Establishment of autophagy model of rat gastric interstitial cells of Cajal induced by glutamic acid |
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| Authors: | TAN Ren-qian ZHANG Zhi NING Hai-en ZHANG Li-min WANG Yuan LING Jiang-hong |
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| Institution: | 1. The First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China;
2. Shanghai Pudong New District Zhoupu Hospital, Shanghai 200120, China |
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| Abstract: | AIM: To establish an autophagy model of glutamic acid-induced rat gastric interstitial cells of Cajal (ICCs) in vitro. METHODS: Glutamic acid was added to the medium of cultured ICCs in vitro at different concentrations (high, medium and low concentrations were 10 mmol/L, 5 mmol/L and 2.5 mmol/L, respectively) for different periods (3 h, 6 h and 24 h). The cell viability was measured by CCK-8 assay. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) in the ICCs after cultured with glutamic acid at different concentrations for different periods was determined by Western blot. The ultrastructure and autophagic vacuoles of ICCs were observed under electron microscope, and immunofluorescence technique was used to measure the fluorescence intensity of autophagic protein LC3 after co-cultured with glutamic acid at medium concentration. RESULTS: Compared with control group, glutamic acid significantly inhibited the viability of ICCs (P<0.01). The ratios of LC3-Ⅱ/LC3-I in medium and high concentration groups at 3 h, 6 h and 24 h were significantly increased as compared with control group and low concentration group (P<0.01), and that in medium concentration group at 3 h was the best to increased the ratio of LC3-Ⅱ/LC3-I (P<0.01). Under electron microscope, the ICCs in glutamic acid group showed obvious autophagic vacuoles and cytoplasmic vacuoles. The fluorescence intensity of autophagic protein LC3 in the ICCs of glutamic acid group was significantly enhanced (P<0.01). CONCLUSION: Glutamic acid successfully induces autophagy in ICCs. The optimum condition of glutamic acid was 5 mmol/L for 3 h. |
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| Keywords: | Glutamic acid Interstitial cells of Cajal Autophagy Microtubule-associated protein 1 light chain 3 |
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