RNA Sequencing Reveals the Regulation Mechanism of Yunnan Baiyao in Treating Skin Infection Caused by Staphylococcus aureus

Yunnan Baiyao is a well-known traditional Chinese medicine that can be formulated into a powder or capsule form. The mechanism by which it exerts its anti-inflammation effect, which is used in skin care products, needs to be further explored. In this study, we established the Staphylococcus aureus-induced mouse skin inflammatory model to investigate the effects of Yunnan Baiyao by the method of RNA-sequencing technology. The mice were randomly assigned to three groups, and those were control, model, and the Yunnan Baiyao-treated (YNtreated) group. Key genes and pathways were identified using bioinformatics analyses. In the study, we obtained 1,053 differentially expressed genes (DEGs) induced by Yunnan Baiyao. The 233 upregulated genes were enriched in 32 GO terms and 5 KEGG pathways, focused on the items, such as wound healing, cell metabolism, and proliferation, indicating the accelerating effects of Yunnan Baiyao on these aspects. The 820 downregulated genes were enriched mainly in the items, including the regulation of inflammation factor production, immune responses, and regulation of structure dermal components. Besides, Yunnan Baiyao reversed the expressions of 277 (201 decreased and 76 increased DEGs, respectively) induced by S. aureus. Ten key regulatory nodes (MMP2, PLK1, CCNB1, TLR4, CDK1, CCNA2, CDC25C, PDGFRA, MYOC, and KNG1) were identified by the construction of the protein interaction network, half of which were related to cell proliferation. VAV1 was another hub node that was affected by Yunnan Baiyao (Top 20). In the study, VAV1 and TLR4 can be considered key module genes in inflammation regulation. In conclusion, this study found that Yunnan Baiyao can significantly relieve inflammatory symptoms by regulating genes and pathways involved in the regulation of inflammation and immune response and also helped to deepen our understanding of the associated molecular mechanisms.


Introduction
Skin inflammation is a relatively common skin condition that is significantly associated with recurrent skin infections, disease severity, and Staphylococcus aureus (S. aureus) colonization [1][2][3]. S. aureus proteins, δ-toxin that stimulates mast cells, α-toxin that damages keratinocytes, phenolsoluble molecules that stimulate cytokine release by keratinocytes, protein A that triggers inflammatory responses from keratinocytes, superantigens that trigger B cell expansion and cytokine release, and proinflammatory lipoproteins that contribute to the intensity of symptoms [4], may be important for inflammation. us, it can be seen that S. aureus is a key component responsible for inflammation and a particularly troublesome microorganism in the field of dermatology. e rapid evolution of whole transcriptome profiling using next-generation high-throughput RNAsequencing (RNA-Seq) has shed light on the complex mechanisms and pathways involved in skin inflammation [5]. Gene expression profiling of RNAs isolated from skin biopsy specimens has revealed significant differences in the expression profiles of patients with skin inflammation, compared with those without, implicating type 2 immune pathways, as well as dysregulation of the skin barrier, through modification of cornification genes and lipid biosynthesis [6,7]. Although many drugs have been used to alleviate skin inflammation [8,9], changes in skin genome expression caused by drug treatment need to be studied further. erefore, there is an urgent need to identify specific treatment response groups so that approval for these drugs can be obtained as soon as possible.
A clinical study conducted in 2019 showed that Yunnan Baiyao can reduce hospital-acquired pressure ulcers caused due to S. aureus infection by suppressing virulence gene expression and biofilm formation [16]. However, the pharmacological assessment is still lacking. In this study, we established a mouse model of intradermal S. aureus infection and analyzed the local response in the untreated and Yunnan Baiyao-treated animals. e transcriptomic profiles of the skin tissues were analyzed and functionally annotated in order to identify critical pathways and pivotal related genes that may be involved in the anti-inflammatory effects of Yunnan Baiyao.

Materials.
e strain of Staphylococcus aureus S1 was purchased from China General Microbiological Culture Collection Center (CGMCC Number: 1.4519), and its identity was confirmed to be Staphylococcus aureus (ATCC Number: 25923). Before use, S. aureus bacteria were streaked onto a nutrient agar (NA) plate and grown overnight at 37°C in a bacterial incubator. Single colonies were picked and cultured in a nutrient broth medium at 37°C in a shaking incubator (200 rpm) overnight (15 h), followed by a 2% subculture at 37°C for 2 h to obtain midlogarithmic phase bacteria. e bacteria were harvested through centrifugation and washed and resuspended in normal saline solution at the concentration of 3 × 10 7 CFU/100 μL [20]. e absorbance at 600 nm was determined to estimate the number of CFU, which was verified through overnight culture on an NA plate.

