Wound healing is subject to sophisticated regulation by a number of key factors. Failure in achieving wound closure may cause delayed healing or wound dehiscence with subsequent severe complications, even death[21–22]. During cutaneous wound healing, keratinocytes make up approximately 90%-95% of the epidermis and act as an innate immune cell for skin homeostasis, which is considered the most important in maintaining structural integrity and chemical barrier function of the skin. Also, it is well established that local anesthetic infiltration and infusion to the wound site, acting directly on the application site, is considered a valuable technique to relieve pain. Ropivacaine, a long-acting regional anesthetic, is commonly selected for postoperative analgesia. In this study, we found that ropivacaine delayed wound closure in vivo. We wanted to further explore the function of ropivacaine on the proliferation and migration of HaCaT cells during wound healing. Our experiments showed that ropivacaine inhibited the PI3K/Akt/mTOR signaling pathways, thus suppressing the proliferation and migration of HaCaT cells. Understanding the long-term impact of ropivacaine on wound healing may help design new therapies aiming at minimizing the risk of adverse events.
Accumulating studies have proved that ropivacaine exhibits anti-proliferative and anti-migratory activities on numerous cells[23–26]. For example, ropivacaine inhibits proliferation and promotes apoptosis of SH-SY5Y cells (human neuroblastoma cell line) in a dose- and time-dependent manner to induce neuronal injury. Besides, ropivacaine also suppresses MSC proliferation and migration by reducing the expression of ICAM-1 via the IκB–NF-κB signaling pathway. In addition, ropivacaine plays an anti-proliferative role in breast cancer cells through regulating the miR-27b-3p/YAP axis. In this study, we found that ropivacaine promoted the apoptosis of HaCaT cells. Apart from this, the study stipulated that proliferation and migration of HaCaT cells were suppressed by ropivacaine in a dose- and time-dependent manner, which may subsequently lead to delayed re-epithelialization, impeding the wound healing progresses.
Furthermore, the mechanisms of the ropivacaine-mediated suppression of proliferation and migration in HaCaT cells were further investigated. Wound healing is regulated by a complex network of signaling pathways stimulated by various alert signaling peptide. The PI3K/AKT/mTOR signaling pathway is one of the numerous molecular signaling pathways that can affect the initiation of wound healing. Notably, accumulating studies have shown that PI3K/AKT/mTOR pathway is involved in the proliferation and migration of keratinocytes[18, 32–34], indicating that it may be associated with the migration and proliferation of keratinocytes induced by ropivacaine. Our research demonstrated that ropivacaine significantly inhibited the activation of the PI3K, Akt, and mTOR proteins, as evidenced by the decreased phosphorylation of these proteins. Additionally, we found that the suppression of proliferation and migration induced by ropivacaine was significantly reversed after intervention with IGF-1, a PI3K/Akt agonist. Therefore, we further confirmed that ropivacaine promotes the proliferation and migration of HaCaT cells by inhibiting the PI3K/Akt/mTOR pathway.
As mediators of migration and proliferation, cytokines and chemokines are involved in all of the wound-healing phase. At the early stage, activated keratinocytes are the principal source of cytokines (e.g., IL-6, IL-10, TNF-α) acting on leukocytes, keratinocytes, and fibroblasts[36, 37]. It is generally known that IL-10 is an anti-inflammatory cytokine, while IL-6 and TNF-α are pro-inflammatory cytokines. IL-10 appears to influence the wound-healing environment by decreasing the expression of pro-inflammatory/profibrotic mediators, promoting angiogenesis[38, 39]. During wound healing, IL-6 affects fibrogenesis, angiogenesis, re-epithelialization and granulation tissue formation[40, 41]. Physiological levels of TNF-α promote cellular migration, proliferation, while persistent and elevated TNF-α is detrimental to angiogenesis and collagen fiber arrangement[38, 42]. Inflammatory cytokines are a necessary component of wound healing; however, which they persist, they may hinder wound healing process. In the present study, we detected mRNA expression and concentrations of IL-6, TNF-α, and IL-10 in the culture supernatants and the results showed that the IL-10 mRNA level was significantly decreased in the ropivacaine-treated group, whereas the mRNA expression of IL-6 and TNF-α was significantly reduced. Following this result, the concentration of IL-6, IL-10, and TNF-α secreted from the HaCaT cells showed the same trends. These findings indicated that ropivacaine contributed to the release of pro-inflammatory cytokines and inhibited the secretion of anti-inflammatory cytokines of HaCaT cells. PI3K/AKT/mTOR pathway activation has been reported to mediate cytokine production and release and has been shown to play an essential role in wound repair[39, 43]. Hence, we performed we applied IGF-1 for estimating intervention effects. The result demonstrated that IGF-1 intervention remarkably reversed the downregulation of IL-10 and the upregulation of IL-6 and TNF-α induced by ropivacaine action, suggesting that PI3K/AKT/mTOR pathway is involved in the process of ropivacaine-induced cytokines expression alterations.