CHEN Wn-Lu,HONG Jia-Na,ZHANG Xin-Ning,EMMANUEL Ibarra-Estrada,WAN Li-Shng,LI Sha-Sha,YAN Shi-Kai,d,XIAO Xu

a.The Second Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510006,China

b.Shenzhen Children’s Hospital of Guangzhou University of Chinese Medicine,Shenzhen,Guangdong 518038,China

c.Institute of Chinese Medicine Sciences,Guangdong Pharmaceutical University,Guangzhou,Guangdong 510006,China

d.School of Pharmacy,Shanghai Jiao Tong University,Shanghai 200240,China

e.Mexican Seed Association,A.C.,Mexico City 01030,Mexico

ABSTRACT Objective To study the common pathogenesis of pneumonia and colitis using modern biological network analysis tools,and to explore the theory that the lung and large intestine are exteriorly and interiorly related.Methods The relevant target genes (hereinafter,“targets”) of pneumonia and colitis were separately queried on the GeneCards database.The main targets of the two diseases were then screened out according to their correlation scores and intersected to obtain those common to the two diseases.Metascape was used to analyze the main and common targets identified,and the Database for Annotation,Visualization and Integrated Discovery (DAVID)was used to enrich and analyze the common targets.Cytoscape 3.7.2 software was used to build the network diagram.Results In total,54 targets,such as TNF,IL-10,IL-6,IL-2,IL-4,TLR4,TLR2,CXCL8,IL-17A and IFNG,etc.,are common to pneumonia and colitis,which are mainly enriched in these processes such as cytokine–cytokine receptor interaction,the Tcell receptor signaling pathway,the Toll-like receptor signaling pathway and the Jak-STAT signaling pathway.The Metascape modular analysis identified 11 modules for pneumonia,six modules for colitis,and two modules for the common targets.Conclusions Pneumonia and colitis have the same pathogenic targets and mechanisms of action and finally interact with each other through inflammatory reactions and immune responses.This provides a probable molecular mechanism that explains the theory that the lung and large intestine are exteriorly and interiorly related.

Keywords Theory of the exterior-interior relationship between the lung and large intestine Pneumonia Colitis Network pharmacology Th17 cell differentiation Inflammatory reactions Immune responses

1 Introduction

The theory that the lung and large intestine are exteriorly and interiorly related (hereinafter,the“Exterior-Interior Theory”) is based on the fact that the Taiyin lung meridian of hand (belonging to the lung collaterals and large intestine) and the Yangming large intestine meridian of hand (belonging to the large intestine collaterals and lungs) form the exterior and interior relationship of thezang-fuorgans through the mutual collaterals of the meridians.This theory was first found in the ancient Chinese medical textYellow Emperor’s Inner Classic(Huang Di Nei Jing,《黄帝内经》) where the relationship between the lung and large intestine is mainly reflected in their physiological interdependence,pathological transmission and changes,and their therapeutic use of each other[1].The lung is considered to be visceral and superior,whereas the large intestine is thefuorgan that occupies the lower part of the body.According to the law of disease transmission and changes,such events occur from the exterior to the interior (i.e.,muscle surface-meridians-sixfu-fivezangorgans),where the large intestine belongs to Yang in the exterior and the lung is Yin in the interior.The exterior-interior relationship of thezang-fuorgans is also related to their anatomical structures.Although the description of the medical anatomy by the ancient Chinese texts is not as accurate and clear as the modern version,it can generally reflect the position and characteristics of thezang-fu organs:the lung is described as being“empty as a honeycomb,with no penetrating orifices under them,full when inhaled,empty when exhaled,with natural news when inhaled”.The shape of each lung is like that of a honeycomb with orifices in it,whereas the large intestine is a hollow body,but the two are similar in shape[2].Moreover,the route of the Taiyin lung meridian of hand passes through the large intestine,and the Yangming large intestine meridian of hand also enters the thoracic cavity.The fact that these two meridians are connected to each other may be the anatomical basis for the Exterior-Interior Theory.The lung governs Qi (i.e.,energy),promote hair growth and Qi descent,and controls water circulation;that is,they essentially regulate the entire body’s Qi activity and the transportation and metabolism of water and liquids.By contrast,the large intestine governs the transmission of body wastes and fluids.When the lung is not invaded by external pathogens,its Qi function of promoting growth and descent is normal,and the function of the large intestine Qi is unobstructed and waste transmission is normal.At the same time,the lung’s descending function and water metabolism function are combined to transport body fluid to the large intestine in order to moisten the intestinal tract and help waste conduction.If the lung’s Qi is abnormal or there is no body fluid to moisten the intestinal tract,then the normal physiological function of the intestinal tract will be affected.Likewise,when the normal physiological function of the intestines is affected,lung Qi stagnation will ensue.The physiology and pathology of the two organs are therefore closely related and influence each other,which is another possible factor for the establishment of the Exterior-Interior Theory.

