NLRP12 promotes host resistance against Pseudomonas aeruginosa keratitis inflammatory responses through the negative regulation of NF-κB signaling.

OBJECTIVE: To investigate the role of NLRP12 in regulating Pseudomonas aeruginosa (P. aeruginosa) keratitis. MATERIALS AND METHODS: Real-Time-PCR and Western blot were performed to measure the NLRP12 level in corneas and bone marrow-derived macrophages (BMDMs) of C57BL/6 (B6) mice. B6 mice received a subconjunctival injection of lentivirus expressing active NLRP12 (NLRP12-lentivirus) or Ctl-lentivirus (as control), followed by infection of P. aeruginosa. The clinical score, slit lamp and bacterial plate count of mice were evaluated. In addition, myeloperoxidase (MPO) was detected to assess the infiltration of polymorphonuclear neutrophil (PMN). Cytokine levels were measured by Real Time-PCR and ELISA. Meanwhile, the bacterial burden was also evaluated. The activation of NF-κB signaling was determined by pIκBα/IκBα levels based on Western blot and NF-κB-dependent Luciferase activity on the basis of Luciferase assays using 293T cells. RESULTS: NLRP12 mRNA and protein levels were decreased in B6 corneas and BMDMs after P. aeruginosa infection. The over-expression of NLRP12 in B6 corneas significantly ameliorated the severity of corneal disease, bacterial burden, PMN infiltration and pro-inflammatory cytokine expression. In vitro analysis demonstrated that the up-regulation of NLRP12 suppressed pro-inflammatory cytokine production and enhanced bacterial clearance in RAW264.7 cells. The protein levels of pIκBα and IκBα were significantly decreased after NLRP12-lentivirus treatment compared with that of Ctl-lentivirus. NF-κB-dependent Luciferase activity was potently inhibited by NLRP12 infected with P. aeruginosa or cotransfected with the downstream signaling molecules including IKKα and IKKß in 293T cells. CONCLUSIONS: NLRP12 decreases the severity of P. aeruginosa keratitis, reduces corneal inflammation and bacterial burden through the down-regulation of the NF-κB signaling pathway.

Protective effects of melatonin on ischemia-reperfusion induced myocardial damage and hemodynamic recovery in rats.

AIM: To investigate the mechanism of melatonin (MT) protection of adult rate myocardial ischemia-reperfusion injury and its influence on rat's hemodynamic recovery. MATERIALS AND METHODS: 48 rats were randomly divided into MT group (n=36) and the control group (n=12), MT group was divided into three sub-groups according to different dosages: Group I (n=12) was administered with 2.5 mg/kg MT; Group II (n=12) was administered with 5 mg/kg MT; Group III (n=12) was administered with 10 mg/kg MT. The electrocardiogram of four groups was observed with the left coronary artery blocked for 10min at first and then reperfused for 15min. Hemodynamic evolving was observed and changes in energy metabolism of rat myocardium were monitored. TUNEL and immunohistochemistry were applied to detect the cell apoptosis index, protein expression of Bcl-2 and Bax. RESULTS: LVDP (left ventricular developed pressure) and ± dp/dt in MT group presented better recovery at various time points than the control group. Among them, Group III had the optimal recovery degree (p < 0.05). After MT administration, ATP content in myocardial cells in MT group was significantly higher than the control group. Compared with the control group, the concentration of mitochondrial MDA and Ca2+ in myocardial cells in MT group showed a downward trend. But its GSH concentration was significantly higher than the control group (p < 0.05). The improvement degree of ATP, MDA, GSH and Ca2+ concentration in Group II over-performed Group I (p < 0.05). MT-intervened myocardial apoptosis index (AI) and Bax positive expression index declined while Bcl-2 positive expression index increased (p < 0.01). CONCLUSIONS: MT effectively inhibited myocardial apoptosis during the myocardial ischemia-reperfusion of rats, protected the structural integrity of mitochondria in myocardial cells, promoted ATP synthesis, and avoided heart damage in many ways. This protection mechanism was related with anti-oxidative damage. Meanwhile, MT could promote the hemodynamic recovery after myocardial ischemia-reperfusion in rats.