Diabetes mellitus (DM) is a metabolic disease caused by multiple etiologies and characterized by chronic hyperglycemia. Further increases in morbidity and mortality from complications of DM are predicted (1). Diabetic skin lesions (2), especially diabetic foot, are of particular concern as they not only reduce the quality of life of patients but also put enormous economic pressure on national healthcare systems. In recent years, the importance of early diagnosis and treatment of diabetes and its complications has become increasingly recognized (3). A series of changes in the skin of diabetes patients before skin injury occurs have aroused great interest in researchers (4). Non-injured skin in diabetes patients may be the basis of skin lesions, and early detection and rapid intervention may reduce or delay the occurrence of skin lesions to a certain extent.

The skin has three layers: epidermis, dermis, and subcutaneous tissue. The integrity of the epidermis and stability of the dermis are the basis for preventing the invasion of external harmful substances. Diabetes damages the structure of dermal collagen, and studies have shown that a higher average score for skin structural defects (5) and inferior biomechanical properties (6) increase the risk for developing DM skin complications. Collagen is the main component of the dermal extracellular matrix (ECM), primarily secreted by fibroblasts and regulated by the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) (7, 8).

MMPs are a family of zinc-dependent endopeptidases that are the most important enzymes for the degradation of the ECM. They play a vital role in both physiological and pathological tissue remodeling. TIMPs are endogenous specific inhibitors of MMPs; for instance, TIMP-2 can inhibit the activity of MMP-2 and TIMP-1 can inhibit that of MMP-9 (9). A puncture biopsy of the wound tissue of a chronic ulcer of diabetic skin showed that the expression of MMP-1, -2, -8, and -9 was increased and that of TIMP-2 was decreased (10). The degradation of ECM regulated by MMPs/TIMPs is a crucial cause of poor wound healing in diabetes patients (11). Although it takes a long time for diabetic skin to be injured, the skin tissue will have been impacted by high glucose levels for a long time, and its structure will have largely changed at an early stage (12). The function of the MMP/TIMP balance has been studied in diabetic poor wound healing, but whether this balance contributes to early changes in diabetic skin is unknown. Therefore, it is of great significance to improve the understanding of early non-injured skin changes in diabetes patients, and early intervention is important for the clinical prevention and treatment of skin lesions.

In the current study, we found that dermal collagen deposition disorder occurred in the skin of some diabetes patients before evident skin injury. Moreover, the cause of the changes in the non-injured skin collagen targeting the balance of MMP2/TIMP2 and MMP9/TIMP1 was investigated.

Dermal collagen deposition disorders in diabetic skin prior to skin injury have increased clinical awareness and caution toward complications of diabetic skin. The collagen deposition disorder can be caused by either decreased synthesis of new collagen or increased collagen breakdown and both. In our study, we found that collagen type I expression was reduced in high glucose-treated HDFs. This implied that the HDFs play a vital role in collagen secretion in diabetic skin. In addition, our subsequent study found that the balance of MMP-2/TIMP-2 and MMP-9/TIMP-1, which can regulate collagen synthesis and decomposition, was disrupted after HDFs were cultured with high glucose. To verify the regulatory relationship among the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratio and skin collagen deposition disorder, rescue experiments were implemented. By inhibiting the activities of MMP2 and MMP9 in diabetic mice, the disorder of collagen deposition was alleviated. Combined with all findings, we have reason to believe that the collagen deposition disorder in diabetic non-injured skin is caused by both decreased synthesis of new collagen and increased collagen breakdown. The imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1 contributes to the skin disorder of collagen deposition in diabetes patients, providing ideas for managing diabetes skin complications early.

The pathogenesis of diabetic skin lesions is complex and has not yet been fully understood. Some researchers believe that the epidermal structural barrier in the skin of diabetes patients is damaged (22) and prolonged hyperglycemic stimulation and insulin deficiency inhibit keratinocyte proliferation and directional migration, interfering with epidermal differentiation (23–26). In addition, the glycosylation of collagen in the dermis makes the ECM hard and brittle, reducing the mechanical competence of the dermis (27). The glycosylated ECM can regulate the morphology and function of surrounding cells, such as by inducing the differentiation and migration of keratinocytes (28) and reducing the adhesion of dermal fibroblasts and their proliferation and migration (27). In this study, it was found that the skin collagen of diabetes patients become disordered and its density was variable, resulting in aged appearance skin (12), which likely changed the mechanical competence of the dermis. Both the destruction of the epidermal structural barrier and the reduction of the mechanical competence of the dermis impair the mechanical stability of skin; decrease skin resistance to physical, mechanical, chemical, and pathogenic microbial invasion; and increase the risk of skin injury. Although the methods for early diagnosis of skin changes in diabetes patients are limited, it is of great clinical significance to improve awareness for the diagnosis of early non-injured skin (29).

