Exploring the active components and potential mechanisms of Qingre Liangxue Decoction in the treatment of psoriasis vulgaris with blood-heat syndrome based on UHPLC-Q-exactive Orbitrap/MS and network pharmacology

doi:10.21203/rs.3.rs-3806518/v1

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Research Article

Exploring the active components and potential mechanisms of Qingre Liangxue Decoction in the treatment of psoriasis vulgaris with blood-heat syndrome based on UHPLC-Q-exactive Orbitrap/MS and network pharmacology

https://doi.org/10.21203/rs.3.rs-3806518/v1

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Background

Psoriasis is a common, chronic, inflammatory papulosquamous skin disease which is difficult to be cured. Qingre Liangxue Decoction(QRLXD), originated from the classic formula Xijiao Dihuang Decoction, as a famous classical prescription for the treatment of psoriasis vulgaris (PV) with blood-heat syndrome in the traditional Chinese medicine (TCM), shows good efficacy in clinical practice. Since the mechanism of action is still unclear, this article was aimed to profile the absorbed prototype constituents in rat serum after its oral administration and explore the molecular mechanism of QRLXD using network pharmacology and molecular docking technology.

Methods

First, chemical components of QRLXD absorbed in vivo were comprehensively analyzed by Ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-Exactive-Orbitrap-MS) method. The Swiss Target Prediction was used to predict corresponding potential target genes of active compounds. Simultaneously, 5 disease databases were used to search and filter psoriasis vulgaris (PV) with blood-heat syndrome targets, and then we constructed a protein-protein interaction (PPI) network. Metascape were utilized for GO and KEGG enrichment analysis. After that, we constructed a network of "QRLXD-compounds-target-pathway-PV" by Cytoscape. Molecular docking simulation was conducted to further refine the drug-target interaction by AutoDock software.

Results

A total of 68 ingredients were identified in vitro and 18 ingredients in vivo, targeting 523 proteins, among which 129 were related to psoriasis. The protein with a higher degree in the PPI network is TNF, ALB, AKT1, STAT3 and EGFR. 1488 GO biological process items and 162 KEGG signal pathways were enriched. GO and KEGG indicated that QRLXD treatment of psoriasis mainly involves protein kinase activity, protein tyrosine kinase activity, phosphotransferase activity, alcohol group as acceptor and other biological processes.The signaling pathways mainly include the pathways in cancer,PI3K-Akt ,MAPK and IL-17 signaling pathways. The main compounds and the target protein had a good binding ability in molecular docking. The molecular docking results revealed that Dibutyl phthalate, Ethyl benzoylformate, Scopoletin, Albiflorin R1, 3-Hydroxycoumarin have higher affinity with TNF, ALB and AKT1.

Conclusion

This study suggests that QRLXD against PV with blood-heat syndrome through multiple components, multiple targets and multiple pathways. This study identified effective compounds of QRLXD in vivo and predicted potential molecular mechanisms of QRLXD in psoriasis treatment, serving as an important basis for the modernization of QRLXD.

Qingre Liangxue Decoction

psoriasis vulgaris

network pharmacology

molecular docking

therapeutic mechanism

Psoriasis is a common, chronic, auto-immune papulosquamous skin disease presenting at any age, and affects approximately 2,3% of the global population^[1]. The classic morphology of psoriasis usually manifests well demarcated erythema covered in silvery scales. Removal of the adherent scales can result in small bleeding points (known as the Auspitz sign)^[2]. The most frequent recognised is psoriasis vulgaris(PV). According to the consensus of TCM, PV mainly include blood-stasis syndrome, blood-heat syndrome, and blood-dryness syndrome. Psoriasis vulgari with blood-heat syndrome is characterized by bright red papules with pruritus. Although the exact pathogenesis of psoriasis is not totally understood, interleukin-17 (IL-17), interleukin-23 (IL-23) and helper T cells type 17 (Th17) axis has been identified as key drivers of PV pathogenesis^[3]. Topical therapy (corticosteroids, vitamin D3 analogues and calcineurin inhibitors), oral systemic therapies(methotrexate, ciclosporin and acitretin)^[4], phototherapy (UVA and UVB) and biologics (anti-TNFα, anti-IL17, anti-IL-12p40 or IL-23p40 and anti-IL-23p19) remain the main therapies for psoriasis. However, none of them can cure psoriasis and these medications had obvious systemic side effects^[5]. Therefore, it is very urgent to develop new anti-psoriatic medicine with low side effects.

Traditional Chinese medicine (TCM) has its unique advantages in disease management because of the characteristics of multiple components, multiple targets and multiple pathways. Qingre Liangxue Decoction(alias: Quyin Granule) has been used to treat psoriasis vulgari with blood-heat syndrome for over 20 years in China-Japan Friendship Hospital. As shown in our clinical observations, QRLXD shows good efficacy in clinical practice and is widely recognized by patients^[6]. However, its active compounds, pharmacological effects and underlying mechanisms have not been studied yet.

