Gene Expression of PSORI-CM01 and Yinxieling in the Treatment of Psoriasis Vulgaris

State Key Laboratory of Dampness Syndrome of Chinese Medicine, e Second Affiliated Hospital of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou 510006, Guangdong, China Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou 510006, Guangdong, China Guangdong-Hong Kong-Macau Joint Lab on Chinese Medicine and Immune Disease Research, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong, China Shanghai Molecular Medicine Engineering Technology Research Center, Shanghai 201203, China Shanghai National Engineering Research Center of Biochip, Shanghai 201203, China

Notably, the TCM treatment of psoriasis begins with bloodrelated factors, which are commonly recognised in the TCM community [3,4]. e PSORI-CM01 formulation was optimised by Professor Guowei Xuan of Guangdong Provincial Hospital of Chinese Medicine, a TCM master, from the clinically proven prescription "Yinxieling" and intended for the treatment of blood stasis syndrome of psoriasis by promoting blood circulation, removing blood stasis, and cooling and detoxifying of the blood. In 1998, Yinxieling tablets (Z20080123, Patent no. CN 105233150A) were approved by the Guangdong Food and Drug Administration, Guangdong Province, China, for prescription use in hospitals. With over 22 years of clinical application in more than 200,000 people, the Yinxieling formulation has been proven to be safe and to have significant treatment effects [5]. PSORI-CM01 (Patent no. CN 101632827A) was optimised by Professor Chuanjian Lu's team of Guangdong Provincial Hospital of Chinese Medicine over ten years ago and has a sound basic experimental research foundation and positive clinical trial results [6][7][8].
With the rapid development of sequencing technology, a large amount of genomic information has been discovered. Gene chip technology is one of the high-throughput detection technologies; it has been widely used in the research of various diseases [9]. In recent years, researchers have used this technique to conduct several studies on the gene expression of psoriasis [10][11][12]. Most of the studies are using gene chip to study the pathogenesis of diseases, and there are a few studies on the target of drug therapy, especially traditional Chinese medicine.
In order to establish a basis for the clinical development of the PSORI-CM01 formula as a safe and effective modern prescription drug, it is necessary to conduct experimental controlled trials to compare the mechanism of action of PSORI-CM01 and Yinxieling in the treatment of patients with psoriasis. We used gene chip technology to analyze a series of differential expression of genes in clinical samples of PBMCs.

Clinical Study.
Blood samples from patients with psoriasis vulgaris and the healthy volunteers were obtained from the Department of Dermatology, Guangdong Provincial Hospital of Chinese Medicine, and used in a randomised, double-blind, and double-dummy clinical study [13]. e diagnostic criteria were in line with those previously published for psoriasis vulgaris [14]. Detailed information on the psoriatic patients, including their demographics, and psoriasis area and severity index (PASI) is provided in Table 1.
e study was performed in accordance with the Declaration of Helsinki and the relevant clinical trial research regulations of China (Trial registration ChiCTR-TRC-14005185 registered on August 8, 2014). All procedures involving human participants in this study were performed in accordance with the ethical standards of the Guangdong Provincial Hospital of Chinese Medicine, Clinical Research Ethics Committee (ethical approval no. B2012-53-03). Before the collection of samples, all patients enrolled in the study were required to sign an informed consent form, and the researchers explained the purpose and treatments in the study to them ( Figure 1).
e Yinxieling tablets and the matching placebo tablets were prepared by the Guangzhou Kangyuan Pharmaceutical Science & Technology Co., Ltd., Guangzhou, Guangdong Province, China (catalogue no. 20151003).

Group Intervention.
Participants in the PSORI-CM01 group received 5.5 g PSORI-CM01 granules twice daily after meals, and the controls received 5.5 g placebo granules three times daily after meals for 12 weeks. e participants in the Yinxieling group received five Yinxieling tablets three times daily after meals, and the controls received five placebo tablets two times daily after meals for 12 weeks. e details of the treatments and procedures have been previously described [13], instruments, reagents, and software. e following reagents, kits, and sets were used in this study: TRI Reagent (Sigma, Germany, catalogue no. T9424), miRNeasy Micro Kit (QIAGEN, Germany, catalogue no. 217084), RNase-Free DNase Set (QIAGEN, Germany, catalogue no. 79254), TIANGEN miRcute miRNA cDNA first-strand synthesis kit (TIANGEN, Beijing, China, catalogue no. KR201), and SYBR Green PCR kit (TIANGEN, Beijing, China, catalogue no. FP411-02). An Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) was used for electrophoresis and an Agilent Scanner G2505 C (Agilent Technologies, Santa Clara, CA, USA) for chip scanning. e following software was used: Feature Extraction (version 10.7.1.1, Agilent Technologies) was used for data extraction, Genespring (version 13.1, Agilent Technologies) for data processing, and the Affymertix GeneChip ® Clariom D array human chip (Affymertix) for transcriptome gene expression levels.

