Effects of Mu-opioid receptor agonists on wound healing of corneal epithelium

Abstract

Purpose : There is a need for topical analgesic drugs after corneal traumas and surgeries. However, scarce data is available about the effects of opioids, which are potent analgesics, and opioid receptors (OPrs) on corneal epithelial healing. We investigated effects of four different μ-OPr agonists (Biphalin, AM94, EM1, EM2) on in vitro wound healing cell culture model of corneal epithelium. Methods : We used immortalized human corneal epithelial cells (HCECs) for experiments. We investigated OPRM1, OPRD1, and OPRK1 gene expressions in HCECs with quantitative polymerase chain reaction (qPCR). Then, we stained μ-OPrs with immunofluorescence antibodies (Figure 1). We measured toxicity of agents in various doses with MTT (n=4). In vitro scratch assay was used to investigate the wound healing effect of OPr agonists (n=8). Cell migration and proliferation were assessed in transwell migration assay (n=20) and Ki67 proliferation assay (n=20), respectively. We used PBS as vehicle and naloxone, non-selective competitive OPr antagonist, to inhibit their OPr dependent effects. All experiments were replicated at least two times and all data was analyzed with two-way ANOVA. Results : We were able to show RNA expressions of OPRM1 and OPRD1 with qPCR in HCECs. MTT assay showed that μ-OPr agonists have no toxic effects on HCECs at 1, 10 and 100 µM concentrations. Therefore 1 µM and 10 µM concentrations are used in all assays. At 1 µM concentration, EM1(selective μ-OPr agonist), EM2(selective μ-OPr agonist) and Biphalin (μ- and δ-OPr agonist) significantly increased relative wound closure(p<0.01) when compared to vehicle. At 1 µM, Biphalin showed significant increase at migration (p<0.001) but not in proliferation; EM1 and EM2 showed significant increase in both migration(p<0.001) and proliferation(p<0.01). AM94(μ-, δ- and κ-OPr agonist) had no significant effects on wound healing, migration and proliferation in both doses. Naloxone significantly decreased positive effects of μ-OPr agonists in all three assays (p<0.05). Conclusions : Our study suggests that selective μ-OPr agonists have positive effects on corneal epithelial wound healing. Positive effects of μ-OPr agonists can be based on increased migration and proliferation of HCECs. We propose selective μ-OPr agonists could be new agents for analgesia after surgeries and traumas of cornea.