Although there are many chemical components in traditional Chinese medicine, only the components absorbed can be the real effective components^[7^]. In order to explore the main components and predicts molecular biological mechanisms of QRLXD treating PV to the greatest extent, we used UPLC-Q‐TOF‐MS to profile and characterize chemical components of QRLXD and the absorbed constituents in rat sample after its oral administration, followed by targets prediction, protein association, GO and KEGG pathway analysis to explore the molecular mechanism of QRLXD using network pharmacology, and finally verified the results with molecular docking, increasing the possibility of the prediction results. In this paper, we explore the main components and predicts molecular biological mechanisms of QRLXD in treating PV, in order to provide relevant references for follow-up research.

The flowchart of the present study is illustrated in Fig. 1.

2.1. Preparation of QRLXD

QRLXD consists of 8 herbs (Table 1) and were purchased from China-Japan Friendship Hospital. To prepare the QRLXD solution, all 8 herbs were soaked with distilled water for 1h .Then the materials of QRLXD were co-decocted in water twice. Water added in the first decoction was 12 times the total weight of the herbs for 1.5h. In the second decoction, it was 10 times the total weight of the herbs for 1h. The two decoctions were then combined and concentrated to 1g/mL and were stored at 4°C.

Table 1

Chinese traditional medical herbs of QRLXD
Chinese name	Accepted scientific name	Plant part	Batch number	Amount (g)
Baiying	Solanum lyratum Thunb.	Herb	220916002	15
Shemei	Duchesnea indica (Andr.) Focke	Herb	221014001	10
Zicao	Arnebia euchroma (Royle) Johnst.	Root	220804001	15
Tufuling	Smilacis Glabrae Rhizome	Root	220602002	15
Baihuasheshecao	Hedyotis Diffusa	Herb	230520002	15
Chishao	Radix Paeoniae Rubra	Root	220909001	15
Mudanpi	Paeonia suffruticosa Andrews	Cortex	220827001	15
Shengdihuang	Rehmanniae Radix	Root	230421002	15

2.2. Materials and Reagents

3,4-Dihydroxybenzoic acid(PCS220412), Gallic acid(PCS221026), Paeonol(PCS220425), Paeoniflorin (PCS220425), 5-O-caffeoylshikimic acid(PCS220314), Desacetyl asperulosidic acid (PCS220620), Rehmannioside D(PCS221110), Paeonolide(PCS221203), Forsythoside E(PCS220806), Oxypaeoniflorin(PCS220412), Benzoylpaeoniflorin(PCS220412), and Caffeic(PCS220412) were purchased from Chengdu HerbSubstance Co.,Ltd.(purity ≥ 98%). Ultra-pure water was prepared using a Milli-Q water purification system (Bedford, France). Acetonitrile and methanol were of UPLC grade (Merck, Darmstadt, Germany). All other reagents were of analytical grade. LC Ultimate 3000 Series System tandem Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific, US) was utilized.

2.3. Animals and treatments

12 SD male rats (220 ± 20g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd [SCXY(Jing)2021-0006] and were kept at an ambient temperature (22–26°C) and humidity (40–70%), under a 12h light-dark cycle, had free access to food and drinking sterile water for 7 days before the experiment. All animal experiment protocols were approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (approval number: BUCM-2023091002-3198).

12 rats were randomly divided into 2 groups (n = 6/group): the normal control (NC) group, the QRLXD group (8.3g/ml). Rats were administered drugs or 0.9% normal saline orally once a day for 3 days. On the 4th day, under sodium pentobarbital, the mice were euthanized by cervical dislocation, and their organs and orbital blood were collected for UHPLC-Q-TOF-MS Analysis.

2.4. Sample Collection and Pretreatment Method

Blood Sample: Blood samples were collected from the orbital blood about 0.3mL at 0.5h, 1h, 1.5h, 2h after the last administration of QRLXD and left to stand at room temperature for 30min in the anticoagulation tube and then centrifuged at 3000 rpm/min for 10 min at 4°C to obtain serum, then 200L methanol was added to each 100µL serum and vortexed for 3 min. Blood samples were centrifuged at 13000 rpm/min at 4°C for 15min. After transferring to a new centrifuge tube, the supernatant was dried with nitrogen at 40°C and the residue was reconstituted in 100µL of methanol. The supernatant was centrifuged at 13000 rpm/min at 4°C for 15min and prepared for UHPLC-Q-TOF-MS analysis.

Organ Sample: Following exsanguination, heart, liver, spleen, lung, kidney, brain were rapidly sampled from the mice, rinsed with normal saline and blotted onto a filter paper. 2.0g of tissues was homogenized in 0.9% sodium chloride solution (1:1, w/v) to prepare the homogenate (0.2 g/mL).Then,organ samples were centrifuged at 13000rpm/min at 4°C for 15min. After transferring to a new centrifuge tube, methanol(1:3, w/v) was added to each 100µL sample.The supernatant was centrifuged at 13000 rpm/min at 4°C for 15min and prepared for UHPLC-Q-TOF-MS analysis.

2.5. UHPLC-Q-TOF-MS Analysis

The HPLC conditions: Chromatographic analysis was performed using a Waters ACQUITY UHPLC BEH C₁₈ Column(2.1×150mm, 2.6µm). The mobile phase consisted of methanol (A) and 0.1% formic acid-water solution (B). The gradient profile was as follows: (0-10min, 2%-50%A;10-25min, 50%-95%A;) with the flow rate kept at 0.3mL/min. The column temperature was 40°C, the volume injected was 3µl.Mass spectrum conditions: Mass spectrometry analysis was performed using a heated electrospray ionization (HESI) source. The full scan range was m/z100-1500Da and the spray voltage was 3.5kV. During the analysis, other operating parameters used for MS analysis were 40arb for sheath gas flow, 10arb for auxiliary gas flow rate, 320°C for spray temperature, 350°C for Aux gas heater temperature. The resolution of MS and MS/MS was set at 70000 and 35000, respectively.