Although published studies have provided modern support for the theory that the lung and large intestine are exteriorly and interiorly related[3-5],the underlying mechanisms involved have not been established.Pneumonia and colitis are common diseases of the respiratory and digestive systems.In view of the observations of many clinical cases of patients with pneumonia caused by colitis or colitis caused byKlebsiella pneumoniae,it is speculated that inflammatory reactions play a role in these processes[6,7].Therefore,pneumonia and colitis were selected for the study of their interrelationship,which could provide a mechanistic explanation for the Exterior-Interior Theory.

The use of modern biotechnology to associate the pathogenesis of the two diseases with the Exterior-Interior Theory would be helpful in providing a scientifically based reference for the treatment of the two diseases with TCM and to control the common occurrence of the diseases according to their common pathological mechanism.Network pharmacology,which is a network analysis method based on bioinformatics and systems biology,can reflect the interaction of disease targets and mechanisms as well as the complex mechanisms of drugs and diseases.It is a new tool for finding drugs for disease treatment and explaining the theories and functions of TCM[8].Therefore,in this study,network pharmacology analysis was used to predict the target genes (hereinafter,“targets”) related to pneumonia and colitis and to explore the common targets and mechanisms of action between the two diseases in order to explain the Exterior-Interior Theory from the molecular level.

2 Materials and Methods

2.1 Establishment of the target gene library

Using “pneumonia” and “colitis” as key words,we searched the GeneCards[9](https://www.genecards.org/) database to collect targets related to each disease.Targets with a reliance score of ≥ 10 were then screened to establish a database of major targets for each disease.The reliance score indicates the correlation score of the target,where the higher the score is,the stronger the correlation between the gene and the disease will be.The targets of pneumonia and colitis in the respective databases were then input into the online Dram Venn Diagram Generator (http://bioinformatics.psb.ugent.be/webtools/venn/),whereupon the common targets of the two diseases were obtained and exported to establish the database of targets common to both diseases.

2.2 Analysis of the major targets

The main targets of pneumonia and colitis were imported into the Metascape[10]database (http://metascape.org/) for polygenic combined analysis and modular analysis.

2.3 Enrichment analysis of the common targets

The common targets were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses through the Database for Annotation,Visualization and Integrated Discovery (DAVID)[11](http://david.abcc.ncifcrf.gov/)to determine the biological processes and pathways in which they were significantly involved.

2.4 Construction of the protein-protein interaction(PPI) network

The common targets were input into the STRING 11.0(https://string-db.org/) database to obtain the PPI map,where “Multiple Proteins” was chosen for the group setting,“Homo sapiens” for the species setting,and default values for the other settings.The interaction network map was constructed with the visualization function of Cytoscape 3.7.2 software[12].

3 Results

3.1 Establishment of target gene libraries and the network map

Using “pneumonia” and “colitis” as key words,we searched the GeneCards database and found 4 562 targets for pneumonia and 4 763 targets for colitis.These main targets were screened by establishing a reliance score of ≥ 10,and target libraries were established for each disease,resulting in 247 targets for pneumonia and 142 targets for colitis.

The targets in the databases of both diseases were intersected on the Venn Diagram Generator website,whereupon 54 common targets were found.These common targets were then input into the STRING database to obtain the PPI diagram,which was saved in TSV format.This file was then imported into Cytoscape 3.7.2 to construct the “common target interaction” network map (Figure1).This map contained 54 nodes,with 817 lines between the nodes.The average node degree was 30.359.The Network Analyzer tool was used to further analyze Figure1,where the following correlations applied:the larger the node,the larger the degree value,and the colder the node tone,the larger the betweenness centrality value;the thicker the connection,the greater the edge betweenness of the line,and the colder the connection tone,the stronger the combine-score between targets.As shown in Figure1,tumor necrosis factor (TNF),interleukin (IL)-10,IL-2,IL-6,IL-4,and other targets have higher degree values in the network,indicating that these nodes have more connections with other nodes.Moreover,TNF,IL-2,albumin (ALB),C-reactive protein (CRP),and tumor protein P53 (TP53) have larger betweenness centrality values in the network,which means that these nodes act as bridges for more number of times in the two-to-two connections of other nodes.

