Oleanolic acid regulates the proliferation and extracellular matrix of keloid fibroblasts by mediating the TGF‐β1/SMAD signaling pathway

Keloid (KD) is a unique pathological fibroproliferative disease that seriously affects the appearance of patients. This study investigated the effect of oleanolic acid (OA) on the proliferation of keloid fibroblasts (KFs) and the expression of extracellular matrix (ECM)‐related proteins.


| INTRODUC TI ON
Keloid (KD) is a pathological scar tissue secondary to skin trauma and postinflammatory hyperplasia of a large amount of connective tissue. 1 Its characteristics include a crab-like growth, often beyond the edge of the original skin lesion, excessive deposition of the extracellular matrix (ECM), and an imbalance between the proliferation and apoptosis of fibroblasts. 2 At present, the pathogenesis of KDs is thought to be related to factors such as inflammation, trauma, immunity, genetics, and tension. Owing to poor responses of KDs to treatment, they remain a challenge in dermatology. 3,4 Oleanolic acid (OA) is a chemical natural product that is widely distributed in green leaf galls of Ligustrum lucidum, Prunella vulgaris, and other plants. OA and its derivatives have a variety of pharmacological activities, including hepatoprotective, anti-inflammatory, antioxidant, and antitumor effects. 5 Studies have shown that OA can inhibit fibrosis by reducing TGF-β1-mediated epithelialmesenchymal transition of renal tubular epithelial cells and improve myocardial fibrosis by inhibiting the activity of the AKT/mTOR pathway. 6,7 OA can also inhibit the proliferation of human bladder cancer cells via the AKT/mTOR/S6K and ERK1/2 signaling pathways and exerts its antitumor effect by blocking the cell cycle and destroying the mitochondrial membrane potential. 8,9 OA acetate can attenuate polyhexamethylene guanidine phosphate-induced pulmonary inflammation and fibrosis in mice. 10 Wojciak-Kosior et al. showed that OA had no toxic effect on normal fibroblasts and only slightly reduced their survival rate in a concentration-and time-dependent manner. 11 Considering that KD is a dermal fibroproliferative disease, it has similar pathological characteristics with fibrosis and tumors. Therefore, to help treat KD, this study explored the effects of OA on the proliferation of KD fibroblasts (KFs) and the expression of ECMrelated proteins and its mechanism of action using in vitro studies.
Before adding TGF-β1 and OA during cell culture, the culture medium was replaced with serum-free DMEM for starvation treatment of KFs.

| Cell proliferation assay
Dissolve 100 mg of OA powder in 10 mL of DMSO. An OA stock solution (8 μL) was added to 1 mL of serum-free DMEM, and the 80 μg/mL solution was diluted to 5, 10, 20, and 40 μg/mL. After KFs culturing in serum-free DMEM for 12, 24, and 48 h, 200 μL MTT working solution was added to each well. An MTT working solution at a concentration of 5 mg/mL was incubated at 37°C and 5% CO 2 for 4 h. The working solution was aspirated from the well, followed by the addition of a dimethyl sulfoxide solubilization solution and incubation on a shaker for 15-20 min in the dark. After the formazan crystals were fully dissolved, 200 μL of the solution was transferred from each well to a 1.5 mL centrifuge tube, followed by the addition of 300 μL of distilled water, and 100 μL of the mixture was transferred to a well of a 96-well plate. The absorbance (optical density, OD) value of each group was read at a wavelength of 570 nm using an automatic enzyme-linked immunosorbent assay analyzer. Each experiment was repeated three times.

| Western blotting
To further simulate the KD microenvironment, in vitro cultured KFs were treated with 10 ng/mL TGF-β1 for 24 h. An equivalent of 20 μg of total protein was resolved by 8% or 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat dry skim milk in Tris-buffered saline with Tween 20. Subsequently, the membranes were probed with primary antibodies overnight at 4°C. Then, the membranes were incubated with a secondary antibody, and immunoreactive bands were developed by electrochemiluminescence (Millipore). The experiments were repeated three times.

| Statistical analysis
Data were analyzed using the GraphPad Prism 7.0 software. A Student's t-test and one-way ANOVA were used for comparison between two groups and among multiple groups, respectively. p < 0.05 was considered statistically significant.

| OA inhibited the intra-and extracellular levels of the FN and procollagen I proteins in KFs
Western blotting showed that compared with those in the control group, in extracellular, the levels of procollagen I and FN in the OA group were significantly decreased in a concentration-dependent manner starting at 5 μg/mL OA (p < 0.01). In cells, the expression of the FN protein showed a decreasing trend at 5 μg/mL OA, and the decrease was the most significant at 20 μg/mL OA (p < 0.01).
However, the expression of procollagen I gradually decreased starting at 10 μg/mL OA (p < 0.05), and the most significant decrease was reached at 20 μg/mL OA (p < 0.01; Figure 2).

