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  • AQP protein expression was determined by Western

    2021-02-22

    AQP4 protein expression was determined by Western blot analysis. Briefly, protein samples (50μg) from brains or astrocytes were separated by 12% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were reacted with a rabbit polyclonal antibody against AQP4 (1:500) and a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Kangchen Biotechnology Inc., Shanghai, China) at 4°C overnight. Following repeated washes, the membranes were sequentially reacted with a horseradish peroxidase-conjugated goat–anti-rabbit antibody (1:200; Zhongshan, Shanghai, China), at room temperature for 2h, and the ECL reagents; finally exposed on an X-ray film to show the AQP4 band (34.8kDa). After being thoroughly washed, the membranes were then reacted with another secondary antibody, a horseradish peroxidase-conjugated goat–anti-mouse IgG (1:2000; Chemicon), to show the GAPDH band (36kDa). The optical densities were quantitatively analyzed with a laser densitometer (UltroScan XL, Pharmacia LKB Co., Sweden). The results of AQP4 expression are reported as the percentage changes over GAPDH. Reverse transcription-polymerase chain reaction (RT-PCR). To determine the mRNA expressions of CysLT1 and Amodiaquine dihydrochloride dihydrate CysLT2 receptors, total RNA was extracted from astrocytes or a rat Amodiaquine dihydrochloride dihydrate using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA synthesis and PCRs were performed as reported [30]. The primer sequences were as the following: rat CysLT1 receptor forward 5′-(+) TCT CCG TTG TGG GTT TCT-3′ and reverse 5′-(+) TAT AAG GCA TAG GTG GTG-3′ (product size 214bp); rat CysLT2 receptor forward 5′-(+) AGC GTT AGG AGT GCC TGG AT-3′ and reverse 5′-(+) CAA GTG GAT GGT CCG AAG TG-3′ (product size 520bp); β-actin forward 5′-(+) TAC AAC CTC CTT GCA GCT CC-3′ and reverse 5′-(+) GGA TCT TCA TGA GGT AGT CAG TC-3′ (product size 620bp), or forward 5′-(+) AAC CCT AAG GCC AAC CGT GAA-3′, and reverse 5′-(+) TCA TGA GGT AGT CTG TCA GGT C-3′ (product size 285bp). Statistical analysis. Data are reported as means±SD. Statistical analyses were performed using one-way ANOVA with Newman–Keuls Post Hoc Multiple Comparison (SPSS 10.0 for Windows, 1999, SPSS Inc., USA). A value of P<0.05 was considered statistically significant.
    Results
    Discussion In the present study, we found that LTD4 induces brain edema, which is associated with BBB disruption and the up-regulation of AQP4 expression, and that CysLT2, rather than CysLT1, receptor may be involved in the up-regulation of AQP4 expression. Our findings provide direct evidence for the CysLTs-induced brain edema as previously reported [10], [11], [12], [13], and further reveal that this type of brain edema may be partly modulated via AQP4. LTD4-induced brain edema seems to include vasogenic and cytotoxic edemas. Vasogenic edema occurs when BBB is disrupted, which permits plasma fluid into the brain extravascular space; while cytotoxic edema occurs when water flows from the vascular compartment through intact BBB and astrocytic foot processes, and accumulates primarily in astrocytes [16]. In the present study, LTD4-induced BBB disruption is evidenced by endogenous IgG exudation in mouse brain, which is attenuated by the selective CysLT1 receptor antagonist pranlukast (Fig. 1A and B). Thus, we propose that LTD4 might induce vasogenic edema via CysLT1 receptor-mediated BBB disruption. This action is supported by the localization of CysLT1 receptor in brain microvascular endothelial cells as reported in human and rat brain [31], [32]. However, pranlukast did not inhibit LTD4-induced increase in brain water content (Fig. 1C) and AQP4 expression (Fig. 2), indicating that it does not inhibit AQP4-related brain edema. AQP4 promotes cytotoxic edema and attenuates vasogenic edema [16], [18], so that LTD4 might induce cytotoxic edema by enhancing AQP4 expression (Fig. 2). This response may not be mediated via CysLT1 receptor.