Sample Collection and
Processing. Ten millilitres of cubital venous blood was collected from each participant and placed in an anticoagulation tube containing heparin. According to the instructions of reagent test kits, peripheral blood mononuclear cells (PBMCs) were separated from the blood samples and stored in a refrigerator at −80°C.

RNA Extraction and Quality Control.
Total RNA extraction from PBMCs was performed using TRI Reagent (Cat# T9424, MilliporeSigma, 2020 Merck KGaA, Darmstadt, Germany) according to the standard operating procedures provided by the manufacturer. e total RNA samples were further purified using a miRNeasy Micro Kit (Cat# 217084, QIAGEN, GmBH, Germany) and an RNase-Free DNase Set (Cat# 79254, QIAGEN, GmBH, Germany).
During each round of RNA extraction, a control total RNA was used as the positive quality control, and RNasefree water was used as the negative quality control. If any degradation of the positive quality control occurred during the process of RNA extraction, the sample would exhibit the same effects. If the negative quality control was contaminated by positive total RNA during the extraction, the sample would also be contaminated by the exogenous total RNA. After the validation of quality control, the integrity of purifying the total RNA was checked using an Agilent Bioanalyzer 2100 electrophoresis analyzer (Agilent Technologies, Santa Clara, CA, USA). Samples which met the RNA integrity number value of ≥6 were loaded onto the chips for analysis.    Evidence-Based Complementary and Alternative Medicine 3 operating procedures specified by the manufacturer; the cRNA produced was labelled using biotin. e biotin-labelled cRNA was added to the cassette chip according to the Affymetrix chip hybridisation procedure.
en hybridisation was carried out in a rolling hybridisation oven (Hybridization Oven 645, Cat# 00-0331-220V, Affymetrix, Santa Clara, CA, USA), at 45°C for 16 h following the standard hybridisation process using the supporting kit (GeneChip ® Hybridization, Wash and Stain Kit, Cat# 900720, Affymetrix, Santa Clara, CA, USA) provided with the expression profile chip. Following hybridisation, the chip was washed using a Fluidics Station 450DX2 (Cat# 00-0335, Affymetrix, Santa Clara, CA, USA) according to the standard operating procedure provided by Affymetrix. e gene chip was scanned using a GeneChip ® Scanner 3000DX2 (Cat# 00-0334, Affymetrix, Santa Clara, CA, US). e original data were read with Command Console Software 3.1 (Affymetrix, Santa Clara, CA, USA). Data quality control was also performed, and data reports were issued by Command Console Software 3.1.
e qualified data was normalised using the gene-level SST-RMA algorithm of the software.

Validation by Quantitative Real-Time Polymerase Chain
Reaction.
e RNA isolated from the PBMC samples of different groups was reverse transcribed directly into cDNA using a SuperScript IV Reverse Transcriptase ( ermo Fisher, Dalian, China) according to the manufacturer's instructions. e PCR amplification conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, and 60°C for 30 s, then annealing at 95°C for 5 s and 60°C for 1 min. e expression levels of genes GPVI, TNFSF4, CXCR3A, CXCR3B, CXCL4L1, and TUBB1 were examined by quantitative realtime polymerase chain reaction (qRT-PCR) using an SYBR Green PCR kit (TaKaRa, Dalian, China). e results were expressed as means ± standard errors of the means. e Student's t-test was used for the statistical analyses. P values <0.05 were considered significant.

