title: Angiotensin-converting enzyme 2 activator diminazene aceturate prevents lipopolysaccharide-induced inflammation by inhibiting MAPK and NF-κB pathways in human retinal pigment epithelium.
abstract: 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 h before stimulated with 5 μg/mL LPS for 24 h. The mRNA expression of inflammatory cytokines, AT1R, and AT2R 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 AT1R, whereas increased Ang-(1-7). Furthermore, DIZE downregulated the phosphorylation of p38MAPK, 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), AT1R, phosphorylation of MAPKs and activation of NF-κB.
  
  
  CONCLUSIONS
  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 p38MAPK, ERK1/2, JNK, and NF-κB pathways.
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. 1057287006@qq.com.
authors: Lifei
authors: L
authors: Tao
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. vivianqiu1988@126.com.
authors: Yiguo
authors: Y
authors: Qiu
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. 399925026@qq.com.
authors: Xinyu
authors: X
authors: Fu
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. 83243034@qq.com.
authors: Ru
authors: R
authors: Lin
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. 710131676@qq.com.
authors: Chunyan
authors: C
authors: Lei
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. 38584586@qq.com.
authors: Jiaming
authors: J
authors: Wang
authors: Department of Ophthalmology, The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, 1 You Yi Road, Yu Zhong District, Chongqing, 400016, China. bolei99@126.com.
authors: Bo
authors: B
authors: Lei
mesh_terms: D000085962:Angiotensin-Converting Enzyme 2; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D004133:Diminazene / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D020536:Enzyme Activators / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D055213:Retinal Pigment Epithelium / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics*; D013997:Time Factors
chemical_list: D016207:Cytokines; D020536:Enzyme Activators; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020929:Mitogen-Activated Protein Kinase Kinases; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2; D004133:Diminazene