Animals and Treatment.
Fifteen Balb/c-Nu nude mice (6-8 weeks, male) with an average weight of 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). 2, 2, 2,-tribromoethanol (98.5%) was purchased from J&K Scientific Ltd. e animal experimental procedures described in the following section were approved by the Ethics Committee of the College of Chemistry and Materials Engineering, Beijing Technology and Business University and were performed in accordance with the relevant guidelines and regulations on the use of laboratory animals at the College of Chemistry and Materials Engineering, Beijing Technology and Business University.
e mouse model of intradermal S. aureus infection was established as previously described [21]. In brief, the nude mice were housed under standard conditions and adaptively fed for a week with standard laboratory chow and then randomly divided into the control, model, and YN-treated groups (n � 5).
e mice in the model and YN-treated groups were each injected subcutaneously with a 100 μL of 3 × 10 7 CFU S. aureus into their right hind limbs. e control mice received the same volume of normal saline. All mice had free access to food and water. Inflammation was observed the day after injection. e inflamed skin area was topically treated with 100 μL of 1 mg/ mL of Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd. Kunming, China), twice daily for three days. e treatment duration with YB was set at 3 days based on published literature [22][23][24]. e mice in the model group were treated with the same dose of phosphate buffered saline (PBS). ree mice from each group were euthanized with an intraperitoneal injection of tribromoethanol (240 mg/kg BW), and the inflamed skin was peeled off. e tissue samples from each mouse were divided into two parts, one for paraffin embedding and hematoxylin-eosin (HE) staining and the other for RNA isolation and transcriptomic analysis. HE-stained sections were observed under a microscope, and the thicknesses of the stratum spinosum and stratum epidermis were measured using the image analysis software program CaseViewer (3DHISTECH. Ltd). e critical information about read mapping, bioinformatics tools, and criteria were based on the literature published before [25]. e data were analyzed on the free online platform of Majorbio Cloud Platform (https://www.majorbio.com). RSEM (https://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene abundances [26]. Statistical analysis of DEGs was conducted using the DESeq2 package. Table S1 represented the trimming and read mapping results of the sequences generated from the skin samples. A P value and the 2

RNA Isolation and
Evidence-Based Complementary and Alternative Medicine fold change (FC) for each gene were calculated to denote its expression difference between libraries. e remaining cDNA samples were returned to the laboratory.

Differentially Expressed Gene Analysis.
e differentially expressed genes (DEGs) in the model group compared to the control group and in the YN-treated group compared to the model group were both screened using P value < 0.01 and | log 2 FC| > 3 as the criteria. For convenience, we defined model DEGs as the differentially expressed genes in the model group compared with the control and YN-treated DEGs as the differentially expressed genes in the YN-treated group compared with the model group. A Venn diagram was drawn to visualize the overlap between the DEGs of both pairs, and two overlapping groups (Mup vs YNdown and Mdown vs YNup) were identified, which attracted our attention.
e Database for Annotation, Visualization and Integrated Discovery (DAVID version 6.8, https://david. ncifcrf.gov/) was used to identify enriched gene ontology (GO) terms and KEGG pathways among the DEGs [27,28]. e GO categories included biological processes (PB), molecular functions (MF), and cellular components (CC). e GO clustering analyses of upregulated and downregulated genes were performed with an enrichment score >2 as the criteria. e KEGG database was used to systematically analyze gene functions and pathways. P value < 0.05 was the threshold for statistical significance.

Construction of the Protein-Protein Interaction Network.
To further investigate the molecular mechanism by which S. aureus induced skin inflammation during development and progression, the protein-protein interaction network (PPI) was analyzed using Cytoscape ver. 3.5.1. Weighted protein-protein interactions of human beings were downloaded from the HPRD (Human Protein Reference Database, https://www.hprd.org/, Release 8). HPRD contains curated proteomic information on human proteins [29,30].
e DEGs were converted from the mouse to the human using the online tool, HCOP: orthology prediction search in the HGNC database (https://www.genenames.org/tools/ hcop/). HGNC is responsible for approving unique symbols and names for human loci, including protein coding genes, ncRNA genes, and pseudogenes, to allow unambiguous scientific communication [31][32][33].
All reactions were run in triplicates, and GAPDH was used as the internal control. e cyclic parameters used were predenaturation at 94°C for 30 s, followed by the PCR reaction (45 cycles of 94°C for 15 s, 60°C for 15 s, and 72°C for 10 s), while fluorescence data were collected at 72°C. e relative gene expression levels were analyzed using the 2 −ΔΔCT method.

