Mechanism of Huashi Xingyu Qingre recipe (化湿行淤清热方) in treating oral lichen planus based on network pharmacology and clinical trial verification

DENG Qianlan,LU Yueting,YAN Lijuan,LU Hualin,JIN Ruizhe,XU Yanzhi,SONG Jing,LIU Tiejun

DENG Qianlan,LU Yueting,LU Hualin,JIN Ruizhe,XU Yanzhi,LIU Tiejun,Department of Stomatology,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050000,China

YAN Lijuan,Department of Stomatology,Hebei Children's Hospital,Shijiazhuang 050000,China

SONG Jing,Department of Stomatology,Shijiazhuang Third Hospital,Shijiazhuang 050000,China

Abstract OBJECTIVE:To identify the main active components and targets of Huashi Xingyu Qingre recipe (化湿行淤清热方,HXQR) and to investigate its mechanism in the treatment of oral lichen planus (OLP).METHODS:The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform was searched to identify the active ingredients and corresponding targets of HXQR.Disease genes were obtained from the GeneCards database,and a “drugdisease regulatory network” was constructed using Cytoscape software and PERL programming language.The STRING database was used to build a protein–protein interaction network.Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)terms were analyzed using R software with a Bioconducter plugin.Finally,the results and the efficacy of HXQR in treating OLP were validated in a clinical trial that included enzyme-linked immunosorbent assay(ELISA) testing and observations of the post-treatment changes in clinical symptoms.RESULTS:HXQR contained 167 active components and 261 targets,with 391 disease targets.The intersection of these two categories in a Venn diagram revealed 57 drugdisease common targets.A compound-target network was constructed and revealed that the six key pharmaceutical ingredients of HXQR were quercetin,luteolin,wogonin,kaempferol,beta-carotene,and baicalein.The protein-protein interaction network mainly involved core proteins such as ALB,interleukin-6,and AKT1.Drug-disease common targets were enriched in 1628 GO terms and 117 KEGG terms,mainly involving inflammatory responses,viral infections,and tumorrelated pathways.ELISA testing indicated that HXQR inhibited the tumor necrosis factor (TNF) signaling pathway by reducing the expression of interleukin-6,matrix metalloproteinase-9,and intercellular adhesion molecule-1.The clinical symptoms of the patients with OLP were significantly improved after 8 weeks of treatment with HXQR.CONCLUSION:HXQR treats OLP by regulating the TNF signaling pathway,resulting in a marked treatment effect with few adverse effects.

Keywords:lichen planus,oral;network pharmacology;clinical trial;Huashi Xingyu Qingre recipe

1.INTRODUCTION

Oral lichen planus (OLP) is an autoimmune inflammatory disease with an unknown etiology and an overall incidence of 0.5%-1.5%,mostly affecting middle-aged women.1The most common clinical manifestations of OLP are reticular,atrophic,and erosive lesions that mainly occur inside the cheek and on the tongue.2OLP has the possibility of cancerization,and is classified as a precancerous state by the World Health Organization.3The pathogenesis of OLP remains unclear.The main treatments used to control the progression of OLP are western medicines,surgical treatment,and physical therapy,but the treatment effect is often not ideal.4

With a history of thousands of years,Traditional Chinese Medicine (TCM) is widely used in the treatment of many kinds of disease due to its low toxicity and good therapeutic effect.5TCM theory categorizes OLP as"mouth sores,mouth erosion",and considers the cause of OLP to be toxic heat and wet accumulation,and blood estrangement.Therefore,the TCM treatment principles of dissolving the moisture,decreasing internal heat,promoting blood circulation,and removing blood stasis were used to create Huashi Xingyu Qingre recipe (化湿行淤清热方,HXQR).HXQR has achieved a good curative effect in the long-term clinical treatment of OLP.The 11 ingredients of HXQR are Huangbai (Cortex Phellodendri Amurensis),Peilan (Herba Eupatorii Fortunei),Sharen (Fructus Amomi),Yiyiren (Semen Coicis),Fuling (Poria),Chantui (Periostracum Cryptotympanae),Chishao (Radix Paeoniae Rubra),Honghua (Flos Carthami),Mudanpi (Cortex Moutan Radicis),Gancao (Radix Glycyrrhizae),Jinyinhua (Flos Lonicerae),and Baixianpi (Cortex Dictamni Radicis).However,the specific mechanism of HXQR in treating OLP has not been clarified.

