A highly sensitive fluorescent probe RN-NA reveals peroxynitrite as a novel biomarker for primary open angle glaucoma.

AIM: To directly quantify peroxynitrite (ONOO-) using a highly sensitive fluorescence resonance energy transfer probe RN-NA, investigate the association between ONOO- and primary open angle glaucoma (POAG), and clarify whether RN-NA could be used as a potential tool for POAG diagnosis. METHODS: Plasma and aqueous humor (AH) samples were collected from POAG patients (n=100, age: 59.70±6.87y) and age-related cataract (ARC) patients (n=100, age: 61.15±4.60y) admitted to our hospital. Next, RN-NA was used to detect ONOO- in plasma and AH samples, and the relationship between ONOO- level and POAG was analyzed using binary logistic regression. Besides, Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO- with the patients' age, intraocular pressure (IOP), and mean deviation of visual field testing. The ONOO- scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine (SIN-1)-induced ocular hypertension in mice (n=7, 6-8wk). Finally, the IOP and ONOO- in both eyes were measured 30min after the last drug treatment. RESULTS: ONOO- levels of AH and plasma were significantly higher in the POAG group than in the ARC group (P<0.01). Additionally, ONOO- levels were closely correlated with POAG in a binary logistic regression analysis [odds ratio (OR)=1.008, 95% confidence interval (CI): 1.002-1.013, P<0.01 for AH; OR=1.004, 95%CI: 1.002-1.006, P<0.001 for plasma]. Pearson correlation analysis showed that ONOO- levels in AH or plasma were positively associated with visual field defects (R=0.51, P<0.01 for AH; R=0.45, P<0.001 for plasma), and ONOO- levels in plasma and AH were correlated in the POAG group (R=0.69, P<0.001). However, administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1 (P<0.05). CONCLUSION: ONOO- levels in AH and plasma, detected by RN-NA, are significantly related to POAG and positively correlated with visual field defects in POAG patients. Hence, ONOO- is a potential biomarker of POAG, especially advanced POAG. Besides, anti-nitration compounds may be novel ocular hypotensive agents based on the animal study.

A bifunctional sensitive fluorescence probe based on pyrene for the detection of pH and viscosity in lysosome.

Lysosome is one of the important organelles in intracellular transport. It plays a significant role in the physiological process. The lysosomal microenvironment affects the functions of lysosome. When the original acidic environment of lysozyme is destroyed or the fluid viscosity increases gradually, various diseases are easily induced. However, most fluorescent probes can only locate in cells. The fewer probes of subcellular organelles were found and their functions are often single. So, it is of great importance to design multifunctional fluorescent probes with the capable of localizing in lysosome. In this study, a novel lysosome probe, 4-(4-Pyren-1-yl-but-3-enyl)-morpholine (PIM), was synthesized using pyrene as a fluorescent group and morpholine as a target group. The introduction of morpholine group made PIM localize in lysosome with high selectivity. The fluorescence will be enhanced with the increased viscosity because of restricting the rotation of CC bond and CN in PIM, and the detecting linear range is from 4.05 cP to 393.48 cP, which qualified the requirement of the viscosity monitoring in body. Meanwhile, the fluorescence intensity of PIM declines with the decrease of pH because the Schiff base of PIM is hydrolyzed, which was affirmed by 1H NMR, LC-MS and fluorescence spectra. Moreover, cell imaging and MTT experiments confirmed that PIM as a novel bifunctional probe can be used to detect pH and endogenous viscosity in lysosome.

Continuous Infusion of Angiotensin IV Protects against Acute Myocardial Infarction via the Inhibition of Inflammation and Autophagy.

Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. Angiotensin (Ang) IV possesses many biological properties that are not yet completely understood. Therefore, we investigated the function and mechanism of Ang IV in AMI in in vivo and in vitro conditions. AMI was performed by ligation of the left anterior descending coronary artery (LAD) in male C57 mice. Ang IV was continuously infused by a minipump 3 d before AMI for 33 d. The neonatal rat ventricular myocytes (NRVCs) were stimulated with Ang IV and cultured under hypoxic conditions. In vivo, Ang IV infusion significantly reduced the mortality after AMI. By the 7th day after AMI, compared with the AMI group, Ang IV reduced the inflammatory cytokine expression. Moreover, terminal deoxyribonucleotidyl transferase- (TDT-) mediated dUTP nick-end labeling (TUNEL) assay showed that Ang IV infusion reduced AMI-induced cardiomyocyte apoptosis. Compared with AMI, Ang IV reduced autophagosomes in cardiomyocytes and improved mitochondrial swelling and disarrangement, as assessed by transmission electron microscopy. By 30th day after AMI, Ang IV significantly reduced the ratio of heart weight to body weight. Echocardiography showed that Ang IV improved impaired cardiac function. Hematoxylin and eosin (H&E) and Masson staining showed that Ang IV infusion reduced the infarction size and myocardial fibrosis. In vitro, dihydroethidium (DHE) staining and comet assay showed that, compared with the hypoxia group, Ang IV reduced oxidative stress and DNA damage. Enzyme-linked immunosorbent assay (ELISA) showed that Ang IV reduced hypoxia-induced secretion of the tumor necrosis factor- (TNF-) ɑ and interleukin- (IL-) 1ß. In addition, compared with the hypoxia group, Ang IV reduced the transformation of light chain 3- (LC3-) I to LC3-II but increased p62 expression and decreased cardiomyocyte apoptosis. Overall, the present study showed that Ang IV reduced the inflammatory response, autophagy, and fibrosis after AMI, leading to reduced infarction size and improved cardiac function. Therefore, administration of Ang IV may be a feasible strategy for the treatment of AMI.

Xanthan gum protects temporomandibular chondrocytes from IL­1ß through Pin1/NF­κB signaling pathway.

Temporomandibular disorder (TMD) is a complicated and multi­factorial disease related to inflammation and cartilage destruction. Intra­articular injection of xanthan gum (XG) has been demonstrated to protect the joint cartilage and reduce osteoarthritis progression. However, the role and mechanism of XG in TMD is still unclear. In the present study, chondrocytes were isolated from rats and identified by immunofluorescence. Cells were stimulated by XG or interleukin (IL)­1ß. Cell viability was analyzed by MTT assay. Tumor necrosis factor α (TNF­α) and IL­6 levels were determined by ELISA. The expression of monocyte chemoattractive protein­1 (MCP­1), inducible nitric oxide synthase (iNOS), collagens, matrix metalloproteinases (MMPs), peptidyl­prolyl isomerase 1 (Pin1) and phosphorylated nuclear factor κB (NF­κB) p65 (p­p65) was analyzed by quantitative PCR or western blotting. MMP activity was assessed by gelatin zymography. Compared with the control, XG treatment partially reversed the IL­1ß­reduced cell viability. In addition, IL­1ß stimulation increased inflammatory cytokine expression, including TNF­α, IL­6 secretion, MCP­1 and iNOS expression, whereas XG treatment reduced the expression of these inflammatory cytokines compared with that of the IL­1ß­stimulated cells. Additionally, XG increased the expression of collagen, but reduced MMP expression and activity as compared with that in the IL­1ß group. In addition, XG treatment prevented the IL­1ß­increased Pin1 and p­p65 expression. These data suggested that XG reduced the expression of inflammatory cytokines and may maintain the balance between collagens and MMPs partially through the Pin1/NF­κB signaling pathway in IL­1ß­stimulated temporomandibular chondrocytes. Therefore, XG may be useful in the treatment of TMD.

Remifentanil Protects against Lipopolysaccharide-Induced Inflammation through PARP-1/NF-κB Signaling Pathway.

Sepsis is a leading cause of death in patients with severe infection worldwide. Remifentanil is an ultra-short-acting, potent opioid analgesic. In the study, we aimed to investigate the role and underlying mechanism of remifentanil in lipopolysaccharide- (LPS-) induced inflammation in human aortic endothelial cells (HAECs). HAECs were pretreated with phosphate-buffered saline (PBS) or remifentanil (2.5 µM) for 30 min, then stimulated by LPS (10 µg/ml) for another 24 h. Poly(ADP-ribose) polymerase 1 (PARP-1) was inhibited by small interfering RNA (siRNA). Superoxide anion production and DNA damage were analyzed by dihydroethidium (DHE) staining and comet assay. The inducible nitric oxide synthase (iNOS), intercellular adhesion molecule 1 (ICAM-1), PARP-1, poly(ADP-ribose) (PAR), and nuclear factor-kappa B p65 (NF-κB p65) expressions were analyzed by RT-PCR or western blotting analysis. NF-κB p65 nuclear translocation was assessed by immunofluorescence. Compared with the control group, pretreatment with remifentanil significantly reduced superoxide anion production and DNA damage, with downregulation of iNOS, ICAM-1, and PARP-1 expressions as well as PAR expression. Moreover, pretreatment with PARP-1 siRNA or remifentanil inhibited LPS-induced NF-κB p65 expression and nuclear translocation. Remifentanil reduced LPS-induced inflammatory response through PARP-1/NF-κB signaling pathway. Remifentanil might be an optimal choice of analgesia in septic patients.