Data Availability.
All raw sequencing reads have been submitted to the NCBI Sequence Read Archive (https:// www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA836085.

Statistical
Analysis. Statistical analyses were performed using GraphPad Prism software version 7.0. Data for multiple comparisons were compared using two-way ANOVA, as indicated in the figure legends. All data were presented as the mean value where error bars indicated standard deviation (SD). e two-tailed Student's test was used to compare two groups, and P < 0.05 was considered statistically significant.

Approval.
e Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA) was closely adhered to in the formulation of all animal experimental procedures. All experimental protocols were approved by the College of Chemistry and Materials Engineering, Beijing Technology and Business University.

Histological Evaluation of the Skin.
e innate immune response against S. aureus infection involves neutrophil recruitment and abscess formation and subsequent inflammation, which results in abscess formation, a hallmark of S. aureus infection. Histological examination of the skin samples from the model group revealed a thickened granular layer and the stratum spinosum in the infected area. Furthermore, the stratum epidermis was uneven in thickness, the junction between the epidermis and the dermis was broken, and the hair follicle and sebaceous gland were also fragmented compared to those in the control group (Figures 1(a) and 1(b)). In addition, there was considerable neutrophil infiltration in the dermis and subcutaneous tissues of the infected skin (Figure 1(b)). In the YN-treated group, the epidermal tissue structure was more intact, and the epidermal cells were arranged neatly, with obvious rete ridges and dermal papillae. Wavy fibrous tissue was visible in the dermis, and the hair follicles and sebaceous glands were complete, dense, and evenly distributed. Furthermore, there was no obvious infiltration of inflammatory cells (Figure 1(c)). Quantitative analysis of the thicknesses of the Evidence-Based Complementary and Alternative Medicine stratum spinosum and epidermis in the different groups showed significant differences between the model group and the control and the YN-treated skin (P < 0.001) for both (Figures 1(d) and 1(e)). Taken together, YB alleviated skin inflammation caused by S. aureus infection.

Identification of DEGs Related to S. aureus Infection and YB Treatment.
e DEGs between the control and inflamed skin samples were screened using P value< 0.01 and |log 2 FC| > 3 as the thresholds. As shown in the volcano plot in Figure 2(a), 638 genes were differentially expressed in the inflamed skin relative to the control, including 324 upregulated and 314 downregulated genes. e heat map of the model DEGs is shown in Figure 2(c). e transcriptomic analysis of the S. aureus infection model was published while this manuscript was in preparation [25]. In brief, 38% of the top 100 upregulated genes in the model group were related to the immune response; apart from eight genes with unknown function, some are involved in encoding metallopeptidases and redox proteins. e top 100 downregulated genes were mainly related to metabolism and cell proliferation. In addition, 16% of the DEGs encoding dermal proteins and nine genes involved in DNA repair were downregulated by S. aureus infection. A clear, brief summary was prepared in the supplemental file.
ere were 1053 DEGs in the YB-treated group relative to the model group, of which 233 were upregulated and 820 were downregulated (Figure 2(b)). e heat map is shown in Figure 2(d).

