Di Zhang,Shengsuo Ma,Jianxin Sun,Bing Yang,Haoming Lin,Meijing Xie,Meina Huang,Guoping Zhao*

1College of Traditional Chinese Medicine,Jinan University,Guangzhou,510630,China.

Abstract Objective:The analgesic effect of Paeonia Lactiflora has been widely accepted in traditional Chinese medicine.But little is known about the potential mechanism.This study aims to elucidate the effective components and analgesic mechanism based on network pharmacology.Methods:TCMSPwas screened to collect the possible active ingredients and their CAS and SMILES was searched in Pubchem and further be used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets.Pain-related molecules were obtained from GeenCards database,and the predicted targets of Paeonia Lactiflora for pain treatment were selected by Wayne diagram.For mechanism analysis,the protein-protein interactions were constructed by String,the GO analysis and KEGG analysis were conducted in DAVID.Results:Through GO analysis and KEGG analysis,we found that the pain related signaling pathways mainly involved in serotonergic synapse,calcium signaling pathway,inflammatory mediator TRPchannels.Using network-based systems biology and molecular docking analyses,we predicted that 11 active ingredients in Paeonia Lactiflora has the analgesic effects with 97 potential targets.PRKCA,CASP3,ALOX15,SLC6A4,PRKCG,ALOX5,PRKCB,ALOX12,EGFR,ADRB2,RYR3,RYR1,NOS2,PTAFR,PRKCQ,and PRKCD were involved in the analgesic effects of Paeonia Lactiflora.Conclusion:Paeonia Lactiflora may alleviate pain through inflammatory mediator regulation of TRP channels,Ca2+signalingpathway and 5-HT receptor.PRKCA,PRKCB,PRKCD，PRKCQ,and PRKCG may be new targetsfor pain treatment.

Keywords:Paeonia lactiflora;Paeonia;network pharmacology;inflammatory mediator regulation of TRP channels

Introduction

Pain is a major clinical problem which could lead to impairing physical function and reduced quality of life,accounting for hundreds of billions of dollars in healthcare costs worldwide[1].The number of patients with pain in China might be above 9000 million,including acute pain aswell as chronic pain.Nowadays,second generation antiepileptic drugs carpentry and pregabalin are first-line drugs for these patients.Tricyclic antidepressants such as amitriptyline,imipramine and nortriptyline,exert analgesic effects through blocking 5-serotonin and norepinephrine reuptake which further inhibit the excitability of neurons.Opioid analgesics such as morphine,tramadol and hydrocodone are also used in clinical.But these drugs have many side effects and long-term use also leads to drug dependence.Therefore,it is necessary to search for new analgesics.

Paeonia Lactiflora is the main herb of Danggui Sini Decoction.It has verified that Danggui Sini Decoction can alleviate neuralgia through inhibiting the activity of TRP pathway and promote the proliferation of Schwann cells,thereby repairing sciatic nerve injury.Paeoniflorin is the main component of Paeonia lactiflora,which can protect Schwann cells from oxidative stress[2],injury and apoptosis[3,4].But little is known about the potential mechanism.This study aims to elucidate the effective components and analgesic mechanism based on network pharmacology.

Network pharmacology, which clarifies the synergistic effects and underlying mechanisms of multi-component and multi-target agents using the analysis of networks,is a suitable approach to measure the efficacy and to reveal the functional mechanisms of multi-target drugs[5].In our study,TCMSP was screened to collect the possible active ingredients and their CAS.The SMILESwas searched in Pubmed and further be used for reverse molecular docking in Swiss Target Prediction database to obtain potential targets.Pain-related molecules were obtained from GeenCards database,and the predicted targets of Paeonia Lactiflora for pain treatment were selected by Wayne diagram.The protein-protein interactions were constructed by String.The GO analysis and KEGG analysis were conducted in DAVID to discuss the potential mechanism of antineuralgic effects of Paeonia Lactiflora.

Materialsand methods

Screening of active components of Paeonia Lactiflora

The main components of Paeonia Lactiflora were identified by searching the pharmacological database and analysis platform of TCM system.The drug components(TCMSP:http://lsp.nwu.edu.cn/index.p h)were screened by combining the oral absorption(OB＜30%)and type drug index(DL＜0.18).And the CAS obtained from the TCMSP,while the drug "SMILES" was found in PubChem.(PubChem:http://www.ncbi.nlm.nih.gov/pccompo und).

Prediction of potential targets for active components

Log on to Swiss Target Prediction(http://www.swisstargetprediction.ch)and ROCS(http://targetfishing.molcalx.com.cn/index.html)and enter"SMILES"of monomer drugs with species limited to"Homo sapiens".The obtained protein target and corresponding Uniprot ID were imported into Excel.

