Regulatory role of ADAMTS8 in proliferation and apoptosis of hypertrophic scar fibroblasts

Abstract

BACKGROUND: Studies have confirmed that A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS8) plays a regulatory role in fibrosis, so it is of great clinical significance to explore the mechanism of ADAMTS8 in hypertrophic scars. OBJECTIVE: To investigate the regulatory effect of ADAMTS8 on hypertrophic scars. METHODS: (1) Immunohistochemical staining was used to detect the expression of type I collagen, type III collagen, alpha-smooth muscle actin and ADAMTS8 in normal human skin and hypertrophic scar tissues. Western blot was used to detect ADAMTS8 protein expression in normal skin and hypertrophic scar tissues. With hypertrophic scar as positive sample and normal skin as negative sample, receiver operating characteristic curve was drawn to analyze the ability of ADAMTS8 to predict and distinguish normal skin from hypertrophic scar. (2) STRING 12.0 platform was used to construct a protein-protein interaction network for ADAMTS8, and GO functional enrichment and KEGG pathway enrichment analyses were performed on the obtained targets. (3) Fibroblasts from human hypertrophic scar tissue were isolated and cultured. The 3rd to 6th generation fibroblasts were divided into three groups: control group (routine culture), Ad-NC group (transfected with empty adenovirus), and Ad-ADAMTS8 group (transfected with adenovirus overexpressing ADAMTS8). CCK-8 assay and EdU staining were used to detect cell proliferation activity, and flow cytometry and TUNEL staining were used to detect cell apoptosis. RESULTS AND CONCLUSION: (1) Immunohistochemical staining showed that the expression of type I collagen, type III collagen and alpha-smooth muscle actin in hypertrophic scars was higher than that in normal skin (P < 0.001), while ADAMTS8 expression was lower than that in normal skin (P < 0.001). Western blot showed that ADAMTS8 protein expression in hypertrophic scars was lower than that in normal skin (P < 0.001). Receiver operating characteristic curve showed that the area under the curve of ADAMTS8 predicting hypertrophic scar was 0.86, indicating that ADAMTS8 has good ability to distinguish hypertrophic scar from normal skin. (2) The top 41 genes were screened through STRING database. KEGG enrichment showed that ADAMTS8 was mainly involved in extracellular matrix receptor interaction, phosphatidylinositol-3-kinase-protein kinase B signaling pathway, efferocytosis and other biological processes and key mechanisms. GO enrichment showed that ADAMTS8 was involved in apoptosis-related pathway enrichment, including negative regulation of fibroblast growth factor receptor signaling pathway, fibroblast growth factor binding, negative regulation of apoptosis and apoptotic process. (3) CCK-8 assay and EdU staining showed that overexpression of ADAMTS8 inhibited the proliferation of hypertrophic scar fibroblasts; flow cytometry and TUNEL staining showed that overexpression of ADAMTS8 promoted apoptosis of hypertrophic scar fibroblasts. (4) These results indicate that ADAMTS8 expression is decreased in human hypertrophic scars, and overexpression of ADAMTS8 can inhibit proliferation and promote apoptosis of hypertrophic scar fibroblasts.

1. Introduction

Scar is the final result of tissue repair and healing after severe skin injury [1]. According to whether the scar tissue is repaired by cells and tissues with the same structure as the original damaged tissue, it can be divided into physiological scar and pathological scar [2]. Pathological scars are further divided into hypertrophic scars that usually do not exceed the original wound boundary and keloids that are invasive and can exceed the original wound boundary [3]. The histological characteristics of hypertrophic scars include abnormal hyperplasia of dermal tissue, collagen fibers parallel to the epidermal surface, excessive production of myofibroblasts and various fibroblast proteins [4], and rich acidic mucopolysaccharides [5]. The pathological mechanism involves excessive deposition of type I and type III collagen [6]. At present, there are many treatment methods for hypertrophic scars, including surgical, physical and pharmacological methods [7], but the effects are limited and adverse reactions are numerous, which has become an increasingly prominent problem in clinical treatment.

The A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family proteins are secreted extracellular proteases with thrombospondin type I motifs [8-9], involved in the maturation of procollagen and von Willebrand factor, as well as proteolysis, cell adhesion, and extracellular matrix remodeling [10-11]. The human ADAMTS family includes 19 proteins [12], and different ADAMTS have different biological functions. For example, in the transverse aortic constriction model, ADAMTS16 expression is elevated, and ADAMTS16 overexpression can activate fibroblasts in vitro [13]; BADSHAH et al. [14] found that normal fibroblasts transfected with ADAMTS8 overexpression plasmid differentiated into myofibroblasts; ZHA et al. [15] found that ADAMTS8 regulates cardiac fibrosis by activating the epidermal growth factor receptor-mitogen-activated protein kinase/protein kinase B-Yes-associated protein signaling pathway. It can be seen that ADAMTS8 plays a role in fibrosis.