Network pharmacology has emerged as a powerful problem-solving method for TCM.6Network pharmacology is characterized by predictability and systematization and is widely used to reveal the biological basis of TCM herbal treatment.7,8Based on the concept of disease-gene-targeting medicine,network pharmacology decodes the potential pharmacological principles of drugs (especially natural products) on a system level,enabling TCM to change from “experiencebased” to “science-based”.9

2.METHODS

2.1.Active components and targets of HXQR

The TCM Systems Pharmacology Database and Analysis Platform (TCMSP;http://tcmspw.com/tcmsp.php) was searched to identify the active components of HXQR that met the criteria of oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18,and the targets of these active components.The TCMSP is a systematic pharmacology platform that integrates the chemical components of drugs and their targets,the targets of related diseases and the interaction network,which is conducive to the analysis of the mechanism of TCM.10OB describes the proportion of the dosage of the given drug entering the human circulation;DL refers to the similarity between the compound and the known drug.Compound targets were processed with PERL programming language(https://www.perl.org/,version 5.30.1.1) to obtain their corresponding gene symbol.

2.2.OLP-related targets

The keyword "oral lichen planus" was imputed into the GeneCards database (http://www.genecards.org)11to retrieve the disease targets.

2.3.Drug-disease common target screening

The targets of the active components in HXQR and OLP were imported into the Venny2.1.0 online tool(https://bioinfogp.cnb.csic.es/tools/venny/).A Venn diagram was created to determine the common targets of the drugs and the disease.

2.4.Construction of the regulatory network

By using PERL programing language (https://www.perl.org/ver.5.30.1),the drug-disease common targets and the corresponding active components were intersected.The drug-disease common targets and corresponding active components were inputted into Cytoscape software (http://cytoscape.org/,ver.3.7.2)12to construct the compound regulatory network of HXQR.The Network Analyzer function was used to analyze the main active TCM components.In the network,"node"represents the number of active components and target genes,and "edge" represents the relationship between the compound and the target gene.

2.5 Construction of the protein–protein interaction (PPI)network

Drug-disease common targets were entered into the STRING database (https://string-db.org/)13to construct the PPI network.The filtration condition was set as a minimum required interaction score of >0.4,and the results were saved in TSV format.The drug-disease common targets were then sorted in accordance with the degree of correlation between proteins using R software(version 3.6.1).

2.6 Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis

R software and the Bioconductor plugin (http://www.bioconductor.org)14were utilized to perform GO and KEGG enrichment analysis.The Bioconductor plugin,which included “org.hs.eg.db”,“clusterProfiler”,“enrichplot”,and “ggplot2”,analyzed the gene enrichment function and provided visual results.The GO terms included biological processes (BP),cellular components (CC),and molecular functions (MF).The filter conditions wereP<0.05 andQ<0.05 for further analysis and the results were outputted in the form of bar charts and bubble charts.

2.7 Clinical trial

2.7.1 Specimen collection

30 patients selected were diagnosed with OLP based on pathological examination in the Stomatology Department of the Fourth Hospital of Hebei Medical University.These 30 patients were included as the OLP group with no systemic diseases,no history of OLP treatment and medication for 3 months,and good periodontal condition,comprising eight men and 22 women with an average age of 49.5 years (range 21-63 years).The control group included 28 healthy subjects.The study was performed in accordance with international ethical standards and was approved by the ethics committee of the Fourth Hospital of Hebei Medical University.All participants provided informed consent.