Screening of pain targets

By inserting the keywords "pain", "ache","soreness" into the GeneCards database(https://www.genecards.org),we searched for the reported pain-related genes,removed the false positive genes.And the common target of Paeonia Lactiflora and pain was screened by Wayne diagram.

Acquisition of uniprot ID

According to the 1.3 Wayne diagram,common targets are obtained and their corresponding Uniprot IDs are searched.Uniprot KB(https://www.uniprot.org/)was used to identify the gene target ID.

Construction of"active component-target"network

Using components and predicted targets of Paeonia Lactiflora,the corresponding relationship between active components and target was established.Active ingredients and targets were used as nodes in the network nodes,and TXT text format was made.Cytoscape 3.5 was imported to construct the "active ingredient-action target network"of Paeonia Lactiflora.

Protein interaction network construction(PPI)

String database (https://string-db.org/) is a database containing known and predicted protein-protein interactions.The 1.3 Wayne diagram was input into String to obtain common targets,and the species was defined as"Homo sapiens"to obtain a protein-protein interaction relationship.The results were saved in the TSV format.

Biological process and pathway analysis

DAVID (https://david.ncifcrf.gov/) can provide large-scale functional annotations of genes or proteins,and can identify the most significant enrichment of biological annotations.The common target obtained in 1.3 was imported into DAVID database,and the species was limited to"Homo sapiens".GO analysis and KEGG pathway analysis were carried out on the target of Paeonia Lactiflora.

Results

The ative components of Paeonia lactiflora

Using TCMSF platform,85 main components of Paeonia Lactiflora were screened.There were 13 monomers meeting the criteria of OB(＞30%)and DL(＞ 0.18).Among them,12 components could be identified by Pubchem,11 monomers could obtain SMILES,and the monomers that could obtain SMILES structure were selected for further study(Table 1).We cannot find the “SMILES” of“Lactiflorin”in Pubchem.

Drug target prediction

The SMILESof 11 targets of Paeonia Lactiflora were input into Swiss Target Prediction and ROCSwebsites,and 438 targets were obtained.A total of 119 targets were obtained by removing repetitive targets.Uniprot IDs of 119 target proteins were identified by Uniprot KB(Table 2).

Construction of activecomponent-target network

The active ingredients and targets of Paeonia lactiflora were imported into Cytoscape(3.7.1)to construct the active ingredient-target network.Nodes of different colors represent active ingredients and targets of Paeonia lactiflora.The blue icon is the compound composition,and the yellow part is the drug target(Figure 1).

Screening of pain targets

By inserting the keywords "pain", "ache","soreness"into the GeneCards database,we searched 10895 pain-related genes.We listed the top 100 genes in the table below(Table 3).As well,we selected the 10895 genes and imported them into Webgestalt for KEGG enrichment(http://www.webgestalt.org/).We selected the top 20 channels to make a volcano map(Table 4,Figure 2).Moreover,inserting the keywords"pain" in KEGG, inflammatory mediator regulation of TRPchannels(map04750)is closely related to the pain.We got the map04750 from the KEGG(Figure 3).

Screening of pain therapeutic targets of Paeonia Lactiflora

The therapeutic target and pain related target of Paeonia Lactiflora were imported into Venny 2.1 to obtain the therapeutic target of Paeonia Lactiflora(http://bioinfogp.cnb.csic.es/tools/venny/index.html).Target No.1-97 was the related target of Paeonia lactiflora for pain treatment,while target No.98-119 was not related to pain treatment(Figure 4).

Construction of protein-protein interaction network and target analysis

Targets of Paeonia Lactiflora for pain treatment were input into String databasewith specieslimited to"Homo sapiens".Among them,the top 20 points in the score of the relationship between nodes are as follows(Table 5,Figure 5).

Table 1 Main active ingredients in Paeonia lactiflora

Table 2 Information of potential targets from Paeonia

Figure1 Network of active ingredient-target of Paeonia lactiflora

Table 3 The top 100 pain-related genes

Table 3 The top 100 pain-related genes(continued)

Table 4 KEGG pathway analysis of pain-rated genes

GO Classification and Enrichment Analysis

The common targets were imported into DAVID database with species limited to"Homo sapiens".The target of Paeonia Lactiflora for pain treatment were used to conduct GO analysis.GO analysis found 52 biological processes, 11 cellular components and 35 kinds of molecular function were involved in the mechanism of analgesic effects of Paeonia Lactiflora(Figure 6).

