from Harbin Shiyi Co.,LTD,Chloral hydrate (Shanghai Yuanye Biotechnology Co.,LTD.);Glutathione kit (Nanjing Chuan-Cheng Institute of Bioengineering,lot number:20190417);Superoxide dismutase Kit (Nanjing Jiancheng Institute of Biological Engineering,batch number:20190404);Malondialdehyde Kit (Nanjing Jiancheng Institute of Biological Engineering,batch number:20190401).

2.2 Animals

Thirty male SD rats of clean grade,weighing (150±20) g;Animal Laboratory of Heilongjiang Academy of Chinese Medicine Sciences (Animal License No.SCXK (Black) 2019-0004);Feeding environment (temperature 20℃±1℃ and humidity 50%±10%);Rats were given standard feed and water.

2.3 Preparation of Denbeyi lung Solution

Dan Bei Yi Fei Fang is composed of Salvia miltiorrhiza,fritilaria ligusticum,Taoren,Dilong,Astragalus membranaceus,Radix Codonopsis,Radix Ophiopogonis,Fructus Schisandrae,Fructus Psoraleae,radix Platycodonis,etc.The decoction pieces were provided by the Bureau of Traditional Chinese Medicine of Heilongjiang Academy of Chinese Medicine Sciences.The decoction was concentrated into a water decoction with a crude drug concentration of 0.6g/mL [26],and the gavage volume of rats was 1mL/100g.

2.4 Model replication and sample preparation

According to their weight,30 rats were randomly divided into 3 groups according to the free sequence,namely the blank group,the model group and the drug administration group.In the blank group,phosphate buffer was atomized in the trachea,and bleomycin (5mg/kg) was atomized in all the remaining rats.One day after modeling,Dan Beyi lung prescription was given for intervention (once a day)for 28 consecutive days [26].Nocturnal urine was collected on the 28th day of the experiment and centrifuged (13000rpm,15min,4℃)for metabonomic analysis.On the 29th day of the experiment,rats were anestheted with 1% pentobarbital sodium,and blood samples were collected from the abdominal aorta of the negative pressure tube of all groups.Part of the blood samples were centrifuged(3500rpm,15min,4℃),and the serum was separated and collected for the detection of biochemical indexes (MDA,GSH,SOD).

2.5 Analytical conditions

Uhplc-q-exactive system (Thermo Fisher Scientific),column temperature 25℃,flow rate 0.2mL/min,mobile phase A (0.1%formic water),mobile phase B (0.1% acetonitrile).Gradient elution conditions:0-2.5min,A:95%;2.5 4 min,A:95% to 45%.4-4.5 min,A:45% to 41%.4.5-5 min,A:41% to 50%.5-9.5 min,A:50-95%.

2.6 Data processing and statistical analysis

The urine sample data was processed using Compound Discoverer 3.0 professional software and converted to AIA format with MZmine open source software for data preprocessing and pattern recognition.With the TTEST<0.05 and the value of Fold change>2 as the differential metabolites,the

H and SOD were significantly reduced in the model group.MDA,GSH and SOD were significantly increased in the treatment group(P<0.05).

Structure identification and structure matching of the screened components are carried out in combination with online databases HMDB (http://www.HMDB.ca/) or Massbank (http://www.massbank.jp/),and the substances related to pulmonary fibrosis are inquired as biomarker,and the metabolic pathways are determined by enrichment analysis.

3.Results

3.1 Effects of Danbeyi Lung Prescription on biochemical indexes of pulmonary fibrosis rats

Oxidative stress injury is an important index to evaluate pulmonary fibrosis.See Table 1 for the intervention effect of Dembeti lung Prescription on pulmonary fibrosis.Compared with the blank group,MDA,GS