3.2 Disease target analysis and network diagram construction

Using the Metascape database,the main targets of pneumonia and colitis were analyzed by polygene combination.Figure2 shows the correlation between the main targets of pneumonia and colitis.In addition to the 54 common targets (purple connection network),there were also many targets that could be enriched into the same pathway (blue connection network).On the basis of this,the common targets of pneumonia and colitis were enriched and analyzed to determine the specific pathways in which they were involved.

Using the DAVID database,GO and KEGG enrichment analyses were carried out on the 54 common targets,whereupon significant enrichment pathways were found.Figure3 depicts selected pathway results.In the Biological Process category,the 54 common targets were enriched in the subcategories positive regulation of immune system process,cytokinemediated signaling pathway,defense response,immune response,etc.In the Molecular Function category,the genes were enriched in signaling receptor binding,cytokine receptor binding,cell activity,protein binding and growth factor receptor binding.In the Cellular Component category,the genes were enriched in the extracellular space,extracellular region,side of membrane,cell surface and external side of plasma membrane.The products of these 54 common genes may exist in the cellular environments indicated above and participate in biological processes like immunity and defense through molecular functions such as signal receptor binding,thereby producing the disease-related effects.

The top 20 KEGG pathways were screened out according to thePvalues (Figure4).It was found that the 54 common targets were mainly concentrated in cytokine-cytokine receptor interaction and the T-cell receptor signaling pathway,Toll-like receptor signaling pathway and Jak-STAT signaling pathway.Various other disease pathways were involved (viz.,inflammatory bowel disease,malaria,Chagas disease,measles,rheumatoid arthritis,hepatitis B,influenza A and toxoplasmosis),indicating that these targets may participate in changes of those pathways as well.

After enriching the pathways of the targets,the MCODE algorithm[13]of Metascape was used to identify the main functional modules of the targets of pneumonia (Figure5) and colitis (Figure6),respectively.Figure5A and 6A are the interactive network maps constructed for the main targets of pneumonia and colitis,from which clustering modules were extracted by the MCODE algorithm (Figure5B and 6B).As shown in Figure5,11 modules were identified for pneumonia,involving mainly chemokines,diseases related to the Toll-like receptor signal cascade reaction,grid protein-mediated endocytosis,immune response,respiratory burst,cell response to interferon,cytokine production,B-cell receptor signal pathway,anti-inflammatory factors,interleukin family signals,Th17 cell differentiation,the Jak-STAT signaling pathway,etc.Six modules were identified for colitis,including mainly active regulation of interleukin,immune response,the Jak-STAT signaling pathway,other disease pathways (cancer,inflammatory bowel disease,tuberculosis),etc.The MCODE clustering analysis comparing the modules of the two diseases revealed that the main targets of pneumonia and colitis were also related to inflammatory reactions,especially interleukin-related reactions,immune reactions,cytokine-mediated signal transduction,and other pathways.Additionally,the fact that there were so many disease pathways in the KEGG enrichment results may be because these targets affect the occurrence and development of those diseases at the same time,although this cannot fully explain how such disease pathways can participate in the pathological mechanism of pneumonia and colitis at the same time.The MCODE algorithm was also used to carry out functional clustering of the 54 common targets (Figure7),which was combined with the enrichment results of the DAVID database.In total,two modules involving 15 targets were extracted.Each module selected the first three related pathways.Module 1 (Figure7,left) selected the Toll-like receptor signaling pathway,Chagas disease (American trypanosomiasis),and positive regulation of DNAbinding transcription factor activity.Module 2 (Figure7,right) selected inflammatory bowel disease,Th17 cell differentiation,and positive regulation of cytokine production.The pathways enriched by this method were basically consistent with the results of the KEGG pathway analysis.