GO and KEGG Pathway Analysis of the YN-Treated DEGs.
e top 20 GO terms associated with the YN-treated DEGs are shown in Figure 3 and are arranged in ascending order of the adjusted P value. e genes involved in these top 20 enriched GO terms are shown in Table 1. As shown in Figure 3(a), the upregulated genes were significantly enriched in GO terms, including cell cycle processes, cell division, and chromosome segregation, suggesting that some of these DEGs may promote cell proliferation by regulating metabolism and the cell cycle. GO clustering analysis of these DEGs on the basis of the enrichment score (ES) > 1 and gene counts (GC) showed consistent results. As shown in Figure 4 Furthermore, genes related to the single-organism cellular process, wound healing genes, such as growth-related factors (e.g., fibroblast growth factor 6, Fgf6), and the genes involved in chloride channels (Clcn1, Clca3b, and Clca1) and potassium ion fluxes (Kcna7, Kcne1l, Kcnj16, and Kcnmb4os2), were increased in the YN-treated group (Table 1). is is consistent with the findings of Sun that wound healing effects of Yunnan Baiyao are partly mediated by the decrease in bioelectricity at the edge of the wound [41]. us, the role of ion fluxes in Yunnan Baiyao-induced wound healing deserves further study. e downregulated genes were significantly enriched in GO terms associated with immune response, such as neutrophil chemotaxis, the chemokine-mediated signaling pathway, regulation of leukocyte migration, and regulation of inflammatory response, which verified the antiinflammatory effects of Yunnan Baiyao (Figure 3(b)). According to GO clustering analysis (Figure 4(b)), chemotaxis had the highest enrichment score and gene count (ES � 6.61, GC � 88). Furthermore, positive regulation of inflammation factor production (ES � 2.85, GC � 43), regulation of immune response (ES � 2.69, GC � 12), and the complement receptor-mediated signaling pathway (ES � 1.99, GC � 4) were also clustered, which further underscored the anti-inflammatory effect of Yunnan Baiyao observed in our study. Ren. et al. showed that Yunnan Baiyao treatment significantly decreased IL-1β, IL-6, TNFα, COX-1, COX-2, and other inflammationrelated genes induced by lipopolysaccharide (LPS) [42]. In addition, Li et al. confirmed the immunosuppressive activity of Yunnan Baiyao in a mouse model of colitis, which was mediated by the downregulation of tumor necrosis factor α (TNFα) and interferon (IFN) c expression in the colonic mucosa and plasma [43]. In our study as well, several TNFα-associated genes were inhibited in the YN-treated group, including Tnfaip8l2, Tnfaip6, Tnfsf14, and Tnfsf13b (Table 1).
us, inhibition of TNFα is a possible mechanism underlying the anti-inflammatory effect of Yunnan Baiyao. e phospholipase A2 (PLA2)/arachidonic acid pathway is a pivotal regulator of inflammatory responses. Arachidonic acid is released from the membrane phospholipids by PLA2 and acts as a substrate for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) in the biosynthesis of eicosanoids (such as prostaglandins and leukotrienes). e inhibition of COXs and LOXs can modulate arachidonic acid metabolism and inhibit inflammation. In fact, several DEGs identified in the YN-treated group are associated with the metabolism of arachidonic acid, although not all of them were among the top 20 GO terms. In particular, arachidonate 5-lipoxygenase activating protein (Alox5ap), arachidonate 5-lipoxygenase (Alox5), phospholipase A2, group VII (Pla2g7), and phospholipase A2, group V (Pla2g5) were downregulated, while arachidonate lipoxygenase (Alox12e) and aldo-keto reductase family 1, member C18 (Akr1c18) were upregulated by Yunnan Baiyao. Likewise, Ren et al. previously showed that the anti-inflammatory effects of Yunnan Baiyao are mediated via the modulation of COX and LOX pathways of arachidonic acid metabolism [11]. Meanwhile, prostaglandin (PG)-associated factors, such as PG-endoperoxide synthase 2, PGI receptor (PGIR), and PGER2, were also repressed by Yunnan Baiyao, which is consistent with the previous reports [44]. He et al. had shown that Yunnan Baiyao ameliorated paw swelling and inflammation in mice by decreasing PGE2 and interleukin 1β levels [44].   Evidence-Based Complementary and Alternative Medicine e toll-like receptors (TLRs) are pattern recognition receptors that initiate innate immune responses to pathogenic infections. Several TLRs recognize S. aureus lipopeptides and lipoteichoic acid (LTA), which then triggers secretion of proinflammatory mediators. In this study, several TLRs and pathway intermediates, such as TLR4, TLR7, TLR8, cathepsin K (CTSK), and LPS binding protein (LBP), were upregulated in the inflamed skin. As shown in Table 1, Yunnan Baiyao treatment downregulated multiple  TLRs (such as TLR9, TLR8, TLR7, TLR4, TLR2, TLR13,  TLR11, and TLR1), which could be the basis of its antiinflammatory effects.
e KEGG pathways associated with the YN-treated DEGs are shown in Figure S1. e upregulated genes were significantly enriched in terpenoid backbone biosynthesis, steroid biosynthesis, and the cell cycle, and the downregulated genes were significantly enriched in inflammation-related pathways, such as the chemokine signaling pathway, cytokine-cytokine receptor interaction, complement and coagulation cascades, and the IL-17 signaling pathway. Inflammatory responses involving IL-17 likely contribute to arthritis, asthma, skin immune reactions, and autoimmune disorders. Genes participating in the IL-17 signaling pathway, including Lcn2, Cxcl5, S100a8, and S100a8 [25], were upregulated in the model group. As shown in Figure S1(b), Yunnan Baiyao can reduce inflammation partly by suppressing the IL-17 signaling pathway.