地表一般分布第四系上更新统～全新统洪积含砾粉土层，厚0.5～1m。下覆基岩岩性主要为下白垩统土黄-红黄色泥岩，局部夹砂岩、泥质粉砂岩及少量泥质砂砾岩，泥岩呈巨厚层状，断层和节理裂隙不发育，地下水埋深23.6m，洞身多处于地下水以下，以渗水、滴水为主，局部竖井段存在线状流水；地下水对混凝土具有硫酸盐型强腐蚀性，对钢筋混凝土结构中的钢筋及钢结构均具中等腐蚀性。该段属极软岩，强风化层厚3～5m，弱风化层厚11～15m，洞身处于新鲜岩体内，泥岩的自由膨胀率一般为50%～60%，属弱膨胀岩，宜产生塑性变形，属Ⅴ类围岩。

2.7.2 Enzyme-linked immunosorbent assay (ELISA)testing

In the morning on an empty stomach,3-mL serum samples were taken from the OLP and control groups,and the supernatant was collected by static centrifugation and stored at－80 ℃.After 8 weeks of treatment with HXQR,blood samples were taken again using the same method.The pre-and post-treatment expressions of interleukin (IL)-6,matrix metalloproteinase (MMP)-9,and intercellular adhesion molecule (ICAM)-1 in the OLP and control groups were detected using an ELISA Kit (Arigo,USA),Table centrifuge (Arigo,USA),TECAN (Switzerland),miniOne centrifuge (Hefei abenson),liquid pipette (Thermo,USA).

Table 1 Pre-and post-treatment serum levels of IL-6,MMP-9,and ICAM-1 ()

Table 1 Pre-and post-treatment serum levels of IL-6,MMP-9,and ICAM-1 ()

Notes:the treatment group included oral lichen planus patients treated with Huashi Xingyu Qingre recipe.This group was instructed to intake the Huashi Xingyu Qingre recipe after a meal in the morning and evening,one dose daily,and return visit once a week,for 8 weeks.The control group included healthy subjects.IL-6:interleukin-6;MMP-9:matrix metalloproteinase-9;ICAM-1:intercellular adhesion molecule-1.Compare with the control group,aP <0.05;compare with the before treatment,bP <0.05.

2.7.3 Clinical efficacy

The OLP group was instructed to intake the HXQR after a meal in the morning and evening,one dose daily,and return visit once a week,for 8 weeks.

The efficacy of HXQR in treating OLP was assessed using the criteria of the Mucosa Committee of Chinese Stomatological Association.The treatment was categorized as markedly effective (congestive or erosive surface of the oral mucosal lesions completely disappeared,white stripes disappeared,and pain completely resolved),effective (decreased range of hyperemia or erosion of oral mucosal lesions,less white stripes,and improved pain),or ineffective (no change or even aggravation of the congestion or erosive surface size of oral mucosal lesions,no change or increase in white stripes,no relief or even aggravation of pain).

2.8 Statistical analysis

Data were analyzed using SPSS 23.0 software (IBM,Armonk,NY,USA).The two-sample pairedt-test was performed to compare the differences between the OLP and control groups.Data were expressed as mean ±standard deviation.The significance level was set asP<0.05.

3.RESULTS

3.1.Active components and potential targets of HXQR

In accordance with the OB and DL screening conditions,167 nonredundant active components and 261 related targets were obtained from the TCMSP.Among them,there were 11 active components in Baixianpi (Cortex Dictamni Radicis),13 in Chishao (Radix Paeoniae Rubra),six in Fuling (Poria),88 in Gancao (Radix Glycyrrhizae),16 in Honghua (Flos Carthami),25 in Huangbai (Cortex Phellodendri Amurensis),17 in Jinyinhua (Flos Lonicerae),six in Mudanpi (Cortex Moutan Radicis),seven in Peilan (Herba Eupatorii Fortunei),eight in Sharen (Fructus Amomi),and six in Yiyiren (Semen Coicis).The proportions of active components in each ingredient of HXQR are shown in Figure 1.Due to the repeated inclusion of the active components in the HXQR ingredients,we drew a Venn diagram to summarize the active components in each ingredient.As shown in Figure 2,the main active components in the HXQR formula were stigmasterol,beta-sitosterol,quercetin,sitosterol,luteolin,and kaempferol.