2.6. Target collection of QRLXD and PV

The potential targets of the compounds were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php)^[⁸^,⁹^] and swisstargetprediction database (http://www.swisstargetprediction.ch/)^[¹⁰^].The obtained targets were imported into UniProt database (https://www.UniProt.org/) for gene name transformation. Genes related to PV were acquired from five databases, including the GeneCards databases (http://www.genecards.org/)^[¹¹^], the OMIM database (http://www.omim.org/)^[¹²^],the Therapeutic Target database(TTD, http://db.idrblab.net/ttd/)^[¹³^], DrugBank database (https://go.drugbank.com/)^[¹⁴^]and PharmGkb database(https://www.pharmgkb.org/)^[¹⁵^],using “psoriasis vulgari” as the keyword. Finally, the gene library of PV was established by eliminating repeated targets. The intersection targets between the PV-related genes and the predicted QRLXD targets were obtained and Jvenny (http://www.bioinformatics.com.cn)^[¹⁶^] was used to construct a Venn diagram for visualization.

2.7. PPI network for core target selection

The potential targets of QRLXD against PV were imported into the STRING database (http://string-db.org)^[¹⁷^]. The species was limited to “Homo sapiens” and the confidence score was set to ≥ 0.4. Cytoscape3.9.1 was used to construct a network of potential key targets and to perform a systematic analysis of the network parameters.

2.8. GO and KEGG enrichment analysis

Metascape(http://metascape.org)^[¹⁸^] was used to analyze the enrichment of GO(gene ontology) annotation in the biological process(BP), cellular component(CC) and molecular function(MF) and KEGG(Kyoto encyclopedia of genes and genomes) pathway. Terms with P < 0.01, minimum count of 3 and enrichment factor > 1.5 were considered significant. Subsequently, GO and KEGG data were uploaded to the Bioinformatics (http://www.bioinformatics.com.cn/) platform for visual analysis.

2.9. Construction of “QRLXD-compounds-target-pathway-PV” network

The components of QRLXD, the common target genes of QRLXD and PV, and the top 20 signaling pathways of the genes by KEGG analysis were selected as nodes. The corresponding relationships were constructed into a visual “QRLXD-compounds-target-pathway-PV” network with Cytoscape 3.9.1 software and use Cyto NCA for topology analysis. The main active ingredients of QRLXD in the treatment of PV with blood-heat syndrome were screened according to degree centrality (DC), closeness centrality (CC) and betweenness centrality (BC).

2.10. Molecular Docking Analysis

The top five compounds from the core QRLXD compounds were selected for molecular docking with the most important proteins from the core targets in PPI for PV treatment. Drug molecule structures were downloaded from PubChem databases (https://pubchem.ncbi.nlm.nih.gov/)^[¹⁹^]. The targets crystal structure was acquired from the PDB protein database (https://www.rcsb.org/)^[²⁰^], imported into AutoDockTools1.5.7, dehydrated, hydrogenated, and used for ligand separation. The PDBIDs were TNF(6Q01), ALB(3MRK) and AKT1 (1UNQ). AutoDockVina and PyMOL software were used for molecular docking and visualization.

3.1. Identification of compounds in QRLXD

A total of 68 chemical constituents were identified, including 20 monoterpenoids, 8 iridoid glycosides, 10 phenolic acids, 4 glycosides, 2 organic acids, 2 phenylethanolsides, 1 penylpropanoid, 2 coumarins, 1 alkaloids, 1 flavonoids, 1 cyclic peptides, 1 naphthoquinone,1 furfural, 4 pentagalloylglucose and 8 other groups, shown in Fig. 2. 13 compounds were confirmed by comparison with reference standards.

3.2. Analysis of Prototype Constituents of QRLXD in Rat Sample

The rat sample was analyzed by UHPLC-Q-TOF-MS method. The BPIs of QRLXD absorbed in vivo are shown in Fig. 3. According to the accurate mass and secondary fragment, the chemical compounds in QRLXD and rat sample were identified through comparison with data of chemical components of QRLXD.18 ingredients were identified in vivo and found the source in Table 3.