4 Discussion

4.1 Common mechanism of pneumonia and colitis

Pneumonia and colitis are both well known to be inflammatory diseases.The results of this study also suggest that their common pathogenic mechanism is bound to be related to inflammatory reactions.One way in which foreign risk factors such as pathogens trigger inflammation is to activate inflammatory corpuscles,that is,macromolecular multiprotein complexes,such as nod-like receptor with pyrin domain-containing 3 (NLRP3) in common targets.Inflammatory bodies of NLRP3 are activated through two steps:first,nuclear factor kappaB (NF-κB)signaling is induced by the activation of Toll-like receptor 4 (TLR4),which leads to increased expression of NLRP3,pro-IL-1 and pro-IL-18,and then indirectly activates NLRP3 through various signals.Excessive activation of the inflammatory corpuscles leads to the activation of caspase-1,which cleaves pro-IL-1 and pro-IL-18 to produce IL-1 and IL-18[14].Such inflammatory body signaling leads to the onset of many diseases.At the same time,inflammatory bodies play an important role in the development of immune function and also lead to the generation of autoimmune diseases[15].It was found that after the administration ofBifidobacteriumto mice with colitis,the activation of NF-κB was inhibited and the secretion of inflammatory cytokines was reduced,thereby reducing the intestinal inflammatory reaction[16].Clinical studies have also shown that the inflammatory reaction and immune function indexes of patients with pneumonia are significantly different from those of healthy subjects[17].The upregulation of other signaling pathways,such as the Toll-like receptor signaling pathway and cytokine activation,has also been found to participate in the induction of inflammation.At the same time,these pathways also participate in the immune response[18-20].

In addition to the abovementioned pathways,Th17 cell differentiation may also play an important role in lung-colorectal diseases.IL-1,interleukin 1 receptor (IL-1R),transforming growth factor (TGF),IL-6,signal transducer and activator of transcription 3(STAT3),IL-17A,IL-22,MHC Ⅱ,CD4,zeta chain of T cell receptor-associated protein kinase 70 (ZAP70),NF-κB,IL-4,IFN,STAT1,IL-2,forkhead box P3(FOXP3),and other target genes and gene products are enriched in this pathway.The pathway itself contains key pathways,such as the Toll-like receptor signaling pathway and Jak-STAT signaling pathway.Helper T-cell 17 (Th17),which is a subgroup of CD4+T cells,participates in the immune response and the pathogenesis of autoimmune diseases and plays an important role in the body’s defense mechanisms.It can be induced and promoted to differentiate by IL-6 and TGF.Furthermore,it activates transcription factors such as STAT3 and promotes the secretion of IL-17 and IL-22 under the action of IL-23.However,when STAT1 is activated,IL-2 and other cytokines negatively regulate the differentiation of Th17 cells and the expression of IL-17[21-23].Therefore,the differentiation of Th cells can affect the inflammation and immunity of the body.The target genes in this study were enriched in the whole process of the Th17 cell differentiation pathway.When the imbalance of Th17 cell differentiation leads to the excessive secretion of IL-17,IL-22,etc.,pneumonia and colitis will occur.Some studies have found that lung inflammation can be relieved by regulating Th17 and oxidative stress reactions in the lung,thus reducing acute lung injury and related airway inflammation in mice[24].LIU et al.[25]proposed that respiratory tract microorganisms regulate the Th17/IL-17 axis to affect the airway inflammatory response,leading to asthma.In addition to Th17 cells being proven to affect the lung,it has also been found that when the differentiation of these cells decreases,the inflammatory reaction and damage caused by colitis are relieved[26,27].Additionally,it was found that the intestinal flora can regulate the immune response of Th17/IL-17[28].

Taken together,the results show that the pathways enriched by the identified targets may mostly lead to pneumonia and colitis through their simultaneous induction of inflammation and the immune response.It is suggested that inflammation and immunity can be taken as key observation points in the clinical treatment of these two diseases.

4.2 Modern studies on the exterior and internal phases of the lung and large intestine

On the basis of the results of this study,we speculate that the Exterior-Interior Theory may be related mainly to inflammation and the immune response.Existing related research also suggests that there may be some correlation with histology and embryology as well as the intestinal flora.