Figure 1 Proportions of active components in each ingredient of Huashi Xingyu Qingre recipe

Figure 2 Venn diagram of the active components in each ingredient of Huashi Xingyu Qingre recipe

3.2 OLP-related target genes

The search of the GeneCards database using “oral lichen planus” as the keyword retrieved 391 disease targets.

3.3 Drug-disease common targets

The Venn diagram included 261 drug targets and 391 disease targets (Figure 3).The intersecting area contained 57 drug-disease common targets.

Figure 3 Venn diagram of drug and disease targets The blue part represents 204 drug targets,the yellow part represents 334 disease targets,and the gray part represents 57 drug-disease common targets.

3.4 Compound-target network analysis

Applying PERL language,we obtained 136 active components and 57 disease-drug common target genes,matched them using Cytoscape software,and created a regulatory network (Figure 4).The network contained 193 nodes and 491 edges.The top six compounds of HXQR with higher targets were quercetin (MOL000098),luteolin (MOL000006),wogonin (MOL000173),kaempferol (MOL000422),beta-carotene (MOL002773),and baicalein (MOL002714).There were 47 targets in quercetin,28 targets in luteolin,16 targets in wogonin,15 targets in kaempferol,14 targets in beta-carotene,and 14 targets in baicalein,indicating that these compounds may play an important role in treating OLP.

3.5 PPI network analysis

The PPI network was obtained through the analysis of the 57 disease-drug common target genes recorded in the STRING database (Figure 5).Each edge represented the interaction relationship between proteins,with more lines representing a stronger correlation.The top 10 core genes were ALB,IL-6,AKT1,VEGFA,EGF,CASP3,JUN,MMP-9,MYC,and EGFR.

Figure 4 Network of active compounds and common targets

3.6 GO and KEGG analysis

APvalue of <0.05 andQvalue of <0.05 were used as the screening conditions for the GO enrichment analysis of the 57 target genes.We obtained 1628 GO terms,comprising 1523 BP terms,33 CC terms,and 72 MF terms.The results showed that HXQR played an important role by regulating a variety of BP (Figure 6).The top five BP were response to metal ion (GO:0010038),response to radiation (GO:0009314),response to lipopolysaccharide (GO:0032496),response to molecule of bacterial origin (GO:0002237),response to nutrient levels (GO:0031667).The top five CC terms(P<0.05) were:membrane raft (GO:0045121),membrane microdomain (GO:0098857),membrane region (GO:0098589),cyclin-dependent protein kinase holoenzyme complex (GO:0000307),serine/threonine protein kinase complex (GO:1902554) (Figure 7).The top five MF terms (P<0.05) were:cytokine receptor binding (GO:0005126),cytokine activity (GO:0005125),receptor ligand activity (GO:0048018),growth factor receptor binding (GO:0070851),activating transcription factor binding (GO:0033613)(Figure 8).

A total of 117 KEGG pathways were obtained through R language operation.The KEGG pathway enrichment analyses were based on aPvalue of <0.05 and aQvalue of <0.05,and the results showed the top 20 signaling pathways (Figure 9).The important signaling pathways involved in the treatment of OLP included the tumor necrosis factor (TNF) signaling pathway (hsa04668) and IL-17 signaling pathway (hsa04657).A schematic diagram of the TNF signaling pathway is shown in Figure 10.

The red rectangles represent the central nodes in the tumor necrosis factor (TNF) signaling pathway.

3.7 Experimental validation

3.7.1 Effect of HXQR in regulating the TNF signaling pathway

The pre-treatment serum levels of IL-6,MMP-9,and ICAM-1 were significantly higher in the OLP group than the control group (P<0.05).Within the OLP group,the serum levels of IL-6,MMP-9,and ICAM-1 were significantly decreased after treatment compared with pre-treatment levels (P<0.05).