Table 3

Identification of chemical constituents of QRLXD absorbed by UPLC-Q-TOF-MS
NO.	Identity	Formula	Adduct Type	Average m/z	RT (min)	MS/MS Spectrum	Source	Category	Distribution
1	sucrose or maltose or palatinose	C₁₂H₂₂O₁₁	[M-H]⁻	341.1089	1.07	119.0330,113.0229,89.0229,71.0136	PSA、RPR、RR、HD、SLT	other	-
1	sucrose or maltose or palatinose	C₁₂H₂₂O₁₁	[M + Na]⁺	365.1052	1.09	365.1054,310.1934,203.0526	PSA、RPR、RR、HD、SLT	other	-
2	8-debenzoylpaeoniflorin	C₁₆H₂₄O₁₀	[M + Na]⁺	399.1260	1.7	237.0736	RPR	monoterpenoids	heart;liver;kidney;blood;
3	1-O-galloyl-β-D-glucose or isomer	C₁₃H₁₆O₁₀	[M-H]⁻	331.0673	1.89	169.0142	PSA	pentagalloylglucose	-
4	tyrosine	C₉H₁₁NO₃	[M + H]⁺	182.0811	1.99	136.0609	PSA	other	heart;spleen;lung;kidney;brain;blood;
5	5-hydroxymethylfurfural	C₆H₆O₃	[M-H]⁻	125.0245	2.21	124.0167,123.0087,97.0295,95.0139, 79.0190,69.0346	RR、PSA	furfural	-
6	scandoside	C₁₆H₂₂O₁₁	[M-H]⁻	389.1087	2.51	209.0455,183.0657,165.0557,147.0451	HD	iridoid glycosides	-
7	gallic acid*	C₇H₆O₅	[M-H]⁻	169.0144	2.57	125.0246,124.0165, 107.0138, 97.0295,81.0346,79.0189,69.0346	RR、RPR、PSA	phenolic acids	-
8	adenosine	C₁₀H₁₃N₅O₄	[M + H]⁺	268.1041	2.58	136.0618,119.0353	RR、PSA	other	liver;
9	1-O-galloyl-β-D-glucose	C₁₃H₁₆O₁₀	[M-H]⁻	331.0673	2.79	213.9121,169.0143,124.0164	PSA	pentagalloylglucose	-
10	1-O-β-D-glucopyranosylp-paeonisuffrone or isomer	C₁₆H₂₄O₉	[M + COOH]⁻	405.1404	3.14	359.1344,197.0819,179.0713,122.0372	RPR、PSA	other	-
11	acetylcatalpol or methyl (1S,4aS,5R,7aS)-1-(β-D-glucopyranosyloxy)-5-hydroxy-7-(hydroxymethyl)-1,4a,5,7a-tetrahydrocyclopenta[c]pyran-4-carboxylate	C₁₇H₂₄O₁₁	[M-H]⁻	403.1248	3.46	195.0659,179.0561	RR	iridoid glycosides	-
12	deacetylasperulosidic acid*	C₁₆H₂₂O₁₁	[M-H]⁻	389.1087	3.78	227.0571,209.0455,191.0356,183.0663, 165.0556,147.0453	HD	iridoid glycosides	Kidney;
13	6-O-galloylsucrose	C₁₉H₂₆O₁₅	[M-H]⁻	493.1205	3.81	331.0667,313.0567,169.0142,125.0244	PSA	pentagalloylglucose	-
14	syringic acid or ethyl gallate	C₉H₁₀O₅	[M-H]⁻	197.0456	3.83	123.0452	SGR、PSA、RPR	phenolic acids	-
15	rehmannioside D^*	C₂₇H₄₂O₂₀	[M + COOH]⁻	731.2261	3.87	685.2211,505.1548,343.1023,341.1100, 323.0983,263.0771,221.0666,181.0506, 179.0560, 161.0456,89.0244	RR	iridoid glycosides	-
16	gentisic acid5-O-glucoside	C₁₃H₁₆O₉	[M-H]⁻	315.0724	3.89	152.0115,108.0217	HD	other	-
17	mudanoside B	C₁₈H₂₄O₁₄	[M-H]⁻	463.1098	3.92	403.0879,373.0779,343.0671,301.0566,241.0354,169.0142,125.0244	PSA	monoterpenoids	-
18	galloylsurose or isomer	C₁₉H₂₆O₁₅	[M-H]⁻	493.1205	4.04	331.0672,313.0567,169.0142,125.0244	RPR、PSA	pentagalloylglucose	-
19	protocatechuic acid^*	C₇H₆O₄	[M-H]⁻	153.0195	4.13	109.0297,108.0217,91.0189	RR、HD、RPR、PSA	phenolic acids	-
20	mudanoside A	C₁₄H₁₈O₉	[M-H]⁻	329.0881	4.45	167.0349	PSA	monoterpenoids	-
21	decaffeoyl verbascoside or forsythoside E^*	C₂₀H₃₀O₁₂	[M-H]⁻	461.1666	4.92	461.1703,315.1088,135.0452,85.0295	RR、PSA	phenylethanolsides	-
