PC12细胞铝染毒后代谢型谷氨酸受体1在PKC和NMDAR表达中的调控作用

王翡; 张婷; 李立荣; 薛星莉; 牛侨

doi:10.13213/j.cnki.jeom.2018.18431

PC12细胞铝染毒后代谢型谷氨酸受体1在PKC和NMDAR表达中的调控作用

Regulation of mGluR1 on expression of PKC and NMDAR in PC12 exposed to aluminum maltolate

摘要

摘要:
目的探讨激动和抑制代谢型谷氨酸受体1（mGluR1）蛋白表达对麦芽酚铝染毒的大鼠肾上腺髓质嗜铬瘤分化细胞株（PC12）蛋白激酶C（PKC）及N-甲基-D-天冬氨酸受体（NMDAR）表达的影响。

方法取对数生长期PC12细胞，分为空白对照组（正常PC12细胞）、激动剂组（100 μmol/L mGluR1激动剂）、抑制剂组（100 μmol/L mGluR1抑制剂）、麦芽酚铝染毒组（200 μmol/L麦芽酚铝）、染铝+激动剂组（100 μmol/L mGluR1激动剂+200μmol/L麦芽酚铝）、染铝+抑制剂组（100 μmol/L mGluR1抑制剂+200 μmol/L麦芽酚铝），染毒时间为24 h。采用实时荧光定量PCR法测定各组PC12细胞中PKC及NMDAR亚基的mRNA表达水平；采用Western blot法测定各组PC12细胞中PKC及NMDAR亚基的蛋白表达水平；采用ELISA法测定各组PC12细胞的PKC酶活性。

结果与空白对照组相比，麦芽酚铝染毒组的PKC、NMDAR1、NMDAR2A和NMDAR2B mRNA表达水平分别下降39%、21%、38%和26%，差异有统计学意义（P < 0.05），其蛋白表达水平分别下降39%、31%、41%和46%，差异有统计学意义（P < 0.05）。染铝+抑制剂组的NMDAR1 mRNA和蛋白表达与麦芽酚铝染毒组相比分别上调32%和36%（P < 0.05）；与麦芽酚铝染毒组相比，染铝+抑制剂组NMDAR2B mRNA表达下调46%，染铝+激动剂组NMDAR2B蛋白表达上调95%，差异有统计学意义（P < 0.05）。与对照组相比，麦芽酚铝染毒组PKC酶活性下降56%，而染铝+激动剂组PKC酶活性相对于麦芽酚铝染毒组上调116%（P < 0.05）。

结论麦芽酚铝可以抑制PC12细胞的PKC和NMDAR各亚基的mRNA和蛋白表达；麦芽酚铝可通过mGluR1调节NMDAR表达和PKC酶活性；mGluR1对NMDAR的调节主要作用于NMDAR1和NMDAR2B。

Abstract:
Objective To investigate the effects of agitating and inhibiting mGluR1 on protein kinase C (PKC) and N-methyl-D-aspartate receptor (NMDAR) expressions in rat adrenal-derived pheochromocytoma cells (PC12) induced by aluminum maltolate.

Methods PC12 cells at logarithmic growth phase were divided into a control group (normal PC12), an agonist group (100μmol/L mGluR1 agonist), an inhibitor group (100 μmol/L mGluR1 inhibitor), an aluminum maltolate exposure group (200 μmol/L aluminum maltolate), an aluminum maltolate + agonist group (200 μmol/L aluminum maltolate + 100 μmol/L mGluR1 agonist), and an aluminum maltolate + inhibitor group (200 μmol/L aluminum maltolate + 100 μmol/L mGluR1 inhibitor), and all groups were treated for 24 h. The mRNA expression levels of PKC and subunits of NMDAR in PC12 were measured by real-time fluorescence quantitative PCR, the protein expression levels of PKC and subunits of NMDAR by Western blot, and the PKC enzyme activity of PC12 by ELISA.

Results Compared with the control group, the mRNA expression levels of PKC, NMDAR1, NMDAR2A, and NMDAR2B of the aluminum maltolate exposure group were decreased by 39%, 21%, 38%, and 26%, respectively (P < 0.05), and the protein expression levels decreased by 39%, 31%, 41%, and 46%, respectively (P < 0.05). The NMDAR1 mRNA and protein expression levels of the aluminum maltolate + inhibitor group were increased by 32% and 36%, respectively, compared with the aluminum maltolate exposure group (P < 0.05). The NMDAR2B mRNA expression levels of the aluminum maltolate + inhibitor group was decreased by 46%, and the NMDAR2B protein expression levels of the aluminum maltolate + agonist group was increased by 95%, compared with the aluminum maltolate exposure group (P < 0.05). Compared with the control group, the PKC enzyme activity of the aluminum maltolate exposure group decreased by 56%, while the PKC activity of the aluminum maltolate+agonist group was upregulated by 116% compared with the maltolate exposure group (P < 0.05).

Conclusion The PKC and subunits of NMDAR mRNA and protein expressions in PC12 cells are inhibited by aluminum maltolate. Aluminum maltolate may regulate the NMDAR expression and the PCK enzyme activity via mGluR1. NMDAR1 and NMDAR2B play roles in the proposed regulation.

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