Angiotensin-converting Enzyme 2 Activator Diminazene Aceturate Prevents LPS-induced Inflammation By Inhibiting MAPK And NF-κB Pathways In Human Retinal Pigment Epithelium

Background: Retinal inflammation is a devastating pathological process in ocular diseases. Functional impairment of retinal pigment epithelium(RPE) is associated with inflammatory retinal diseases. Enhancing the protective axis namely ACE2/Ang-(1-7)/Mas by activation of ACE2 presents anti-inflammatory properties. We investigated whether diminazene aceturate(DIZE), an angiotensin-converting enzyme 2(ACE2) activator, prevented lipopolysaccharide(LPS)-induced inflammatory response by activating the protective axis and whether the effect was mediated by inhibiting the mitogen-activated protein kinase(MAPK) and the nuclear factor-κB(NF-κB) pathways.Methods: Cell counting kit-8(CCK-8) assay and real-time PCR were used to determine the optimum concentration and incubation time of DIZE. ARPE-19 cells and primary cultured human retinal pigment epithelia(hRPE) were incubated with or without 10 μg/mL DIZE for 6 hours before stimulated with 5 μg/mL LPS for 24 hours. The mRNA expression of inflammatory cytokines, AT1 R and AT2 R was analyzed. The protein level of inflammatory cytokines, Ang II and Ang-(1-7) was detected. Phosphorylation of p38 MAPK, extracellular signal-regulated kinase(ERK)1/2, c-Jun n-terminal kinase(JNK) and phosphorylated transcription inhibition factor-κB-α(p-IκB-α) were measured. Inhibitors of MAPKs and NF-κB were added to verify the involvement of these pathways. A small interfering RNA(siRNA) targeted to ACE2 and a selective Ang-(1-7) antagonist A779 was used to confirm the role of ACE2 and the involvement of ACE2/Ang-(1-7)/Mas axis.Results: DIZE remarkably increased the expression of ACE2 and inhibited the expression of IL-6, IL-8, and MCP-1 at both mRNA and protein levels in both RPE cell lines stimulated with LPS. Inhibitors of p38, ERK1/2, JNK and NF-κB significantly decreased LPS-induced overproduction of IL-6, IL-8, and MCP-1. DIZE reduced the expression of Ang II and AT1 R, whereas increased Ang-(1-7). Furthermore, DIZE down-regulated the phosphorylation of p38 MAPK, ERK1/2, JNK and the activation of NF-κB upon stimulation with LPS. Downregulating ACE2 and pre-treatment with A779 abrogated the effects of DIZE on production of cytokines, the expression of Ang II, Ang-(1-7), AT1 R, phosphorylation of MAPKs and activation of NF-κB.Conclusion: DIZE inhibits LPS-induced inflammatory response by activating ACE2/Ang-(1-7)/Mas axis in human RPE cells. The protective effect is mediated by inhibiting the p38 MAPK, ERK1/2, JNK and NF-κB pathways.