Introduction

Stroke is the third leading cause of death and the most frequent cause of permanent disability worldwide1, and inflammation appears to be critical in the pathogenesis of ischemic stroke and other forms of ischemic brain injuries. The inflammatory response has a detrimental role in cerebral ischemia/reperfusion (I/R) injury pathogenesis2. The association between inflammation and cerebral I/R outcomes has ensured considerable and continued interest in the development of antiinflammation-oriented therapies for mitigating I/R-induced brain damage. In the brain, microglia and monocyte-derived macrophages are the key players in the immune response after stroke47. The platelet suspensions (3.6 × 108 cells/ml) were treated with 1 μg/ml collagen and 2 μM platonin or PBS for 3 min in a separate vial. The suspensions were incubated for 5 min and 100 μM DMPO was added before the ESR analysis was conducted. The ESR spectrometer was operated at a 20-mW power, 9.78-GHz frequency, 100-G scan range, and receiver gain of 5 × 104. Moreover, a Fenton reaction solution (50 μM FeSO4 and 2 mM H2O2) was pretreated with a solvent control (PBS) or platonin (5 and 10 μM) and vitamin C (100 μM) for 3 min.

Cell culture and platonin treatment

The BV2 and RAW 264.7 cell lines were maintained in DMEM supplemented with 10% heat-inactivated FBS, penicillin G (100 units/ml), streptomycin (100 mg/ml), and L-glutamine (2 mM) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. For platonin treatment, platonin was dissolved in PBS. The cells were pretreated with 5 or 10 μM platonin for 30 min and then stimulated with or without LPS (1 μg/ml) for the indicated time.

Measurement of NO concentration

The nitrite concentration was determined using the Griess reagent (1% sulfanilamide and 0.1% naphthalenediamine in 2.5% phosphoric acid) as an indicator of NO production48. In brief, cells were plated at 8 × 105 cells/well into a 6-cm dish in DMEM with 10% FBS. The cells were pretreated with platonin (5–10 μM) for 30 min and then stimulated with or without LPS (1 μg/ml) for 24 h. After incubation, 150 μl of the culture supernatants was mixed with 150 μl of the Griess reagent. The absorbance of the mixtures was measured at 550 nm on a microplate reader.

Western blotting

To determine the level of COX-2, IL-1β, iNOS, and JNK phosphorylation in the whole cell lysate, protein samples were separated through 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane by using a Bio-Rad semidry transfer unit (Hercules, CA, USA). The membranes were then blocked with TBST (10 mM Tris-base, 100 mM NaCl, and 0.01% Tween 20) containing 5% BSA for 1 h at room temperature and probed with the various specific primary antibodies. After several washes, the membranes were incubated with the HRP-linked secondary antibody (1:3000 in TBST) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence system. The ratios of the semiquantitative results were obtained by scanning the reactive bands and quantifying the optical densities on a videodensitometer and Bio-profil Biolight software (version V2000.01; Vilber Lourmat, Marne-la-Vallée, France).

Statistical analysis

Data are expressed as the means ± SEM, accompanied by the number of observations. The normality of the data was first tested using the Kolmogorov–Smirnov test. The continuous variables were compared using analysis of variance. When the analysis indicated significant differences among group means, each group was compared using the Newman–Keuls method. P < 0.05 was considered statistically significant.

Additional Information

How to cite this article: Sheu, J.-R. et al. A novel indication of platonin, a therapeutic immunomodulating medicine, on neuroprotection against ischemic stroke in mice. Sci. Rep. 7, 42277; doi: 10.1038/srep42277 (2017).

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