Data Analysis.
Preliminary gene-level differential expression analysis was performed using Transcriptome Analysis Console Software (Affymetrix, Santa Clara, CA, USA). Genes with P value <0.05 and fold change ≥2 were screened as differentially expressed genes and enriched by the DAVID Gene Functional Classification Tool (v6.8 http:// david.abcc.ncifcrf.gov). e pathways with a P value <0.05 were selected for further analysis. e FDR-BH adjusted P values obtained by t-test and fold change values were used for differential gene screening. e screening criteria were a fold change value of up-or downregulation >2 and an FDR-BH P value <0.05. Paired-sample t-tests were performed to compare the intragroup difference before and after treatment within groups A and B, whereas two-sample independent t-tests were performed to compare the intergroup differences. e FDR-BH values were calculated after sorting the P values in ascending order. e screened differentially expressed genes were clustered by unsupervised hierarchical cluster analysis using the online OmicShare Tools (https:// www.omicshare.com/tools/) and displayed as volcanic and heat maps. To determine the biological functions or signalling pathways affected by the differentially expressed genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using the online string and metascape tools (https://string-db.org/ and https://metascape.org/gp/index.html#/main/step1, resp.).

Quality Control of Gene Chip Data.
Combined with information from the clinical samples, the results of the Agilent microarray analysis of the 51 samples (21 pairs of samples from patients with blood stasis syndrome of psoriasis, including 12 samples before and after treatment with prescription PSORI-CM01 and nine samples before and after treatment with Yinxieling) and 9 samples before and after placebo treatment in the controls showed that the quality of the original data was well controlled (Figure 2).

Screened Differentially Expressed Genes between Groups.
As shown in Table 2, comparing the genes with an FDR_BH <0.05 and fold change >2, between patients in the PSORI-CM01 group before treatment and the controls, a total of 178 differentially expressed genes were found, including 147 upregulated and 31 downregulated, whereas comparing the genes between patients in the PSORI-CM01 group after treatment and the controls, a total of 908 differentially expressed genes were found, of which 758 were upregulated and 150 were downregulated. However, comparing the genes before and after treatment within the PSORI-CM01 group, a total of 668 differentially expressed genes were found, of which 445 were upregulated and 223 were downregulated. Contrarily, comparing the genes between patients in the Yinxieling group before treatment and the controls, a total of 276 differentially expressed genes were found, of which 246 were upregulated and 30 were downregulated, whereas comparing the genes between patients in the Yinxieling group after treatment and the controls, a total of 732 differentially expressed genes were found, including 475 upregulated and 257 downregulated. However, comparing the genes before and after treatment within the Yinxieling group, a total of 657 differentially expressed genes were found, of which 168 were upregulated and 489 were downregulated.

Differences in Gene Expression Profiles between Patients in the PSORI-CM01 Group, Yinxieling Group, and the Controls.
e differences in gene expression in the PSORI-CM01 group before treatment versus the control, in the PSORI-CM01 group after treatment versus the control, and in the PSORI-CM01 group before treatment versus PSORI-CM01 group after treatment were compared and displayed in volcanic maps and heat maps (Figure 3). e differences in gene expression in the Yinxieling group before treatment versus the control, the Yinxieling group after treatment versus the control, and the Yinxieling group before treatment versus the Yinxieling group after treatment were compared and displayed in volcanic maps and heat maps ( Figure 4). In the volcano plot, red is the upregulation of genes and green is the downregulation. e larger the fold change value, the larger the variable indicating the amount of difference in gene expression between the two groups. In the heatmap plot, each small square represents each gene, and its color represents the expression level of the gene. e greater the expression level, the darker the color (red for upregulation, green for downregulation). Each row represents the expression level of each gene in different samples, and each column represents the expression level of all genes in each sample.

Venn Analysis of Differentially Expressed Genes.
Results of the comparison of differentially expressed genes using Venn analysis showed that there were 113 differentially expressed genes between the PSORI-CM01 group before treatment (PSORI-CM01 group 0 weeks) and the healthy control group. After 12 weeks of treatment, there were 892 differentially expressed genes between the PSORI-CM01 group (PSORI-CM01 group 12 weeks) and the healthy control group. e intersection after the two comparisons can be seen as a total of 35 common differentially expressed genes that existed in both differential gene comparisons at the same time ( Figure 5(a)). In comparison, there were 113 differentially expressed genes between the Yinxieling group before treatment (Yinxieling group 0 weeks) and the healthy controls, 724 differentially expressed genes between the Yinxieling group after treatment (Yinxieling group 12 weeks) and the healthy controls, and 39 common differentially expressed genes both before and after treatment in the Yinxieling group ( Figure 5(b)). Twenty-three genes were differentially expressed in both groups after treatment, compared to the healthy controls, and they overlapped completely ( Figure 5(c)). Seventy-eight genes were not differentially expressed in the PSORI-CM01 group, and 74 were not differentially expressed in the Yinxieling group after treatment, compared with the healthy controls. Among these genes, 72 were common to two groups, which were the genes on which the two drugs acted jointly. Additionally, compared with the healthy controls, six genes, PTX3, ALAS2, Skerber, Toby, HBB, and HBA1 were not differentially expressed in the PSORI-CM01 group but were in the  Evidence-Based Complementary and Alternative Medicine Yinxieling group; compared with the healthy controls, two genes (doyzor and mokari) were not differentially expressed in the Yinxieling group but were in PSORI-CM01 group ( Figure 5(d)).

