羟基红花黄色素A对谷氨酸诱导损伤神经元过氧化物酶体增殖物激活受体γ表达的影响

The effects of hydroxysafflor yellow A on PPARγ expression in glutamate-induced neuron damage

目的探讨羟基红花黄色素A(HSYA)拮抗谷氨酸诱导神经元损伤的保护机制。方法采集经体外原代培养第7天的Sprague Dawley胎鼠皮质神经元,通过形态学观察、噻唑蓝法、Westernblotting法分别检测过氧化物酶体增殖物激活受体γ(PPARγ)和磷酸化PPARγ(pPPARγ,即PPARγ的失活形式)表达水平,以探讨HSYA抗谷氨酸诱导神经元损伤的保护机制。结果经谷氨酸处理后神经元发生肿胀、部分崩解、死亡。与正常对照组相比,谷氨酸不同剂量组(1.00、5.00和10.00mmol/L组)的神经元存活率(66.60%、33.40%和21.60%)和PPARγ蛋白表达水平显著降低,对照组灰度值:1.06±0.18,谷氨酸5.00和10.00mmol/L组灰度值分别为:0.32±0.09、0.28±0.07(均P=0.000),pPPARγ蛋白表达水平明显升高,灰度值分别为:0.37±0.05、1.83±0.17和1.75±0.21(均P=0.000)。HSYA与谷氨酸共同处理后神经元形态明显改善、细胞相对存活率提高,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组分别为33.40%、35.30%、51.00%和72.50%(P=0.742、0.033、0.002),PPARγ蛋白表达水平呈现逐渐升高趋势,对照组,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组灰度值分别为:1.20±0.16、0.25±0.06、0.39±0.10、0.41±0.12、0.37±0.08,但差异未达到统计学意义(均P>0.05),pPPARγ蛋白表达水平降低,对照组,谷氨酸5.00mmol/L,HSYA0.01、0.10和1.00mmol/L组灰度值分别为:0.51±0.14、1.91±0.25、1.70±0.26、1.25±0.23、0.85±0.19,(P=0.022、0.004、0.000)。结论 HSYA对由谷氨酸诱导的神经元损伤具有保护作用,其机制与抑制PPARγ蛋白磷酸化即抑制PPARγ活性有关。

Objective To investigate the effect of hydroxysafffor yellow A on PPARγ expression in glutamate-induced neuron damage. Methods Primary cultured cortical neurons of 7 d embryonic Sprague Dawley rat was prepared. Morphological observation and MTT method were used to detect cell damage; western blotting was chosen to examine PPARγ and p-PPARγ expression （inactivated form of PPARγ） in total protein, in order to explore the protective effect of HSYA on glutamateinduced neuron damage. Results After treated with glutamate neurons were swelling, disintegrated or dead in morphological observation. Compared with the control group, cell survival rate in the groups treated with different dosage of glutamate （1.00, 5.00 and 10.00 mmol/L） was all decreased （66.60%, 33.40% and 21.60%; P = 0.004, 0.000, and 0.000, respectively）. In comparison with the control group, the expression of PPARγ in total protein decreased, the gray value in control group was 1.06± 0.18, while in glutamate 5.00 mmol/L and 10.00 mmol/L groups was 0.32 ± 0.09 and 0.28 ± 0.07, respectively （P = 0.000, for all）. The expression of p-PPAR3＇ increased, the gray value was 0.37 ± 0.05 in control group, and 1.83 ± 0.17 and 1.75 ±0.21 in glutamate 5.00 mmol/L and 10.00 mmol/L group （P = 0.000, for all）. HSYA improved the morphology and cell survival rate of glutamate-induced neuron damage. The cell survival rate in glutamate 5.00 mmol/L group and that treated with HSYA 0.01 mmol/L, 0.10 mmol/L and 1.00 mmol/L was 33.40%, 35.30%, 51.00% and 72.50%, respectively （P = 0.742, 0.033, and 0.002, respectively）; HSYA slightly increased PPAR3＇ expression but without statistical significance, the gray value was 1.20 ± 0.16 in control group, and was 0.25 ± 0.06, 0.39 ± 0.10, 0.41 ± 0.12 and 0.37 ± 0.08 in glutamate 5.00 mmol/L group, and glutamate 5.00 mmol/L group treated with HSYA 0.01 mmol/L, 0.10 mmol/L or 1.00 mmol/L （P = 0.094, 0.145 and 0.576, respectively）; HSYA obviously reduced p-PPARγ expression, the gray value in control group was 0.51 ± 0.14, in glutamate 5.00 mmol/L group and was 1.91 ± 0.25, 1.70 ± 0.26, 1.25 ± 0.23 and 0.85 ± 0.19 in glutamate 5.00 mmol/L group treated with HSYA 0.01 mmol/L, 0.10 mmol/L or 1.00 mmol/L （P = 0.022, 0.004 and 0.000, respectively）. Conclusion HSYA can protect the glutamate-induced neuron damage, and the mechanism involves the inhibition of phosphorylation of PPAR%