22	p-hydroxybenzoic acid	C₇H₆O₃	[M-H]⁻	137.0244	4.96	137.0249,119.0138	SLT、PSA	phenolic acids	liver;spleen;blood;
23	galloyl desbenzoyl paeoniflorin or isomer	C₂₃H₂₈O₁₄	[M-H]⁻	527.1408	5.27	527.1407,497.1302,451.1270	RPR、PSA	monoterpenoids	-
24	8-epiloganic acid	C₁₆H₂₄O₁₀	[M-H]⁻	375.1299	5.32	213.0770,169.0869,151.0765,119.0348,113.0244,89.0244	RR	iridoid glycosides	heart;spleen;
25	unknown	C₁₇H₂₄O₁₂	[M-H]⁻	419.1198	5.67	401.1078,357.1222,239.0558,221.0457,195.0662,177.0556	HD	-	-
25	unknown	C₁₇H₂₄O₁₂	[M + Na]⁺	443.1159	5.68	263.0529,203.0527,185.0424	HD	-	-
26	p-hydroxy-cinnamic acid	C₉H₈O₃	[M-H]⁻	163.0401	5.75	119.0502,93.0345	DIF、HD	penylpropanoid	spleen;
27	asperuloside acid	C₁₈H₂₄O₁₂	[M-H]⁻	431.1198	5.77	431.1232,251.0550,225.0760,165.0558	HD	iridoid glycosides	-
28	10-acetyl scandoside	C₁₈H₂₄O₁₂	[M + Na]⁺	455.1158	5.80	275.0524,249.0730,185.0424	HD	monoterpenoids	-
29	oxypaeoniflora^*	C₂₃H₂₈O₁₂	[M + Na]⁺	519.1472	5.96	519.1473,381.1159,357.0938,323.0732,219.0625,185.0420	RPR、PSA	monoterpenoids	-
30	oxypaeoniflora isomer	C₂₃H₂₈O₁₂	[M + COOH]⁻	541.1564	5.99	495.1511,477.1395,465.1383,333.0987	RPR	monoterpenoids	-
31	unknown	C₂₆H₂₈O₁₄	[M-H]⁻	563.1378	6.00	563.1617	HD	-	-
32	quinic acid derivative	C₁₆H₁₈O₉	[M-H]⁻	353.0882	6.14	191.0561,135.0452	HD	other	-
33	3-hydroxycoumarin	C₉H₆O₃	[M + H]⁺	163.0389	6.16	117.0333, 107.0490, 89.0385	PSA	coumarins	liver;brain;blood;
34	chlorogenic acid	C₁₆H₁₈O₉	[M + H]⁺	355.1021	6.24	191.0562	RPR	phenolic acids	-
35	brevifolin carboxylic acid	C₁₃H₈O₈	[M-H]-	291.0150	6.54	247.0247	DIF	phenolic acids	-
36	catalpin	C₂₂H₂₆O₁₂	[M + H]⁺	483.1471	6.61	483.1468	RR	iridoid glycosides	-
37	albiflorin R1 or isomer	C₂₃H₂₈O₁₁	[M + COOH]⁻	525.1614	6.62	167.035	RPR	monoterpenoids	blood;
38	paeoniflorin^*	C₂₃H₂₈O₁₁	[M + Na]⁺	503.1521	6.65	503.1519	RPR、PSA	monoterpenoids	liver;spleen;kidney;brain;blood;
			[M + H]⁺	481.1702	6.66	197.0810,179.0703,121.0647,105.0335
			[M + COOH]⁻	525.1614	9.15	449.1443,327.1087,165.0556,121.0294
			[M-H + Cl]-	515.1325	9.19	449.1433
39	apiopaeonoside	C₂₀H₂₈O₁₂	[M + COOH]⁻	505.1567	6.97	293.0885,233.0666,165.0556,150.0324	PSA	glycosides	-
40	paeonolide^*	C₂₀H₂₈O₁₂	[M + Na]⁺	483.1471	6.98	483.1452,317.0843	PSA	glycosides	-
41	deacetylasperuloside	C₁₆H₂₀O₁₀	[M-H]⁻	371.0985	7.21	249.0613,231.0506,121.0295	HD	iridoid glycosides	-
42	6'-O-β-glucopyranosylalbiflorin	C₂₉H₃₈O₁₆	[M + COOH]⁻	687.2152	7.28	611.1968,593.1880,519.1724, 489.1617,479.1532,121.0295	RPR、PSA	monoterpenoids	-
43	ethyl benzoylformate	C₁₀H₁₀O₃	[M + H]⁺	179.0702	7.34	161.0595,133.0648,105.0699,79.0541	PSA	other	heart;blood;
44	moudanpioside Ⅰ	C₂₃H₂₈O₁₁	[M-H]⁻	479.1556	7.44	121.0294	RPR、PSA	monoterpenoids	-
45	albiflorin	C₂₃H₂₈O₁₁	[M + Na]⁺	503.1521	7.49	503.1519,341.1000,219.0630	RPR	monoterpenoids	-
46	4-methoxycinnamic acid	C₁₀H₁₀O₃	[M + H]⁺	179.0702	7.51	133.0649,77.0384	HD	penylpropanoid	heart;blood;