Pathway Enrichment Analysis for Differentially Expressed
Genes. KEGG enrichment analysis was performed using DAVID online tools, to determine the biological functions or signalling pathways affected by the differentially expressed genes. e results showed that the top ten major signalling pathways targeted by PSORI-CM01 treatment included haemostasis, PI3K-Akt signalling pathway, G alpha (s) signalling events, neutrophil chemotaxis, formation of fibrin clot (Clotting Cascade), platelet activation, response to mycotoxin, factors involved in megakaryocyte development and platelet production, Rho GTPase effectors, and leukocyte differentiation (Figures 6(a) and 6(b)). However, the top ten major signalling pathways targeted by Yinxieling   (Figures 6(c) and 6(d)). In addition, results of Venn analysis on the signalling pathways in two treatment groups showed that haemostasis and Rho GTPases were the common signalling pathways of drug action shared by the two groups ( Figure 6(e)).

Verification of Selected Genes.
Using qRT-PCR, we verified the six differentially expressed genes obtained from the microarray analysis. As shown in Figure 7, the expressions of GPVI, TNFSF4, CXCR3A, CXCR3B, CXCL4L1,  and TUBB1 were in accordance with the results of the microarray analysis.

Discussion
Psoriasis is a chronic proliferative skin disease with immune abnormalities that is determined by multiple genes and induced by various environmental factors. Because it is inherited, difficult to treat, and easy to relapse, there is no modern medical treatment or radical cure for it. Most current allopathic therapies have significant toxic or side effects or both and thus cannot be used for long-term treatment. TCM has accumulated significant experience in the treatment of psoriasis. TCM syndrome differentiation and treatment can relieve symptoms, delay progression, reduce recurrence, and improve quality of life, and it has few side effects, which make it suitable for long-term treatment.
Results of differentially expressed genes from Venn analysis between the groups showed that, compared with the controls, 78 genes in the PSORI-CM01 group and 74 in the Yinxieling group were not differentially expressed after treatment, of which 72 genes were common in the two groups; the results indicated that these were the genes on which the two drugs acted jointly. Notably, GPVI is a gene related to platelet coagulation and inflammation, and the activation of GPVI can lead to the ubiquitination of platelet protein in humans [17]. GPVI is the major signal receptor of collagen, which can bind to the D1 domain in GPVI through the glycine-proline-hydroxyproline sequence and can trigger thrombosis and stabilise thrombi by binding fibrin [18,19]. e researchers also concluded that glycoprotein IIb/IIIa (GPIIb/IIIa) on platelets, α]β3, and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells mediate platelet stable adhesion [20]. e tumour necrosis factor superfamily 4 (TNFSF4) is involved in the pathogenesis of various autoimmune diseases, such as rheumatoid arthritis and psoriasis [21]. e genes CXCR3A, CXCR3B, and CXCL4L1 are closely associated with angiogenesis, whereas CXC chemokine receptor 3 (CXCR3) can  Figure 5: Venn analysis of differentially expressed genes between groups. (a) Differentially expressed genes between the PSORI-CM01 and the control groups before and after treatment; (b) differentially expressed genes between the Yinxieling and the control groups before and after treatment; (c) differentially expressed genes before and after treatment, common between PSORI-CM01 and Yinxieling groups, compared with the control group; and (d) differentially expressed genes, common between the PSORI-CM01 and Yinxieling groups before treatment that showed no significant difference after treatment compared with the control group. mediate the antiangiogenic effect and chemotactic activity of platelet factor 4 variant (CXCL4L1) [22]. Nomura et al. used microarray technology to compare the differences of gene expression levels between psoriasis patients and atopic dermatitis patients; it was found that there were 62 differentially expressed genes in the lesions of patients with psoriasis, among which the gene encoding CXC chemokine was significantly upregulated [23]. In addition, abnormal expression of TUBB1 is closely related to thrombocytopenia in congenital and acquired platelet diseases and affects platelet function by altering microvascular formation [24]. erefore, we speculated that the genes mentioned above might be involved in the pathological mechanism and treatment process of psoriasis. When compared with the controls, six