Table 1 Results of biochemical indexes of pulmonary fibrosis rats

Table 2 Urine biomarkers of rats with pulmonary fibrosis

3.2 Metabonomic study

This research adopts the latest UHPLC -Q -Exactive system,sensitivity to e -10,thus obtaining data is very huge,dryness of omics data,first of all,smooth,chromatographic peak alignment process,then using pattern recognition technology to huge amounts of data dimension reduction processing,reflected the 2 d data of main branch information and images,the principal component analysis diagram (figure 1).As can be seen from the figure,the data of the three groups were grouped significantly,and the administration group was between the blank group and the model group,which proved that Dan Beyi Lung Recipe had significant curative effect,and the quality control data were aggregated together,which proved that Dan Beyi lung recipe had good stability.Furthermore,partial least 20% discriminant analysis oPLS-DA was used to specifically analyze the difference ions between the blank group and the model group,and the data far away from the origin in S diagram and VIP diagram were selected as the difference ions for further screening (Figure 2 and Figure 3).The biomarkers of pulmonary fibrosis rat urine were identified by comparing the standard composition map included in HMDB database,and the 12 potential biomarkers obtained through Metaboanalyst4.0 (www.metaboanalyst.ca) platform were analyzed for pathway enrichment,so as to generate the metabolic pathway enrichment analysis diagram related to pulmonary fibrosis (Figure 4).Finally,a metabolic pathway map based on urine biomarkers of pulmonary fibrosis rats was constructed (Figure 5).

Figure 1 PCA scores in urine of rats with pulmonary fibrosis Blank group,▲model group,◆ Drug delivery group,QC

Figure 2 OPLS-DA-VIP analysis of urine in rats with pulmonary fibrosis

Figure 3 OPLS-DA-S analysis of urine in rats with pulmonary fibrosis

Figure 4 Enrichment analysis of urine metabolism pathways in pulmonary fibrosis rats

Figure 5 Urine metabolic pathways of pulmonary fibrosis rats Biomarkers,m etabolites,metabolic enzyme

4.Discussion

In this study,the pulmonary fibrosis rat model was firstly replicated by the classical endotracheal nebulized bleomycin method,and the blood biochemical indexes of the pulmonary fibrosis rats showed significant differences.After the administration of Danbeit lung prescription,all biochemical indexes showed a trend of callback,which proved that Danbeit lung prescription had a significant effect on pulmonary fibrosis.Twelve rat urine biomarkers related to pulmonary fibrosis were further studied by using non-targeted metabolomics technology,and the biological significance of their metabolic pathways or related metabolic enzymes proved that they were closely related to pulmonary fibrosis.

The metabolites of arginine involve many pathways.Creatine is involved in the production of ATP,while ornithine can be converted to puthumine and arginine to promote cell proliferation.Ornithine can also be converted to proline and hydroxyproline to form fibrotic collagen.We found that the levels of puthumine,arginine,and 4-hydroxyproline were increased in fibrotic rats compared with normal subjects.In addition,we found that fumaric acid and aspartic acid levels were reduced and they entered the tricarboxylic acid (TCA) cycle,thus using arginine metabolism as a reparative pathway.

The TCA cycle involves oxidation and glycolysis to produce acetylcoa (after pyruvate dehydrogenase converts pyruvate to acetyl-coA)and the production of flavin adenine dinucleotide (FADH2) and NADH as electron transport chains.Therefore,it is an important pathway for cell energy production.We found a significant increase in the metabolite Cis -aconite acid.The TCA circulating metabolite fumarate was significantly reduced.This may indicate downregulation of TCA circulation in rats with pulmonary fibrosis.

Glutamate,glutamine and aspartic acid are involved in several reactions that produce intermediates of the TCA cycle.Glutamic acid is converted to -ketoglutaric acid,the TCA circulating intermediate,and aspartic acid is converted to oxaloacetic acid,the TCA circulating intermediate,and -ketoglutaric acid is converted to glutamic acid.We found that aspartic acid levels were decreased and glutamate levels were increased in pulmonary fibrosis rats compared with those in the blank group.These data indicate a change in the level of these responses,which may affect the TCA cycle.Glutamate is also converted to glutathione,an antioxidant that has been linked to pulmonary fibrosis.We found that compared with the blank group,the level of glutathione disulfide in the lungs of the pulmonary fibrosis rats was increased and its oxidative counterpart glutathione disulfide was decreased,which may reflect the result of the body's fight against oxidative stress in pulmonary fibrosis.