47	moudanpioside E	C₂₄H₃₀O₁₃	[M-H]⁻	525.1614	7.52	449.1455,327.1085,177.0553,165.0556,121.0295,123.0452	RPR、PSA	monoterpenoids	blood;
48	caffeic acid^*	C₉H₈O₄	[M + H]⁺	181.0494	7.71	163.0390,135.0440,107.0490	SGR、RR	phenolic acids	-
49	5-O-caffeoylshikimic acid^*	C₁₆H₁₆O₈	[M-H]⁻	335.0774	7.72	179.0349,161.0244,135.0452	SGR	flavonoids	-
50	suffruticoside B/D	C₂₇H₃₂O₁₆	[M-H]⁻	611.162	7.81	611.1622,445.0989,301.0573,283.0454, 211.0250,169.0142,165.0557,125.0245	PSA	glycosides	-
51	scopoletin^*	C₁₀H₈O₄	[M + H]⁺	193.0496	8.03	193.0499,178.0259,150.0312,137.0597,133.0284,105.0032	RPR	coumarins	heart;lung;brain;blood;
52	moudanpioside D	C₂₄H₃₀O₁₂	[M + COOH]⁻	555.1719	8.13	479.1566,327.1082,165.0556,151.0401	PSA	monoterpenoids	-
52	moudanpioside D	C₂₄H₃₀O₁₂	[M + Na]⁺	533.1627	8.14	533.1625,371.1098	PSA	monoterpenoids	-
53	suffruticoside A/C	C₂₇H₃₂O₁₆	[M-H]⁻	611.162	8.41	611.1614,445.0988,301.0572,169.0142,165.0556,125.0244	PSA	glycosides	-
54	paeoniflorin derivative	C₁₁H₁₆O₃	[M + H]⁺	197.1172	8.51	179.1077,161.0960,137.0603,135.1168,123.1167,109.0648	RPR	monoterpenoids	-
55	resacetophenone	C₈H₈O₃	[M-H]⁻	151.0401	8.67	135.0089	PSA	phenolic acids	-
56	galloylpaeoniflorin or isomer	C₃₀H₃₂O₁₅	[M-H]⁻	631.1673	8.86	631.1669,613.1561,509.1290,491.1197, 463.1256,399.0932, 375.0715,331.0676,313.0564,271.0460,241.0350, 211.0249,169.0139,125.0244,121.0296	RPR、PSA	monoterpenoids	-
57	acetoxypaeoniflorin	C₂₅H₃₀O₁₂	[M + COOH]⁻	567.1721	9.28	491.1544,121.0294	PSA	monoterpenoids	-
58	benzoic acid	C₇H₆O₂	[M-H]⁻	121.0295	9.37	121.0292	RPR、PSA	organic acids	heart;liver;spleen;lung;kidney;brain;blood;
59	lactiflorin	C₂₃H₂₆O₁₀	[M + COOH]⁻	507.1509	9.77	339.1083,121.0294	RPR	monoterpenoids	-
60	ellagic acid	C₁₄H₆O₈	[M-H]⁻	300.9992	10.17	300.9989, 257.0085,229.0141,201.0193,185.0245	RPR、PSA	phenolic acids	-
61	16,23-oxide-22,26-epimino -cholest -22(N),23,25-3-ene-3β-ol-3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→6)-β-D-galactopyranosid	C₄₅H₆₉NO₁₇	[M + H]⁺	896.4631	11.27	896.4620	SLT	alkaloids	-
62	cyclo hexaleucyl or isoleucyl	C₃₆H₆₆N₆O₆	[M + H]⁺	679.5110	11.69	679.5111,661.5009,435.3366	RPR	cyclic peptides	heart;liver;spleen;lung;kidney;brain;blood;
63	acetovanillone or paeonol^*	C₉H₁₀O₃	[M + H]⁺	167.0702	12.53	149.0599,122.0681,121.0647, 110.0363, 106.0412,105.0448,84.9596,78.0463	RPR、PSA	phenolic acids	-
64	benzoylpaeoniflorin or isomer^*	C₃₀H₃₂O₁₂	[M + Na]⁺	607.1782	12.70	607.1777,485.1431,105.0336	RPR、PSA	monoterpenoids	-
65	mudanpioside B/J	C₁₄H₆O₈	[M-H]⁻	629.1879	12.71	553.1738,431.1344,413.1247, 165.0556,121.0295	PSA	monoterpenoids	-
66	corchorifatty acid F	C₃₁H₃₄O₁₄	[M-H]⁻	327.2179	15.25	211.1340,171.1026,85.0296	RR	organic acids	-
67	dibutyl phthalate	C₁₈H₃₂O₅	[M + H]⁺	279.1588	21.24	149.0236,121.0285,57.0703	RR	other	brain
68	des-O-methyllasiodiplodin	C₁₆H₂₂O₄	[M + Na]⁺	301.1408	22.16	149.7017	AEJ	naphthoquinone	heart;spleen;lung;brain;blood;