genes, PTX3, ALAS2, Skerber, Toby, HBB, and HBA1 were not differentially expressed in the PSORI-CM01 group but were in the Yinxieling group. Pathway enrichment analysis demonstrated that these genes are primarily correlated with the metabolism of glycine, serine, and threonine. e result of Venn analysis on the signalling pathways in two drugs groups showed that haemostasis and Rho GTPases were the common pathways targeted by the two treatments. Studies of haemostasis on patients with psoriasis show that there are varying degrees of abnormalities in blood coagulation and fibrinolysis [25][26][27][28]. A recent study demonstrated that psoriasis is linked to increased cardiovascular risks. Since chronic skin-specific inflammation may promote atherosclerosis, myocardial infarction or stroke may also be correlated with haemostatic disorders; there were dysfunctions of fibrinolysis and thrombosis in patients with psoriasis [29]. Rho GTPases are a major branch of the Ras superfamily and play an important role in the signal transduction pathway of cells [30]. Since the overexpression of the Rho protein is related to the initiation and development of tumours, Rho GTPases, which are involved in cytoskeleton remodelling, cell movement, gene transcription regulation, and cell cycle regulation, can play a role in the proliferation and apoptosis of tumours [31]. Psoriasis is an end-point disease similar to epithelial-mesenchymal transition (EMT), which is a process that transforms epithelial cells into fibroblast-like cells, and the extracellular-signalregulated kinase, Rho, and glycogen synthase kinase-3 can promote the EMT process in psoriatic keratinocytes [32].
Some researchers have used gene chip technology to study psoriasis, mainly including the pathogenesis mechanism of psoriasis. Bowcock et al. collected samples from 15 patients with psoriasis and found that there were 177 gene expression differences between lesions and nonlesions [33]. Another research used gene chip technology and real-time quantitative PCR to detect the expression of G-protein coupled receptors in lesions and nonlesions of psoriatic patients [12]. Our study is based on the clinical efficacy of   Figure 6: Pathway enrichment analysis for differentially expressed genes between groups. (a) Analysis of regulated differential pathways between PSORI-CM01 and the control group; (b) analysis of differentially expressed genes between the PSORI-CM01 and the control groups before treatment that showed no significant difference after treatment; (c) analysis of regulated differential pathways between the Yinxieling and the control group; (d) analysis of differentially expressed genes between the Yinxieling and the control groups before treatment that showed no significant difference after treatment; and (e) Venn analysis of differentially expressed pathways between the PSORI-CM01 and Yinxieling groups.
traditional Chinese medicine prescription before and after treatment to explore the differences of gene expression in PBMCs by utilizing gene chip technology.
ere are some limitations in this article, such as the small size of the original sample due to the limitation of the clinically available sample size. is study followed a rigorous screening method and strictly controls the sample quality, so the final sample size that meets the conditions is low. In the future, our research can extend the clinical data collection time and continue to increase the sample size for   related research. We will continue to study microRNA gene chip and perform correlation analysis results between the gene chip and microRNA gene chip.

Conclusions
In conclusion, through the comparison of gene expression profiles in PBMC between patients with psoriasis vulgaris before and after treatment with PSORI-CM01 or Yinxieling formulations and the controls and through pathway enrichment analysis of differentially expressed genes, we found that the two drugs had positive effects on psoriasis vulgaris primarily by the regulation of pathways related to platelet activation, aggregation, and blood coagulation. is study clarified the mechanisms of the two Chinese herbal compound prescriptions used in the treatment of psoriasis and laid a foundation for the future study.
Data Availability e datasets used and/or analyzed during the current study would be available from the corresponding author on reasonable request.

Consent
Each patient enrolled in the study was required to sign an informed consent form. All participants were informed of the purpose of the study and the treatments before their blood samples were collected.

Conflicts of Interest
e authors have no conflicts of interest to declare.