Centella triterpenes cream as a potential drug for the treatment of hypertrophic scar through inhibiting the phosphorylation of STAT3: A network pharmacology analysis and in vitro experiments

Hypertrophic scars (HS) often affect the normal function and appearance of the skin and bring adverse effects to the body and mind of patients, being a challenge in the fields of burns and plastic surgery as well as rehabilitation. Despite significant efficacy of centella triterpenes cream for treating HS clinically, its pharmacodynamics and molecular targets are still unclear. Therefore, the network pharmacology analysis combined with in vitro cell molecular biology experiments was used to explore the mechanism and targets of centella triterpenes cream treating HS in this study.


| INTRODUC TI ON
Hypertrophic scars (HS) are raised scars formed by the deposition of excess connective tissue in the primary wound area during wound healing.Generally, HS occurs in tight skin areas following skin trauma, burns, or surgical incisions, usually accompanied by abnormal hyperpigmentation. 1,2HS causes pain or itching, and if it develops over joints, potentially limits motion and thus needs medical attention. 3, a condition affecting the normal function and appearance of the skin, leads to adverse effects on patients' physical and psychological health.The treatment for HS is still a major challenge in burn and plastic surgery as well as rehabilitation.The commonly used means for treating HS included cryosurgery, laser, surgery, and drugs. 4,5rticosteroid injections are often applied to smooth and soften scar tissue and relieve pain and itching.Bleomycin or 5-fluorouracil (5-FU) can smooth scars and reduce itching and pain by destroying overgrown cells.However, 5-FU and corticosteroids will also affect normal skin, resulting in apoptosis and necrosis of normal cells and consequently causing side effects such as skin atrophy, telangiectasia, and local necrosis. 6inese medicinal preparations show good therapeutic potential for scar treatment with high safety and no damage to normal skins. 6Centella triterpenes tablets and cream, 7,8 Bahen Zhiyang Ruanhua Rugao, 9 and moist exposed burn ointment 10 have been confirmed to have good efficacy for treating HS.Compared with other medicines, the composition of centella triterpenes cream is simple; Centella asiatica, composing of asiaticoside (AC) and a small amount of other triterpenoid saponins, is primarily responsible for the effect of centella triterpenes cream. 11Centella asiatica extracts have pharmacological activities such as anti-inflammatory, 12,13 antibacterial, 14 antioxidant, 15 neuroprotective, 16 and wound healing. 17 the Asian region, Centella asiatica is mainly used for the treatment of skin and vascular diseases. 18Early studies have pointed out that Centella asiatica can improve collagen synthesis 19 and microcirculation function 20 in the process of wound healing, so it is commonly used for the treatment of burns, HS, eczema, psoriasis, gastric ulcer, and gastric mucosal lesions. 21AC as the most active major component of Centella asiatica 18 can inhibit pigment deposition at scar-forming sites. 8Despite the confirmed efficacy of centella and its active ingredients on HS by a great many researches, the potential therapeutic mechanism and molecular targets are still lack of experimental support.In this study, network pharmacology analysis and cell/molecular biology experiments were employed to systematically investigate the potential mechanism and molecular targets of AC (the main active ingredient of centella triterpenes cream) in the treatment of HS.

SwissTargetPrediction
(http://www.swisstarge tpred iction.ch/), 24d TargetNet (http://targe tnet.scbdd.com/). 25The search results of each database were merged, and the duplicate items were removed.Referring to the list of human-derived genes obtained from the UniProtKB database (https://www.uniprot.org/help/unipr otkb), the protein names of potential targets of the compounds were converted to gene names and then put them in order.

and Online
Mendelian Inheritance in Man (https://www.omim.org/). 28The obtained data from each database were merged, and the duplicates were removed.Subsequently, a Venn diagram of AC targets and HS targets was plotted.

| Protein-protein interaction analysis and core gene screening
The obtained common targets of AC-HS were uploaded to the STRING 11.0 protein-protein interaction (PPI) database via StringApp of Cytoscape3.9.0. 29Specifically, the organism was set as "nismN.CITE" and other parameters were set as default values to generate PPI network diagram.Through analyzing the connection between each target in the PPI network using Cytoscape3.9.0, the degree of nodes of each target was calculated; the color and size of to reducing p-STAT3 in HSFs, AC significantly inhibited the cell viability and migration of HSFs and downregulated the protein levels of TGF-β1, COL 1, FN 1, and α-SMA.