| Effect of OA on the expression of the MMP-1 and α-SMA proteins in KFs
Western blotting showed that compared with that in the control group, the expression of MMP-1 in KFs increased and that of α-SMA decreased at the OA concentration of 5 μg/mL, with a statistically significant change at 20 μg/mL OA (p < 0.05; Figure 3). Meanwhile, the FN protein showed a decreasing trend at the OA concentration of 5 μg/mL, and the decrease was the most significant at 20 μg/mL OA (p < 0.05; Figure 4).

| OA affects TGF-β1-induced expression of MMP-1 and α-SMA in KFs
The results of Western blotting showed that the expression of the MMP-1 and α-SMA proteins in the TGF-β1 group was significantly higher than that in the control group (p < 0.01). Compared with that in the TGF-β1 group, the expression of the MMP-1 protein in the TGF-β1 + OA group increased starting at the OA concentration of 5 μg/mL, and the most significant increase was observed at 20 μg/ mL OA (p < 0.05). The expression of α-SMA began to decrease at the OA concentration of 5 μg/mL, and the decrease was the most significant at 20 μg/mL OA (p < 0.01; Figure 5).

| OA inhibits TGF-β1-induced phosphorylation of SMAD2 and SMAD3 in KFs
Western blotting showed that the levels of P-SMAD2 and P-SMAD3 in the TGF-β1 group were significantly higher than those in the control group (p < 0.01). Meanwhile, the levels of P-SMAD2 and P-SMAD3 in the TGF-β1 + OA group were significantly lower than those in the TGF-β1 group (p < 0.01; Figure 6).

| DISCUSS ION
KD is characterized by excessive collagen deposition due to an imbalance between the production and degradation of the ECM. 12 In the process of KD tissue proliferation, the synthesis of type I and III collagens increases from the translation stage to the protein synthesis stage. It has been suggested that a large number of fibroblasts activate type I and III procollagen mRNA expression in the endoplasmic reticulum to produce type I and III procollagens. Procollagens are secreted into the ECM in the form of type I and type III collagens after being modified by prolyl 4-hydroxylase activity. 13 TGF-β1 is the only cytokine capable of accelerating the maturation of the epithelial cell layer. 14  In this study, KFs were further stimulated with TGF-β1, and the extracellular levels of FN and procollagen I were significantly increased, while OA could significantly reduce their levels. However, the expression of procollagen I was significantly increased after stimulation with TGF-β1 and was significantly downregulated after each OA concentration increment. We suggested that scar proliferation may mainly be caused by increased extracellular secretion of F I G U R E 4 Effects of OA on TGF-β1-induced intra-and extracellular levels of the FN and procollagen I proteins in KFs. Cell: cell lysate; Med: medium. # p < 0.05, ## p < 0.01 vs. control. *p < 0.05, **p < 0.01 vs. TGF-β1. As expected, the levels of P-SMAD2 and P-SMAD3 significantly increased under TGF-β1 stimulation compared with those in the control group and were significantly reduced by OA suggesting that OA may regulate the ECM production in KD via the TGF-β1/ SMAD signaling pathway. These results provide a molecular and theoretical basis for the use of OA in the treatment of KD.
There are limitations to this study. First, there was no comparative observation of the progression of normal fibroblasts. Second, the findings were not verified by in vivo experiments. In the future, after understanding the inhibitory effect of OA on the function of KFs in vitro and its mechanism of action, we intend to study the antiscarring effect of OA in vivo using an animal model to provide a more robust theoretical basis for the treatment of KD.

| CON CLUS ION
Our results suggest that OA can inhibit the proliferation of KFs and reduce the deposition of ECM via the TGF-β1/SMAD pathway.
These findings suggest that OA may be an effective drug for the prevention and treatment of KD.

AUTH O R CO NTR I B UTI O N S
Yinli Luo: Conceptualization, visualization; writing-review and editing. Zhehu Jin: Conceptualization, data curation, writing-review and editing. Longquan Pi: Conceptualization, data curation, writingreview and editing. Dongming Wang: Data curation. Xinghua Yuan: Data curation. All authors have read and approved the final manuscript.

FU N D I N G I N FO R M ATI O N
Funding not received for the study.

CO N FLI C T O F I NTER E S T S TATEM ENT
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 AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

E TH I C S S TATEM ENT
Not required.