Figure 2 Pain-related pathway volcano map

Figure3 Inflammatory mediator regulation of TRPchannels(＊Red font part is pain related)

KEGGAnalysis

Common targets were imported into DAVID database with species limited to"Homo sapiens".KEGG enrichment analysis was carried out for Paeonia Lactiflora.KEGG enrichment analysis involves 37 pathways.The first 10 pathways areselected and plotted in Figure 7.The possible analgesic mechanisms of Paeonia Lactiflora include:nitrogen metabolism (12/7.9%), ovarian steroidogenesis(4/4.6%),steroid hormone biosynthesis(7/4.6%),bile secretion (7/4.6%),serotonergic synapse(8/5.2%),morphine addiction (7/4.6%).;calcium signaling pathway(9/5.9%);inflammatory mediator regulation of TRP channels(6/3.9%).The pain-related pathways are serotonergic synapse,calcium signaling pathway,inflammatory mediator regulation of TRP channels.We can find these in the pathways above,which are PRKCA,CASP3,ALOX15,SLC6A4,PRKCG,ALOX5,PRKCB,ALOX12,EGFR,ADRB2,RYR3,RYR1,NOS2,PTAFR,PRKCQ,PRKCD(Table 6).

Figure 4.Targets of Paeonia lactiflora for pain

Table 5 Score of Inter-node correlation

Discussion

Figure 5 Interaction diagram of target proteins of Paeonia lactiflora(＊Line thickness indicates the strength of data support)

Figure 6 GOAnalysis of target genes of Paeonia lactiflora

Paeonia Lactiflora is a component of Danggui Sini Decoction.Danggui Sini Decoction is used to cure various kinds of pain,such as diabetic neuralgia,postpartum abdominal pain and limb pain,dysmenorrhea,migraine.Therefore,it is necessary to study the mechanism of Danggui Sini Decoction in the treatment of pain.There were 11 monomersof Paeonia lactiflora,including paeoniflorgenone,paeoniflorin_qt,albiflorin_qt, Mairin, (+)-catechin, paeoniflorin,palbinone, kaempferol, beta-sitosterol, Sitosterol,benzoyl paeoniflorin,Lactiflorin.A total of 119 targets were obtained by removing repetitive targets.Therapeutic targets of Paeonia Lactiflora were 97.Through KEGG analysis of 10895 pain targets,we obtained 20 related pathways,containing metabolic pathways,pathways in cancer,microRNAs in cancer,human cytomegalovirus infection,alzheimer disease,adrenergic signaling in cardiomyocytes,fluid shear stress and atherosclerosis,estrogen signaling pathway,dopaminergic synapse,relaxin signaling pathway,carbon metabolism,glutamatergic synapse,cholinergic synapse, chagas disease,AGE-RAGE signaling pathway in diabetic complications,phosphatidylinositol signaling system,inflammatory mediator regulation of TRP channels,glycerophospholipid metabolism, prostate cancer,circadian entrainment.Only inflammatory mediator regulation of TRP channels are bound with the pain(Figure 3).Through GOanalysis and KEGGanalysis of 97 targets,we found that the pain related signaling pathways mainly involved in serotonergic synapse,calcium signaling pathway,inflammatory mediator regulation of TRP channels.PRKCA,CASP3,ALOX15,SLC6A4,PRKCG,ALOX5,PRKCB,ALOX12,EGFR,ADRB2,RYR3,RYR1,NOS2,PTAFR,PRKCQ,and PRKCD were involved in the analgesic effects of Paeonia Lactiflora.

Figure 7 KEGG pathway analysis of target genes of Paeonia lactiflora

Table 6 Pain-related pathway and corresponding gene of Paeonia lactiflora

It has been manifested that the principal parts of paeonia Lactiflora can treat pain.Paeoniflorin can alleviate persistent spontaneous pain and hypersensitivity caused by bee venom,relieve pain caused by formalin, and significantly improve the secretion of inflammatory factors such as IL-1βand TNF-αand iNOS induced by Parkinson's disease[6].Qiang-Song Wang held that paeoniflorin and albiflorin could inhabit the expressions of iNOS,COX-2,IL-6,and TNF-α[7],which are bound up with the pain[8].Paeoniflorin can check down-regulate the expression of TRPV1 protein[9].Qing-ping Li held that paeoniflorin can inhibit plantar incision-induced microglia TLR4/MMP-9/2/IL-1β signalling pathway and suppress postoperative pain[10].Zhi-Li Huang considered that paeoniflorin exerted analgesic effects via adenosine A1Rs and might be of potential use in the treatment of neuropathic pain[11].Yi-Xin Fan demonstrated that paeoniflorin promoted the efflux of HSP70 from the cytoplasm into the extracellular environment as well as induced SOCS3 expression in a TLR4-dependent manner [12].Dan-Li Zhou considered that paeoniflorin is an effective drug for the treatment of neuropathic pain in rats via inhibiting the phosphorylation of ASK1[13].Jian-Yu Zhou found that both paeoniflorin and albiflorin could inhibit the activation of p38 mitogen-activatedprotein kinase (p38 MAPK)pathway in spinal microglia and subsequent unregulated proinflammatory cytokines interleukin-1β(IL-1β)and tumor necrosis factor-α (TNF-α),and Albiflorin suppressed the overelevated expression of phosphorylation of c-Jun N-terminal kinases(p-JNK)in astrocytes,and decreasing the content of chemokine CXCL1 in the spinal cord [14].Catechin,(-)-Epicatechin-3-O-β-d-allopyranoside, suppressed inflammation and inflammatory pain and adjuvant-induced arthritis[15].Kishore found that kaempferol attenuated oxidative stress mediated release of pro-in-flammatory cytokines which might be responsible for diabetes-induced nerve damage[16].Mumtaz Ali concluded that overall analgesic activity of the Kaempferol seems to involving COX-2 inhibition and activation of cholinergic receptors[17].