Conclusion:
STAT3 can be activated in HS.AC may exert its pharmacological effects of inhibiting TGF-β1 signal transduction and regulating extracellular matrix remodeling in HS by inhibiting STAT3 phosphorylation.However, the specific molecular mechanism of AC remains to be verified through further experiments.the circles in the network were adjusted according to the degree of nodes.

| Construction of "drug-component-target" network diagram
According to the data entry requirements of cytoscape software, 30 the data of the above steps were sorted out and imported into cytoscape 3.9.0 to generate "drug-component-target" network.Later, the network relationship between AC and common targets was analyzed, and the analysis result was saved.Then, the shape and color of each node were adjusted to visually display the connection of "drug-component-target".

| Cell culture
Primary human hypertrophic scar fibroblasts (HSFs) and normal fibroblasts (NFs) were isolated and cultured by a tissue culture method of Ti et al. 31 HS tissue and normal skin tissue were deriving from patients receiving scar repair surgery in our hospital.The cells were grown in a high-glucose DMEM containing 10% fetal bovine serum (Gibico, USA) and penicillin-streptomycin solution (Gibico, USA).

| Cell transfection and grouping
STAT3 siRNA (si-STAT3) and its negative control (siNC) were designed and synthesized by Shanghai GenePharma Co., Ltd.HSFs were seeded in a six-well plate at the density of 5 × 10 4 cells/well, followed by cell culture performing in an incubator with constant temperature, 5% CO 2 , and saturated humidity at 37°C.After 24 h, the si-STAT3 and siNC were transfected into HSFs according to the instruction of Lipofectamine™ iRNAiMAX kit (Invitrogen, America).
On completion of 24-h transfection, the medium was changed by a new one and 20 μM AC was added into the new medium for 24-h culture.
The cells were divided into the control group (HSFs without any treatment), AC group (HSFs with AC treatment), AC + siNC group (HSFs with AC treatment and transfection of siRNA negative control), and AC + si-STAT3 group (HSFs with AC treatment and si-STAT3 transfection).

| MTT assay
HSFs were seeded in 96-well plates at a density of 5 × 10 3 cells/ well, and after static culture for 24 h, the cells were treated by AC.
After AC treatment, the culture supernatant was aspirated, 10 μL of the MTT working solution (0.5 mg/mL, GBCBIO Technologies Inc., Guangzhou) was added, and the cells were cultured in an incubator for 4 h.After completion of the culture, MTT supernatant was aspirated and 150 μL of DMSO was added to each well.The formazan product was shaken for 15 min in the dark until it was completely dissolved.Later, the optical density at 570 nm was detected with a microplate reader, and cell viability was calculated based on OD.

| Western blot
NFs or HSFs were seeded in six-well plates at the density of 1 × 10 5 cells/well.After drug administration, the cells were washed with PBS twice and RIPA lysate was added to extract total protein.
Next, the concentration of protein sample was detected using BCA kit (Beyotime Biotechnology, Shanghai).Subsequently, the target protein was separated by vertical electrophoresis using 10% SDS-PAGE gel and 5% concentrated gel at a constant pressure of 90 V for 120 min.Later, the target protein was transferred onto PVDF membranes via wet transfer method at a constant current of 300 for 90 min.The PVDF membrane was blocked with 5% skimmed milk (Beyotime Biotechnology, Shanghai) blocking solution for an hour and a half at room temperature.Then, the membranes were incubated with primary antibodies anti-STAT3, anti-p-STAT, antitransforming growth factor-β1 (TGF-β1), anti-collagen I (COL 1), anti-fibronectin 1 (FN1), anti-alpha-smooth muscle actin (α-SMA,) and β-actin (1:1000, Cell Signaling Technology, Inc., America) at 4°C overnight.Next, the membranes were incubated with the secondary antibodies for 1 h at room temperature.After that, the protein bands were visualized using electrogenerated chemiluminescence (ECL, GBCBIO Technologies Inc., Guangzhou).The statistical analysis for gray value of target proteins was performed by Image J. The relative expression of target protein in cells of each group was calculated using β-actin as the internal control.