3.7.2 Clinical observations

After 8 weeks of treatment,the outcome was categorized as markedly effective in 18 cases,effective in 10,and ineffective in two.The total effective rate was 93.3%.HXQR had a significant clinical effect on OLP.Typical cases are shown in Figure 11.

Figure 5 Protein-Protein Interaction network

Figure 6 Top 20 biological process terms

Figure 7 Top 20 cellular component terms

Figure 8 Top 20 molecular function terms

Figure 9 Top 20 Kyoto Encyclopedia of Genes and Genomes pathways

Figure 10 TNF signaling pathway

Figure 11 Pre-and post-treatment photographs of typical cases of oral lichen planus

4.DISCUSSION

At present,the pathogenesis of OLP is still unclear.However,OLP is widely considered to be an immunemediated inflammatory disease.In TCM,OLP belongs to the category of "mouth sores,mouth erosion".From the perspective of TCM,OLP is divided into the dampnessheat accumulation type,Qistagnation and blood exhaustion type,QiandYindeficiency type,and liver and kidneyYindeficiency type.The most common types of OLP are dampness-heat accumulation andQiandYindeficiency.Using the TCM principle of “removing dampness,promoting blood circulation and removing blood stasis,clearing heat and decrease internal heat”,HXQR is composed of 11 Chinese herbal medicines,such as Huangbai (Cortex Phellodendri Amurensis),Chishao (Radix Paeoniae Rubra),Peilan (Herba Eupatorii Fortunei),and Gancao (Radix Glycyrrhizae).As the “king medicine” in the formula,Huangbai (Cortex Phellodendri Amurensis) has the function of clearing heat and cooling blood,reducing fire,and detoxification.Chishao (Radix Paeoniae Rubra) serves as the “minister medicine” and has the effects of activating blood circulation,dispersing stasis,and analgesia.Peilan(Herba Eupatorii Fortunei) invigorates the spleen,relieves heat,and participates in the anti-inflammatory response.Licorice relieves pain and protects and repairs the mucosa.

In our study,network pharmacology revealed that the 11 TCM ingredients of HXQR had 136 active components and 58 corresponding disease targets.According to the compound-target network analysis,each effective active component acted on at least one target gene,and each gene was regulated by at least one active component,indicating that HXQR treated OLP in a multi-component and multi-target way.The active components of HXQR with the largest number of targets were quercetin(MOL000098),luteolin (MOL000006),wogonin(MOL000173),kaempferol (MOL000422),betacarotene (MOL002773),and baicalein (MOL002714).Quercetin,luteolin,and kaempferol were widely distributed in HXQR.Quercetin had anti-inflammatory,anti-viral,and analgesic effects,and is used to treat various kinds of inflammation.15,16Quercetin inhibits the production of MMP-9,and reduces the production of IL-6,ICAM-1,and other inflammatory factors and chemokines by blocking the NF-kB signaling pathway.17It has been proved that quercetin reduces human oral keratinocyte injury induced by lipopolysaccharides.18Luteolin is a natural flavonoid with strong antioxidant and anti-inflammatory effects,19and has been used to treat a variety of inflammatory diseases.20-22In addition,luteolin inhibits the proliferation of oral squamous cell carcinoma.23Wogonin has a variety of pharmacological effects,including anti-inflammation,which inhibit the mucosal inflammatory response and maintain membrane function.24Wogonin reduces the expression of TNF and MMP-9 by inhibiting the activation of NF-kB.25Kaempferol and quercetin induce CASP3-dependent cell apoptosis.26Kaempferol protects the vascular endothelial function by reducing oxidative stress and inflammation.27Beta-carotene prevents the occurrence of OLP and reduces the number of micronucleated exfoliated cells in OLP lesions.28,29Baicalein negatively regulates the NF-kB signaling pathway,inhibits the production of TNF and IL-6,and reduces the inflammation caused by OLP.30Through network pharmacology,we predicted the active components in HXQR.The mechanism of these compounds in the treatment of OLP by reducing inflammatory response,inhibiting apoptosis and alleviating oxidative stress has been verifiedin vivoandin vitroexperiments.

