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The Role of JNK/C-Jun Signal Pathway in Regulating DDAH/ADMAN/NO System after the Administration of ANGⅡ

Author: ZengWenZuo
Tutor: YangTianLun; XiaZuo
School: Central South University
Course: Clinical
Keywords: Angiotesin Ⅱ HUVECS JNK/c-Jun ADMA DDAH NO
CLC: R363
Type: PhD thesis
Year: 2012
Downloads: 143
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Abstract


Objective:The purpose of this study was to discuss the change of JNK/c-Jun pathway after the administration of ANG Ⅱ in cultured human umbilical vein endothelial cells(HUVECs), and its role in regulating SREBP-1and DDAH/ADMA/NO system. This study is expected to explore a new viewpoint for the mechanism of ANG II in regulating plasma NO level and endothelial function.Method:HUVECs was purchased from the Cell Culture Center of the Xiangya Medical School(Changsha, China) and cultured in the appropriate environment.Then HUVECS were induced by incubation with different concentrations of ANG Ⅱ. The expression of p-JNK and p-c-Jun was analyzed by western blot, to see which concentration of ANG Ⅱ can lead to greatest change in p-c-Jun and p-JNK expression, and this concentration was marked as the optimal concentration. Then HUVECs was incubated with ANG II containing medium, of the optimal ANG Ⅱ concentration, for different durations, The expression of p-JNK and p-c-Jun was analyzed by western blot to see which duration could result in p-c-Jun and p-JNK expression, and this duration was marked as the optimal duration HUVECs were treated with ANG Ⅱ containing medium, of the optimal concentration and for the optimal duration, then the expression of p-JNK, p-c-Jun, SREBP-1and DDAH-1was analyzed by western blot. ADMA content and DDAH activity in the supernatant was analyzed by high-performance liguid chromatography (HPLC).NO level in the supernatant was indirectly determined by the nitrate and nitrite contents, using Griess reagent. Then HUVECs was exposed to JNK inhibitor SP600125, while incubated with ANG Ⅱ. The expression of p-JNK, p-c-Jun, SREBP-1and DDAH-1was analyzed by western blot. ADMA content and DDAH activity in the supernatant was analyzed by high-performance liguid chromatography (HPLC).NO level in the supernatant was indirectly determined by the nitrate and nitrite contents, using Griess reagent.Results:Western blot showed that after incubation with different concentration of ANG Ⅱ for varied durations, the expression of p-JNK and p-c-Jun has been significantly elevated in HUVECS compared to the control group. After the blockade of JNK pathway using SP600125,western blot showed that there was no significant change in the expression of SREBP-1compared to the SP600125untreated group. However, the expression of DDAH-1was significantly decreased compared to the SP600125untreated group. After the administration of the JNK inhibitor SP600125, the ADMA level in the supernatant was significantly decreased compared to the SP600125untreated group, while DDAH activity and NO content in the supernatant were significantly elevatedConclusions:1.ANG II can lead to the activation of JNK/c-Jun signal pathway in HUVECS.2. JNK/c-Jun signal pathway does not regulate the expression of SREBP-1.3. JNK/c-Jun signal pathway paticipates in the regulation of DDAH-1in HUVECS after the administration of ANG Ⅱ.4. JNK/c-Jun signal pathway is involved in regulation of total DDAH activity, ADMA content, NO level in HUVECS by ANG Ⅱ.

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