| Scratch wounding experiment
A six-well plate was prepared for seeding HSFs at 1 × 10 5 cells/well.
After a single cell layer was formed, the cells were scratched with a sterile pipette tip.Subsequently, the cell monolayer was washed three times with PBS to remove detached cells.After culturing using a serum-free medium, the cells were treated with AC.At 12, 24, and 48 h of drug treatment, the samples were observed under a microscope, and nine fields of view of each sample were randomly selected for photographing.Scratch width at different time points was measured by Image J, followed by the calculation of the cell migration rate.

| Statistics analysis
According to the results of normality test and homogeneity of variance test, the SPSS26.0software was employed for one-way analysis of variance of cell viability, and the nonparametric test for the analysis of the relative protein expression and cell migration rate.
p < 0.05 indicated a significant difference.Besides, all statistical plots were drawn using GraphPad Prism 6.0.

| Screening for the common targets of drug-disease
In this study, we obtained a total of 134 potential pharmacodynamic targets of AC and 2333 targets of HS.The Venn diagram of AC targets and HS targets demonstrated a total of 50 common targets (Figure 1) and the specific names of these 50 targets were listed in Table 1.

| Construction of a "drug-component-target" network
Subsequently, cytoscape3.9.0 software was applied to analyze the "drug-component-target" network of AC-HS, and the analysis data were exported (Figure 2).In the network, the green hexagon indicated Centella asiatica, the lake green diamond represented AC and madecassoside, and the purple circles were the common targets of AC-HS and madecassoside-HS.

| Construction of the protein-protein interaction (PPI) network and screening of the core genes
The obtained 50 common targets were uploaded to the STRING 11.0 database, and a total of 178 edges were obtained (Figure 3A).
Each circle called "node" represented a common target; the node degree value indicated the number of nodes interacting with other nodes, and the larger the value indicated more nodes interacting with this node.As shown in Figure 3A, the size and color depth of circles were changed with the node degree value of the target; the larger node degree value indicated the larger circle and the deeper color.According to node degree, the top five genes consisted of JUN, STAT3, ESR1, HSP90AA1, and MAPK14 in sequence (Figure 3B).

| p-STAT3 protein level and cell viability in hypertrophic scar fibroblasts are significantly upregulated
The expression levels of STAT3 were detected in NFs and HSFs.
The detection results revealed few differences between NFs and HSFs in terms of the STAT3 protein expression, but compared with NFs, HSFs exhibited significantly increased p-STAT3 (Figure 4A).In addition, the results of MTT assay showed that the cell viability of HSFs was much higher than that of NFs (Figure 4B).

| Asiaticoside reduces p-STAT3 protein expression and inhibits cell viability and migration in hypertrophic scar fibroblasts
Western blot analysis showed a decrease in STAT3 protein levels

| Asiaticoside reduces the protein expression levels of collagen I, fibronectin 1, alpha-smooth muscle Actin and transforming growth factor in hypertrophic scar fibroblasts
The main components of scars include COL 1, FN1, α-SMA, and TGF-β1 is the main driving factor of tissue fibrosis.In this study, the western blot outcomes displayed that, in comparison with the control group, the protein levels of COL 1, FN1, α-SMA, and TGF-β1 in the AC group and AC + siNC group were significantly downregulated.The protein levels of COL 1, FN 1, α-SMA, and TGF -β1 were markedly lower in AC + si-STAT3 group than in the AC + siNC group (Figure 6A,B).

