The mechanism of growth enhancement of P2X₇ receptor on primary cultured rat cortical astrocyte

Purpose The purpose of the present study was to investigate whether ATP could promote reactive astrogliosis and the role of purine'receptor P2X ₇ in the process. Methods Separation and culture of primary astrocytes (As) from cortices of Sparague-Dawley rats were conducted,and the purity was checked by immuno- fluorescence. 5-BrdU. incorporation and flow cytometer were used to detect the proliferation of As after treatment with ATP of different concentrations. Real-time RT-PCR was used to measure the changes of GFAP mRNA, P2X₇mRNA, TGF-β1mRNA expression after ATP treatment. The expression of GFAP protein was analyzed by Western blot. ELISA was used to detect the amount of TGF-β1 protein in the supernatant. Results Separation and culture of primary As were successful, the purity of As reached 99%. The proportion of As at s-phase was significantly enhanced after 100 μmol/L ATP treatment. 5-BrdU incorporation showed enhanced positive mitosis ratio after 100 μmol/L ATP treatment. These results indicated that ATP at concentration of 100 μmol/L might induce the proliferation and hypertrophy of As in vitro. ATP at concentration of 500 μmol/L could promote the expression GFAP mRNA and P2X₇ mRNA. The content of GFAP was elevated in 2 days with 2′-3′-O-(4-benzoylbenzoyl)-adenosine-5′ -triphosphate(BzATP), a selective agonist of P2X₇ receptor, at concentration of 50 μmol/L, 75 μmol/L and 100 μmol/L respectively in the culture media with Ca²⁺. After cultured with Ca²⁺ and BzATP for 2 hours, TGF-β1 mRNA reached the first peak, and then reached the second peak at 12 h. TGF-β1 protein in the supernatant reached the first peak at 8 h, and another peak at 48 h. TGF-β1 neutralizing antibody with concentration of 6 μg/mL can partly inhibit GFAP expression induced by P2X₇ receptor. Conclusions ATP promotes proliferation and hypertrophy of primary cultured of As,mimicking the change as reactive astrogliosis in vivo. P2X₇ activated by BzATP may also induced the same morphological As. BzATP combined with Ca²⁺ plays an important role in enhancing the transcription of TGF-β1 mRNA and release of TGF-β1 protein into the supernatant.