The GO analysis results showed that the common targets in the Cell Component category were enriched in the subcategories extracellular space,side of membrane,cell surface,external side of plasma membrane,etc.At the same time,they were also located on organelles such as the endoplasmic reticulum,Golgi apparatus and lysosomes.This suggests that pneumonia and colitis may have high similarity in terms of their cellular composition.LIU et al.[29]used histology and flow cytometry to observe the morphology and proliferation and apoptosis of the lung and large intestinal cells and found that there was no morphological difference between these cells in the early embryo,which supports the GO results.Moreover,modern research studies have shown that the epithelial tissues of the lung and large intestine all originate from the endoderm of gastrula,and the tracheal tissue originates from the plica of gastrula[30].The same tissue source and structure will also render the lung and large intestine physiologically closely related.FU et al.[31]carried out clinical trials on 60 patients with bronchial asthma and 60 healthy controls to observe the effect of body surface temperature changes on the interstitial structure of the lung and large intestine in the patients.Their results showed that the temperature difference in the experimental group was statistically significant.The authors surmised that there is an intermediate structure between the lung and large intestine that acts as a reaction area of bronchial asthma and will affect the large intestine.

FU[32]established a rat model of allergic asthma complicated with intestinal flora imbalance and found that the TLRs/NF-κB signal transduction pathway participated in the immune regulation of the lung and intestinal tract at the same time.Consistent with the KEGG pathway enrichment results,it is speculated that the intestinal flora can regulate the Toll-like receptor signaling pathway and other related pathways to affect pneumonia and colitis,suggesting that the proof of the relationship between the lung and large intestine is not only reflected in the embryology of hair tissue but also in the changes of the intestinal flora.In the lung,the microbial flora is made up mainly ofBacteroides,SclerodermaandProteusspecies,which is similar to that of the intestinal tract,and the flora in the two organs become more similar with time.Some scholars have proposed a“lung-intestinal axis” on the basis of this,the theoretical connotation of which is similar to the Exterior-Interior Theory[33].The intestinal flora plays important roles in the human body,such as aiding in digestion and nutrient absorption,substance metabolism,immunity and other processes.Its dynamic balance can be compared with the Yin-Yang balance of TCM.Usually,the species type and quantity of intestinal microorganisms are relatively constant,maintaining the normal physiological function of the body.However,when inflammation and immune responses occur,changes of the intestinal flora can also ensue,which will lead to the development of diseases[1].WU et al.[34]found that intestinal anaerobic bacteria may affect the immune response by regulating changes in inflammatory factors (e.g.,IL-10 and IL-17) in the lung of mice,thus affecting the lung injury caused by the influenza virus.LIU et al.[35]tested the feces of both mice with influenza and control mice and found that the influenza group had an obvious imbalance of the intestinal flora and weight loss.LI et al.[36]studied the effect of the influenza virus on the intestinal flora and showed that the virus could cause significant changes in theEscherichia coli,

LactobacillusandBifidobacteriumcontents.Thus,the influenza virus can change the intestinal flora,and intestinal flora disorder will in turn lead to digestive tract complications such as diarrhea,affecting the symptoms of primary respiratory diseases.In general,imbalance of the intestinal flora will lead to the development of diseases,which in turn will also change the intestinal flora further.

4.3 Summary and future prospects

Although TCM has a long history and clear curative effects,their mechanisms of action have yet to be completely clarified.The biological networks elucidated through modern biological research are consistent with the complex mechanisms of TCM and can provide a reference for them to a certain extent.The exterior and interior relationship of the lung and large intestine is one of the basic theories of TCM.The clinical application of this theory is extensive and its source has a long history,but it lacks modern theoretical support.Our study relied on modern biological network technology,where network pharmacology tools were selected to predict the relationship between pneumonia and colitis,revealing 54 targets and multiple enriched pathways in common between the diseases.In the study of pathways,it was found that the inflammatory and immune responses may eventually affect each other.Therefore,combined with the research results of modern medicine on the lung and large intestinal tissue and embryology,anatomy,intestinal flora changes,molecular mechanisms and other aspects,it was confirmed that the Exterior-Interior Theory is still applicable to this day.However,the existing research on the lung and large intestine has not yet formed a systematic framework,suggesting that the mechanisms underlying the theory of the exteriorinterior relationship between the lung and large intestine still have to be proven through modern studies.

Acknowledgements

We thank for the funding support from the Guangdong Provincial Key Construction Unit Project of Traditional Chinese Medicine Pediatrics(Guangdong Traditional Chinese Medicine Office Letter [2018] No.202).

Competing Interests

The authors declare no conflict of interest.