| DISCUSS ION
According to PPI network analysis results, JUN, STAT3, ESR1, HSP90AA1, and MAPK14 were the top five genes in terms of node degrees.In other words, these five genes have more interaction with other common targets of AC-HS.Besides, the node degrees of JUN and STAT3 were the highest, both of which were 24.Wound healing involves three stages: inflammation, proliferation, and remodeling. 33AT3 is activated in the first stage, 34 and under normal conditions, STAT3 plays a positive role in cell survival and proliferation.35 However, the continuous activation of STAT3 is closely correlated with tissue fibrosis.36 A previous research has pointed out abnormally increased expression and phosphorylation of STAT3 in keloid tissue (another type of abnormal scar). 37Unfortunately, there are few studies on STAT3 and HS.In this research, the phosphorylation level of STAT3 was observed to be significantly higher in HSFs than in NFs by western blot assay, indicating that STAT3 may be abnormally activated in both abnormal keloids and HS.
Subsequently, based on the findings of network pharmacology analysis, we explored the effect of AC on the STAT3 expression and activation in HSFs.Briefly speaking, AC could significantly reduce tion of keloid fibroblasts. 38On the basis of the results of this study, it could be preliminarily concluded that the inhibitory effect of AC on the viability of HSFs was associated with its role in reducing the phosphorylation level of STAT 3.
HS is a pathological result of uncontrolled fibrous tissue proliferation after skin injury.Essentially, HS is a dermal form of fibroplasia disorder. 39During wound healing, the continuous proliferation and activation of myofibroblasts in the regenerated skin tissues are the primary drivers of HS. 40 Cell migration is the main form of fibroblast movement, 41 and cell scratch assay is the most commonly used experimental method for detecting cell migration. 42In this paper, after treatment of AC or si-STAT3 in HSFs, the healing rate of cell scratches was decreased in HSFs after AC treatment or si-STAT3 transfection, and the healing rate was further decreased after the combination of AC treatment and si-STAT3 transfection (Figure 5C).
It is reported that STAT3 is involved in cell migration, cell proliferation, gene activation, regulation of actin cytoskeletal recombination, and other cellular activities. 43Our experiments determined that STAT3 regulated the migration of HSFs, and collectively, AC not only reduced the phosphorylation of STAT3, but also effectively inhibited the migration of HSFs.
TGF is one of the key signaling molecules activated in fibrosis. 44udies have shown that TGFβ stimulates local and bone fibroblasts to differentiate into myofibroblasts, allowing an increase in the number of myofibroblasts in HS; actin filaments in myofibroblasts contribute to wound contraction and the production of tension on the wound. 45According to previous studies, TGFβ can activate STAT3 7][48] Western blot analysis in this study showed that both AC and si-STAT3 could significantly downregulate the TGF-β1 protein level in HSFs, indicating a negative regulatory effect of AC on the expression and signaling of TGF-β1 in HSFs.
However, this negative regulatory effect and its mechanism need to be further verified.Furthermore, after a combination of AC and si-STAT3 treatment, the protein level of TGF-β1 was further downregulated.In a nutshell, STAT3 in HS may have a certain regulatory effect on TGF-β1 signal, and the specific molecular mechanism remains to be further explored.
During the proliferation stage of wound healing, the recruited fibroblasts synthesize extracellular matrix (ECM) and serve as a scaffold of tissue repair. 49Excessive collagen deposition, altered collagen remodeling and contraction play a key role in HS formation. 50stological studies have stated that HS is mainly characterized by the overproduction of a variety of fibroblast proteins (including FN1). 51As for α-SMA, it is a marker of mature myofibroblasts, and Arti V Shinde et al. suggested that α-SMA might significantly affect the remodeling and contraction of ECM. 52In another study, STAT3 is involved in the regulation of ECM and cell proliferation in HS fibroblasts. 53Based on the above literature, we investigated the effect of AC on COL 1, α-SMA and FN1 in HSFs by western blot assay.Shortly speaking, both AC and si-STAT3 could significantly downregulate the protein levels of COL 1, α-SMA and FN1 in HSFs, and the combination of AC and si-STAT3 had better effect.
Nevertheless, the molecular mechanism of STAT3 in regulating COL 1, α-SMA, and FN1 in HS needs further experimental investigation.

| CON CLUS ION
In

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors report no conflict of interest.