According to the PPI network analysis,HXQR played a therapeutic role in 57 targets,including ALB,IL6,AKT1,VEGFA,EGF,CASP3,JUN,MMP-9,MYC,and EGFR.As a pro-inflammatory factor,IL-6 regulates the immune and inflammatory responses,and its expression levels are increased in the oral cavity and serum of patients with OLP;31thus,IL-6 is used as an indicator to monitor the OLP disease status and evaluate the therapeutic effect.32,33EGF and EGFR are increased in patients with OLP compared with normal controls and play an important role in the carcinogenesis of OLP.34,35When CD8+T cells bind to keratinocytes,they activate the caspase signal transduction pathway and lead to the apoptosis of keratinocytes.The high expression of CASP3 is related to the apoptosis of keratinocytes in OLP.36,37Regulated by Th9/IL-9 and TNF-α,a high expression of MMP-9 mediates the destruction of the basement membrane in OLP and the migration of intraepithelial T lymphocytes,resulting in disease progression.38,39AKT1,protein kinase B,and PI3K/AKT/MTOR signaling abnormalities regulate the local immunity of OLP,40and miR-128b regulates MMP-2 expression and promotes the apoptosis of keratinocytes through the PI3K/AKT/MTOR signaling pathway.41MYC is involved in the malignant transformation of OLP.42These results indicate that HXQR is a multitarget treatment for OLP.

To further study the mechanism of action of HXQR in the treatment of OLP,we used R language to carry out GO and KEGG enrichment analysis of drug-disease common targets,and conducted clinical trials to verify the results.The main results of the GO analysis were response to lipopolysaccharide,response to molecule of bacterial origin,cytokine receptor binding,cytokine activity,receptor ligand activity,growth factor receptor binding,and other molecular processes.KEGG enrichment analysis showed that the main common drugdisease targets were inflammatory immunity,viral infection,and tumor-related pathways,among which the TNF signaling pathway was the key pathway by which HXQR treated OLP.TNF is a key regulator of the inflammatory response,and its pro-inflammatory effect is manifested by changing the expressions of leukocyte adhesion molecules,pro-inflammatory factors,and MMPs,including ICAM-1,VCAM-1,IL-6,and MMP-9.43,44It has been proved that TNF-α upregulates the secretion of MMP-9 in the T cells of OLP lesions.45Immunohistochemical results show that compared with normal skin,patients with OLP have higher positive expression rates of TNF and ICAM-1,and the expression of these factors are significantly correlated.46Clinical trials have shown that NF-kB-dependent TNF and IL-6 are significantly increased in patients with OLP.47Our ELISA test further showed that HXQR significantly reduced the expressions of IL-6,MMP-9,and ICAM-1 in the TNF signaling pathway.In conclusion,the TNF signaling pathway may be the mechanism by which HXQR treats OLP.Our clinical observations showed that HXQR had a significant effect on OLP,and significantly improved the clinical outcome of patients with OLP.Furthermore,HXQR had minimal toxicity compared with adrenocortical hormone,immunosuppressant drugs,retinoic acid,and other drugs.

OLP is an immune-mediated inflammatory mucosal disease with unknown etiology that may be caused by multiple genes and factors.TCM treatment emphasizes the combination of various herbs that workviathe overall regulation of many aspects.Through the combination of TCM network pharmacology and clinical trials,we elucidated the potential mechanism of HXQR in the treatment of OLP.However,the components of TCM prescriptions are complex,and further research is needed to confirm our findings.

In conclusion,HXQR is widely used in clinical practice and contains many components with pharmacological activities.Through the network pharmacology of TCM,we constructed the drug-disease network,PPI network,and identified the main active components and targets of HXQR.The GO and KEGG enrichment analysis showed the mechanism of HXQR.The clinical trial showed that HXQR significantly improved the clinical symptoms of OLP and effectively reduced the serum levels of IL-6,MMP-9,and ICAM-1 by regulating the TNF signaling pathway.In summary,HXQR treats OLP by reducing the inflammatory response through the TNF signaling pathway.