The synthesis and degradation of ECM are primarily regulated by the balance between MMPs/TIMPs that destroys the stability of the ECM and promotes the occurrence of skin injury. The ratio of MMPs/TIMPs is unbalanced in the skin tissue of diabetes patients. The imbalance of MMPs/TIMPs in diabetes may be related to continuous pro-inflammatory and -fibrotic factors being secreted by tissues and cells under high glucose. Conversely, the disrupted balance of MMPs/TIMPs may be caused by the formation of early glycosylation products (30). Generally, MMPs can be classified into four types according to substrate specificity: stromeolysins (MMP-3, -10, and -11); membrane-type MMPs (MT-MMPs, MMP-14–17, -24, and -25); collagenases (MMP-1, -8, -13, and -18), which degrade fibrous collagen into shorter fragments; and gelatinases (MMP-2 and -9), which degrade type IV collagen and gelatin (31, 32). The dermal ECM primarily consists of type I followed by type III collagen. Its stability is greatly affected by the synthesis and decomposition of type I collagen. In addition to being catabolized by collagenase, its decomposed fragments can be further degraded by MMP-2 and -9 (33, 34). Besides, canstatin, a type IV collagen fragment catabolized by MMP-2 and -9, is reported to regulate the migration and secretion of fibroblasts (35). Thus, the function of MMP-2 and -9 also plays a role in regulating the synthesis and decomposition of type I collagen, and their aberrant expression may change the stability of skin collagen.

Increased expression of MMPs has been reported in chronic wounds. Besides, some studies have reported that the expression of MMP-1, -2, and -9 increased in non-injured skin of diabetes patients (12, 36, 37). Unlike these studies, considering the interaction of MMPs and TIMPs, we focused more on its balance. Furthermore, to understand the effect of the disturbance of this delicate balance of MMPs/TIMPs, in vivo experiments were conducted on the regaining of this balance in the present study.

Previous studies indicate that MMPs can act multifunctionally in the regulation of inflammation, wound repairing, and wound healing (38, 39). Chronic nonhealing wounds in DM go through multiple stages. The skin goes from normal in the non-diabetes stage to non-injured in the early diabetes stage; the progression is from slight injury to wound formation and finally to nonhealing, accompanied by the persistent abnormal expression of MMPs. The loss of the balance of MMPs/TIMPs inhibits the overexpression of MMPs selectively and can promote the healing process in wounds (40, 41). Similarly, determining whether early intervention of the overexpression of MMPs can prevent or reduce the occurrence of skin injury to reduce the formation of wounds requires further exploration. The present study provides insights into this to some extent.

Although we observed that collagen deposition disorders were alleviated after inhibiting the activity of MMP-2 and -9 in the skin of diabetic mice, the details of this mechanism were not elucidated, which serve as a basis for our future studies. In addition, as a result of experimental condition limitations, there was a lack of morphological and mechanical analyses of disordered collagen fibers in the skin of diabetes patients, limiting our understanding of the stability change of the skin structure to some degree.

According to national legislation/guidelines, specifically the Administrative Regulations of the People’s Republic of China on Human Genetic Resources (http://www.gov.cn/zhengce/content/2019-06/10/content_5398829.htm, http://english.www.gov.cn/policies/latest_releases/2019/06/10/content_281476708945462.htm), no additional raw data are available at this time. Data of this project can be accessed after an approval application to the China National Genebank (CNGB, https://db.cngb.org/cnsa/). Please refer to https://db.cngb.org/, or email: CNGBdb@cngb.org for detailed application guidance. The accession code CNP0000999 should be included in the application.

The studies involving human participants were reviewed and approved by Wuhan Union Hospital Ethics Committee (REC 08/H1202/137). The patients/participants provided their written informed consent to participate in this study.

PZ, CY, and SZ performed experiments. Z-XK and D-XC generated the mice. PZ and CY analyzed data and interpreted results of experiments. Y-QL and QL conceived and designed research, prepared figures, and drafted manuscript. All authors contributed to the article and approved the submitted version.

We would like to thank the National Natural Science Foundation of China (No. 81900432 to QL) for funding.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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