Analysis of the Model-Related DEGs Affected by YB
Treatment. e Venn diagram in Figure 5(a) shows the overlapping DEGs of the model and YN-treated groups. A total of 201 genes that were upregulated in the model group relative to the control were suppressed in the YN-treated animals and were designated as Mup-YNdown ( Figure 5(a)). Furthermore, 72 of these Mup-YNdown genes encode immune response-related proteins, of which eighteen are involved in the immune system process, nine (S100a8, S100a9, Amica1, Ccl6, Ccl9, Cxcr2, C5ar1, ITGAM, and Trem1) are related to neutrophil chemotaxis, seven (Cd163, Hp, Lbp, Serpina3a, Saa1, Saa2, and Saa3) are involved in acute-phase response, and five (Oas1a, Oas1f, Oas1g, Tlr7, and Tlr8) are involved in defense response to virus. Forty-nine genes with the largest decrease in transcript levels encode proteins with known functions in metabolism and cell proliferation, while  Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine  (Table S2). Seventy-six genes that were downregulated in the model group were activated upon YB treatment ( Figure 5(a)) and were designated as Mdown-YNup. As shown in Table S3, 26 Mdown-YNup genes encode proteins with known functions in metabolism and cell proliferation, of which eight (Cdc20, Ccna2, Ccnb2, Ccnb1, Plk1, Cdk1, Cdc25c, and Bub1) are involved in cell cycle and cell division, seven genes (Pif1, Neil3, PARPBP, Foxm1, Hist1h3g, Rad54b, and Hist1h2ag) encode proteins involved in DNA repair, and 19 genes encode proteins essential for transport and cell mitotic cell cycle process cell cycle process cell division chromosome segregation mitotic nuclear division single-organism cellular process regulation of nuclear division lipid biosynthetic process regulation of microtubule polymerization or depolymerization lipid metabolic process microtubule cytoskeleton organization involved in mitosis mitotic spindle assembly regulation of mitotic nuclear division sterol biosynthetic process kinesin complex microtubule     e kinesin family members, including Kif4, Kif22, Kif20a, Kif14, Kif18b, and Kifc5b, were repressed in the model group and upregulated by Yunnan Baiyao. e kinesin superfamily proteins (KIFs) transport organelles, protein complexes, and mRNAs to specific destinations in a microtubule-dependent and ATP-dependent manner [48], which correlates with the proproliferative effect of Yunnan Baiyao. ree genes were downregulated in both the model and YN-treated groups ( Figure 5(a)  GO enrichment analysis showed that the Mup-YNdown genes were most significantly enriched in terms associated with the regulation of inflammatory response, leucocyte chemotaxis, positive regulation of defense response, and positive regulation of immune response ( Figure 5(b)), and GO clustering analysis showed high ES and gene counts for acute-phase response, chemotaxis, and response to cytokine, and the regulation of inflammation factor production had a high ES and large count ( Figure 6). However, the Mdown-YNup showed significant enrichment of terms associated with cell division, proliferation, and cellular matrix formation ( Figure 5(c)), and GO clustering further identified cell cycle and chromosome segregation. Taken together, Yunnan Baiyao mitigates S. aureus-induced skin inflammation by targeting multiple pathways.