3.3. Network Pharmacology Analysis

3.3.1 Potential Targets of the Active Ingredient of QRLXD

Based on the results of UPLC-ESI-MS/MS, 523 compound-related targets of QRLXD were acquired through database. A total of 1312 psoriasis disease targets were obtained from the GeneCards, OMIM, Therapeutic Target, DrugBank and PharmGkb databases, after removing the repeated gene targets. Altogether 129 overlapping targets of QRLXD and PV were visualized by a Venn diagram (Fig. 4A). The 129 overlapping genes were selected for protein-protein interaction (PPI) analysis by the STRING database and constructed by Cytoscape3.9.1 software. The PPI network formed 129 nodes and 1815 edges (Fig. 4B). Select targets with Degree ≥ 61 as the core target, and TNF, ALB and AKT1 were considered to be key targets(Fig. 4C).

3.3.2. GO and KEGG pathway enrichment analysis

To further explore the potential mechanisms of QRLXD in the treatment of PV, we performed a GO and KEGG pathway enrichment analysis of the common targets. GO enrichment analysis showed that 1488 items in three categories were enriched : 1281 biological processes (BP), 116 molecular functions (MF), and 91 cellular components (CC). The top 10 significantly enriched terms in the three categories (P < 0.05) were shown in Fig. 5A. The most highly enriched BP terms were selected as positive regulation of phosphorylation, positive regulation of protein phosphorylation, response to hormone, protein phosphorylation, positive regulation of transferase activity, etc. Highly enriched MF terms were identical protein kinase activity, phosphotransferase activity, alcohol group as acceptor, kinase activity, endopeptidase activity, peptidase activity, etc. The CC terms included side of membrane, membrane raft, membrane microdomain, lytic vacuole, lysosome, etc. Moreover, the KEGG enrichment analysis showed that the active components absorbed from QRLXD affected PV mainly through 162 signaling pathways. As shown in Fig. 5B, the top 20 enriched items included pathways in cancer,PI3K-Akt signaling pathway, Lipid and atherosclerosis, MAPK signaling pathway, IL-17 signaling pathway,etc.

3.2.3. QRLXD-compounds-target-pathway-PV network analysis

The “QRLXD-compounds-target-pathway-PV” network was constructed using Cytoscape 3.9.1 software, according to the target prediction results, and the active compounds of QRLXD absorbed in vivo; the core bioactive compounds included Dibutyl phthalate, Ethyl benzoylformate, Scopoletin, Albiflorin R1 and 3-Hydroxycoumarin (ranked by degree value). The degree value of network nodes is then analyzed using Network Analyzer. It is found that there are 169 nodes and 664 edges. (Fig. 6,Table 4)

Table 4

The top 5 degree of compounds in network diagram
No.	Drug	DC	BC	CC
1	Dibutyl phthalate	32	3731.1953	0.4169
2	Ethyl benzoylformate	27	2748.7458	0.4068
3	Scopoletin	20	2108.4958	0.3898
4	Albiflorin R1	18	1410.3782	0.3844
5	3-Hydroxycoumarin	17	736.9761	0.3862

3.3. Molecular docking verification and analysis

The docking results of the top five core active ingredients (Dibutyl phthalate, Ethyl benzoylformate, Scopoletin, Albiflorin R1, 3-Hydroxycoumarin) and the top three core targets (TNF:tumor necrosis factor, ALB:Albumin, AKT1:serine/threonine kinase 1), were shown in Fig. 5. Smaller binding energy leads to a more stable conformation. A docking score lower than − 5kcal/mol indicates that the ligand and protein can bind, while a docking score lower than − 7kcal/mol indicates that the binding activity is strong. The docking results showed that the core active ingredients had a favorable docking activity with the target proteins and were visualized in 3D(Fig. 7A-G).

Psoriasis is an inflammatory skin disease that is associated with many other medical conditions, and affects over 60 million adults and children worldwide^[²¹^]. Due to the safety and drug resistance, it is limited to use western medicines for long-term treatment of psoriasis. TCM has irreplaceable advantages in the treatment of chronic and complex diseases^[²²^,²³^]. For orally administrated TCM recipe, profiling the absorbed constituents is critical to understand the pharmacodynamic material basis and the therapeutic mechanism of TCM formulas. Qingre Liangxue Decoction, originated from the classic formula Xijiao Dihuang Decoction, improves clinical symptoms of psoriasis significantly. This study explores the effective ingredients and potential effects of QRLXD in the treatment of PV through UHPLC-Q-TOF/MS, network pharmacology and molecular docking technology.

A total of 18 prototype components were identified in QRLXD in rat samples after oral administration of QRLXD, with the core components being Dibutyl phthalate, Ethyl benzoylformate, Scopoletin, Albiflorin R1 and 3-Hydroxycoumarin. Pharmacological research has shown that scopoletin and 3-Hydroxycoumarin belong to the coumarin compound family and possess antioxidant, anti-tumor, antiviral, anti-inflammatory, and neuroprotective effects. Scopoletin can induce cell apoptosis, regulate cell cycle, inhibit cancer cell proliferation, and angiogenesis by activating AMP-activated protein kinase (AMPK), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase/Akt (PI3K/Akt), and signal transducer and activator of transcription 3 (STAT3), thereby exerting anti-inflammatory effects. Dibutyl phthalate downregulates the PI3K/AKT/mTOR signaling pathway to enhance cellular autophagy. Our results suggested that DBP and DEHP exposure at least partially activated the NOD2/RIP2/NF-κB signal pathway in grass carp hepatocytes, and caused inflammatory response and apoptosis. In terms of hepatotoxicity, there was synergistic relationship between DBP and DEHP^[²⁴^]. Albiflorin R1 inhibits the proliferation of J774 macrophages and exhibits immunomodulatory and anti-inflammatory effects. Ethyl benzoylformate belongs to the class of α-keto esters, which undergo alcohol oxidation, decarbonylation, and decarboxylation reactions, thereby promoting the mutual reactions between Chinese medicinal materials and exerting therapeutic effects on psoriasis.