F I G U R E 4
and p-STAT3 level in the AC + si-STAT3 group in comparison with the AC + siNC group.AC treatment significantly inhibited the expression of p-STAT3 protein in HSFs (Figure 5A).According to the outcomes of MTT assay, HSFs in the AC and AC + siNC groups exhibited obviously declined cell viability compared with those in the control group; in comparison with the AC + siNC group, the cell viability was markedly decreased in the AC + si-STAT3 group F I G U R E 1 Common targets of asiaticoside and hypertrophic scars.TA B L E 1 Gene names of common targets.Gene names Common targets of asiaticoside and hypertrophic scars PTPN1, DNMT1, F2R, ACHE, AR, TLR9, HSD11B1, HMGCR, ESR1, HSP90AA1, NF2, PRPS1, MAPK9, PCNA, CCNE1, ZEB2, MUC1, ITPKC, HBEGF, TRIM21, BTK, RARB, NR1I3, NR3C2, HBB, HFE, NFATC1, TUT1, MMP2, HLA-E, PAH, MMP1, FCGR2A, BCL2L1, HSD11B2, JUN, PTPA, F2, STAT3, CA2, PLCG1, P4HB, ACP1, BDNF, CAT, MAPK14, MTOR, NGF, PDPK1, STX1A F I G U R E 2 A "drug-component-target" network diagram.F I G U R E 3 Protein-protein interaction (PPI) network.(A) The diagram of protein-protein interaction (PPI).The larger node degree value meant the larger circle and the deeper color.(B) Top 13 genes according to the node degree value.Significantly upregulation of the STAT3 protein level and cell viability in hypertrophic scar fibroblasts.(A) The protein expression levels of STAT 3 and p-STAT 3 were determined by western blot.NFs, normal fibroblasts; HSFs, hypertrophic scar fibroblasts.(B) Cell viability was measured by the MTT assay.**p < 0.01 versus NFs.(Figure 5B).Furthermore, the scratch assay results showed that AC deceased the migration abilities of HSFs, and the migration was further inhibited after AC treatment and si-STAT3 transfection.With the prolongation of the drug administration, the scratch width in each group was gradually reduced, with the smallest in the control group (Figure 5C,D).

F I G U R E 5
Asiaticoside reduces p-STAT3 protein expression and inhibits cell viability and migration in hypertrophic scar fibroblasts.(A) The protein expression levels of STAT3 and p-STAT3 in each group were determined by western blot; (B) MTT was applied to test the cell viability of each group; (C,D) Cell migration ability was examined by the scratch assay.**p < 0.01. the STAT 3 phosphorylation level in HSFs (Figure 5A).Either si-STAT3 or AC could effectively inhibit the viability of HSFs, and the cell viability was further decreased after si-STAT3 transfection and AC treatment.Studies have confirmed that STAT3 inhibitors can effectively inhibit abnormal behaviors such as excessive prolifera- summary, this study has been performed based on the results of network pharmacology research and previous literature.After confirming the abnormal activation of STAT3 in HSFs, we establish a si-STAT3 cell model using siRNA interference technology.This model serves as a positive control to evaluate the efficacy of AC and investigate the effect of AC on HSFs.Briefly speaking, AC may regulate the generation and remodeling of ECM in HSFs by inhibiting the phosphorylation of STAT3 and the transduction of TGF signal in HSFs, thereby resulting in the decreased expression levels of COL 1, α-SMA, and FN1 proteins.However, the specific mechanism of AC in HS requires further experimental verification.

F I G U R E 6
Asiaticoside decreases the protein expression levels of collagen I, fibronectin 1, alpha-smooth muscle Actin and transforming growth factor-β1 in hypertrophic scar fibroblasts.(A,B) The protein levels of collagen I (COL 1), fibronectin 1 (FN 1), alpha-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1) in each group were detected by western blot.**p < 0.01.