Protein-Protein Interaction Network.
e background PPI network was established using the control vs. model DEGs, followed by the inclusion of the model vs. YN-treated DEGs and their first neighbors in order to identify the target genes of Yunnan Baiyao.
ere were 687 nodes (DEGs with a degree higher than 20) in the YN-treated target network that were mapped to the model network (Figure 7(a)). Clustering analysis based on the function of the nodes reveals 11 clusters, of which 4, 6, 9, 7, 2, and 1 were the main mapped targets of the nodes in the YN-treated network in that order (Figure 7(b), and detailed information is presented in Table S4 in supplemental file). e nodes in these clusters were mainly related to cell growth and death (Cluster 4), immune system (Cluster 6), apoptosis (Cluster 9), signal transduction (Cluster 2), and genetic information processing (Cluster 1) based on the KEGG pathway analysis. ese network results were consistent with that of GO analysis. e top 20 nodes of the model and YN-treated networks were screened based on the degree of topology, of which MMP2, PLK1, CCNB1, TLR4, CDK1, CCNA2, CDC25 C, PDGFRA, MYOC, and KNG1 were common to both (Table S5 in Supplemental file) and possibly targeted by Yunnan Baiyao. Furthermore, these ten nodes were related to the cell cycle (5 nodes), oocyte maturation (4 nodes), cell division (4 nodes), protein ubiquitination (3 nodes), and ATP binding (3 nodes). Among the top 20 hub nodes, TLR4 (toll-like receptor 4) and VAV1 (VAV guanine nucleotide exchange factor 1) are the key inflammation genes. TLR4 is a member of the toll-like receptor family, which plays an important role in pathogen recognition and innate immune activation [49]. In many types of animals, TLRs are highly conserved in structure and function across species [49,50], although they differ in their expression patterns and recognize distinct pathogen-related molecules [51]. TLR4 recognizes LPS of most Gram-negative bacteria and is the key driver of LPS-induced inflammation [52,53]. VAV1, a member of the VAV gene family, is the guanine nucleotide exchange factor (GEFs) for GTPases of the Rho family, which activates actin cytoskeleton rearrangement [54,55]. Furthermore, the Vav1-encoded protein is involved in the mechanical peristalsis and migration of neutrophils in inflammatory microvessels and plays a role in the development and activation of T cells and B cells [56]. In addition, CCNA2, CCNB1, PLK1, CDK1, and CDC25 C, which are related to the cell cycle or other proliferation-associated processes, were also altered to some extent by Yunnan Baiyao. Taken together, Yunnan Baiyao can regulate S. aureus-induced skin inflammation through immune activation and cell proliferation.

qRT-PCR Confirmation of Differential mRNA Expression.
Expression of MMP2, TLR4, CDK1, and VAV1 was further validated by qRT-PCR and were coincident with RNA-seq data (shown in Figure 8). us, both approaches showed a strong correlation.

Conclusion
In conclusion, we performed a comprehensive bioinformatics analysis on transcriptome data of skin tissue samples obtained from groups of infected animals and Yunnan Baiyao-treated infected animals. e pivotal DEGs and pathways were identified and screened to provide a theoretical basis for the molecular mechanisms involved in skin inflammation and potential drug target discovery. e results showed that Yunnan Baiyao could regulate the inflammatory pathway by changing the expression of various genes so as to alleviate inflammation. Our findings provide insights into the molecular mechanisms underlying bacterial infection-induced skin inflammation and discovery of potential drug targets.

Data Availability
All raw sequencing reads have been submitted to the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under the BioProject accession number PRJNA836085.  (Table S1-S6, Table I, and Table  II), and a brief result of the analysis of DEGs in the inflamed skin samples relative to the normal skin as listed in the following paragraph, which were published before. Plus, Figure I, Figure II, Table I, and Table II were belonging to the brief analysis published before. Besides, a graphic bbstract was added in the Supplementary Material too. Figure S1. e results of the KEGG pathway data of upregulated (A) and downregulated (B) YN-treated DEGs. Figure I. e top 20 GO terms (A) and KEGG pathways (B) of different expressed genes enrichment in RNA-sequencing (P value ≤ 0.05). Gene counts: the number of target genes in each GO term or the KEGG pathway. Rich factor: the ratio of the number of target genes divided by the number of all the genes in each GO term or the KEGG pathway. e size of pot indicated the gene counts, and the color reflected the different P value. Figure II. GO clustering results of DEGs with a criterion of the enrichment score >1. A, upregulated. B, downregulated. Counts represent the gene number enriched in each term. e enrichment score was obtained according to the built-in program in DAVID, which ranks the biological significance of gene groups based on the overall P value of all enriched annotation terms. e higher the enrichment score is, the more important the term is. Table S1. Summary of trimming and read mapping results of the sequences generated from the skin samples. Table S2. 201 genes upregulated in the model while downregulated by Yunnan Baiyao (known as Mup-YNdown). Table S3. 76 genes downregulated in the model while upregulated by Yunnan Baiyao (known as Mdown-YNup). Table S4. Detailed information of YN-treated nodes mapped in each cluster. Table S5. e top 20 nodes in the Model net and YNtreated net.