Based on the absorbed components, network pharmacology was used to analyze the key components and target proteins of QRLXD in treating psoriasis, and a comprehensive analysis of the “QRLXD-compounds-target-pathway-PV” network and PPI results identified TNF, ALB, AKT1, STAT3, and other proteins as potential key targets for therapeutic effects. TNF activates downstream kinases to modulate protein phosphorylation and promote pro-inflammatory effects^[²⁵^]. Inhibiting TNF-α expression can reduce the downstream inflammatory cascade reactions in psoriasis and alleviate the condition, improving the quality of life in psoriasis patients. ALB is the most abundant protein in plasma and can affect the activation of transcription factor nuclear factor-κB by increasing intracellular glutathione levels, mediating the signal regulation of inflammatory cells and inhibiting inflammation. The circulating concentrations of albumin are response to inflammation, for the albumin with low levels indicative a wide range of conditions, including presence, persistence or improvement of malnutrition, infections and cancer^[²⁶^]. AKT1, through the activation of the PI3K/Akt signal pathway, participates in physiological processes such as cell proliferation, differentiation, and apoptosis, inhibiting the proliferation of psoriatic keratinocytes. STAT3 is highly expressed in psoriatic skin lesions^[²⁷^]. Various cytokines such as IL-6, IL-20, IL-22, and growth factors, when bound to their receptors, activate STAT3 phosphorylation, inducing the differentiation of T cells into Th17 cells and the secretion of interleukin-17A and interleukin-22, thereby exacerbating psoriasis. Inhibiting STAT3 phosphorylation and reducing IL-17 production are important approaches for treating psoriasis. VEGFA regulates kinase activity, thereby controlling cell proliferation and enhancing microvascular permeability, which contributes to the treatment of psoriasis. The IL-17 signaling pathway activates keratinocytes to secrete cytokines and interleukins, and it synergistically activates the MAPK and NF-κB signaling pathways with TNF-α. Molecular docking simulations confirmed that the binding energies between the core targets and core components were all less than − 5 kcal/mol, indicating good binding affinity. Notably, Ethyl benzoylformate, Scopoletin and Albiflorin R1 showed higher binding activities with TNF, ALB, suggesting their close correlation with the occurrence and development of blood-heat type psoriasis.

In this study, by integrating UHPLC-Q-TOF-MS/MS, network pharmacology, and molecular docking simulations, the potential pharmacological and molecular mechanisms of QRLXD in treating psoriasis vulgaris with blood-heat syndrome were systematically revealed. The key active substances in QRLXD includes Scopoletin, Ethyl benzoylformate, Albiflorin R1, 3-Hydroxycoumarin and Dibutyl phthalate, which operated mainly through regulating targets and pathways related to inflammation, tumor invasion, metastasis and apoptosis, through multiple pathways and targets. This study provides initial insights into the potential effective components and mechanisms of the Qingre Liangxue Decoction in the treatment of psoriasis vulgaris with blood-heat syndrome.

In summary, the present study first identified the chemical components in QRLXD and the effective compounds of QRLXD in vivo based on UHPLC-Q-TOF-MS/MS. Combining UHPLC-Q-TOF-MS/MS, network pharmacology, molecular docking, the method was constructed to systematically expose the potential pharmacological and molecular mechanism of action of QRLXD in PV treatment. Our research indicated that Scopoletin, Ethyl benzoylformate, Albiflorin R1, 3-Hydroxycoumarin and Dibutyl phthalate may be the main active compounds of QRLXD in PV treatment, and act on the protein target TNF, ALB and AKT1 through the pathways in cancer, PI3K/Akt signaling pathway. Generally, the findings will provide a theoretical basis for the application of QRLXD and for further research on the mechanism of QRLXD in the treatment of PV.

TCM Traditional Chinese medicine

AKT1 Serine/threonine kinase 1

TNF Tumor necrosis factor

BC Betweenness centrality

CC Closeness centrality

DC Degree centrality

GO Gene ontology

KEGG Kyoto Encyclopedia of Genes and Genomes

PPI Protein-protein interaction

UHPLC-Q-TOF/MS Ultra high-performance liquid chromatography equipped with quadrupole time of flight and mass spectrometer

Author contributions

Jiayi Liu conceived and designed the study. Jiayi Liu and Rui Cai contributed to the reagent preparation and data analysis. Xiangnan Zhou performed the experiments and analyzed the data. Jiayi Liu and Doudou Wu wrote and revised the manuscript. Yanping Bai supervised the study. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

This work was supported by the National Science Foundation of China (82074445).

Contributor Information

Yanping Bai, Email: [email protected].

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Exploring the active components and potential mechanisms of Qingre Liangxue Decoction in the treatment of psoriasis vulgaris with blood-heat syndrome based on UHPLC-Q-exactive Orbitrap/MS and network pharmacology

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1. Preparation of QRLXD

2.2. Materials and Reagents

2.3. Animals and treatments

2.4. Sample Collection and Pretreatment Method

2.5. UHPLC-Q-TOF-MS Analysis

2.6. Target collection of QRLXD and PV

2.7. PPI network for core target selection

2.8. GO and KEGG enrichment analysis

2.9. Construction of “QRLXD-compounds-target-pathway-PV” network

2.10. Molecular Docking Analysis

3. Results

3.1. Identification of compounds in QRLXD

3.2. Analysis of Prototype Constituents of QRLXD in Rat Sample

3.3. Network Pharmacology Analysis

3.3.1 Potential Targets of the Active Ingredient of QRLXD

3.3.2. GO and KEGG pathway enrichment analysis

3.2.3. QRLXD-compounds-target-pathway-PV network analysis

3.3. Molecular docking verification and analysis

4. Discussion

5. Conclusions

Abbreviations

Declarations

Author contributions

Declaration of competing interest

Data availability

Funding

References

Additional Declarations

Supplementary Files

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