The mechanism of pain isexceptionally complicated,which is still an unsolved mystery.However,inflammation-regulated TRP pathway plays an important role in peripheral neuralgia[9].The TRP channels are located on the cell membrane of non-selective-cation channels.TRP family includes TRPV1-4,which are sensitive to heat pain,TRPA1 and TRPM8,which are sensitive to cold,and TRPV4 and TRPA1,which are sensitive to mechanical pain[18,19].Moreover,5-HT plays an important role in pain.The peripheral pronociceptive role of 5-HT is well established to date, and central serotonin neurotransmission is also implicated in the modulation of pain signals[20].Serotoninergic 5-HT2and 5-HT7receptor agonists have repeatedly been shown to promote motor function recovery in adult rodents after spinal cord injury[21].Furthermore,Ca2+participates in many physiological and biochemical reactions,and its mechanism is very complex.It can regulate the activity of various enzymes,maintain the integrity and stability of cells.The instantaneous increase of Ca2+concentration can activate a series of calcium-sensitive signaling pathways,such as activating protein kinase,causing protein phosphorylation,thus activating downstream signaling molecules,leading to the synthesis of new proteins for gene transcription.Ca2+signaling pathway can activate TRP pathway.Current clinical use of Ca2+antagonists can effectively alleviate pain [22].Inflammation-regulated TRP pathway embraced TRP family,5-HT receptor,and Ca2+signaling pathway (Figure 3).KEGG enrichment analysis of Paeonia Lactiflora involves 37 pathways.The pain-related pathways are Serotonergic synapse,Calcium signaling pathway,Inflammatory mediator regulation of TRP channels,which accorded with the pathway above in 2.4.Therefore,we can hold that Paeonia Lactiflora may alleviate pain through inflammation-regulated TRPpathway.

The first line drugs include tricyclic antidepressants(TCAs),selective serotonin norepinephrine reuptake inhibitors(SSNRI),anticonvulsants,and topical lidocaine.The clinical applications of gabapentin,pregabalin are quite extensive.The second line drugs include opioids,tramadol.Due to the side effects of opioids,it limits the usage on the treatment of neuropathic pain.The third line drugs include antiepileptic drugs such as citalopram,paroxetine,mexiletine,and others.But drug dependence and addiction limit the use of analgesics.It is imperative to find new analgesics.The principal parts of paeonia Lactiflora can treat pain.PRKCA,PRKCB,PRKCD,PRKCQ,PRKCG,EGFR,ALOX5,ALOX12,ALOX15,NOS2,RYR1,RYR3 ADRB2,CASP3,SLC6A4,and PTAFR were involved in the analgesic effects.EGRF has been regarded as the standard first-line treatment of advanced non-small cell lung cancer in patients[23].CASP3 is intently related to apoptosis[24].That ADRB2 activation inhibits apoptosis can be used to guide clinical trials of ADRB2 antagonist propranolol as potential life-extending therapy for prostate cancer[25].Nitric oxide is an endogenous molecule that plays pivotal physiological and pathophysiological roles[26].The ryanodine receptor calcium release channel(RYR1,RYR3)is central to cytoplasmic Ca2+signalling in skeletal muscle,the heart,and many other tissues,including the central nervous system,lymphocytes,and stomach[5].Whether they can treat pain still requires generous basic experiments.

Protein kinase C(PKC)proteins are a group of well-conserved,intracellular signaling enzymes expressed in all cells and tissues[27].At the intersection of the second messenger and the activation of intracellular proteins lies PKC,a family of serine/threonine kinases[28].PKC proteins play crucial parts in serotonergic synapse,calcium signaling pathway,and inflammatory mediator regulation of TRP channels.PRKCA,PRKCB,PRKCD，PRKCQ,and PRKCG may be new pain-curing targets.

Conclusion

This study showed that 11 active components of Paeonia Lactiflora could act on multiple targets to treat pain,mainly involving three main signaling pathways:TRP signaling pathway,Ca2+signaling pathway and 5-HT receptor.PRKCA,PRKCB,PRKCD,PRKCQ,and PRKCG may be new targets for pain treatment.

Acknowledgements

This work was supported by the National Natural Science Foundation of China(Grant No.81874404).