| 陈子昂,宋宗工,王 昊,苗 壮,蒋彦峰,薛东波.沉默MAPK1对牛磺胆酸钠诱导的胰腺腺泡细胞内胰蛋白酶原的细胞内激活及自噬流的影响[J].,2018,(16):3012-3016 |
| 沉默MAPK1对牛磺胆酸钠诱导的胰腺腺泡细胞内胰蛋白酶原的细胞内激活及自噬流的影响 |
| Effect of Silenced MAPK1 on the Intracellular Activation of Trypsinogen and Autophagic Flux in Taurolithocholic-induced Acute Acinar Cells |
| 投稿时间：2018-01-22 修订日期：2018-02-28 |
| DOI：10.13241/j.cnki.pmb.2018.16.003 |
| 中文关键词: 沉默MAPK1 自噬 急性胰腺炎 |
| 英文关键词: Silenced MAPK1 Autophagy Acute pancreatitis |
| 基金项目:国家自然科学基金项目(81370566) |
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| 中文摘要: |
| 摘要 目的：探讨沉默丝裂素活化蛋白激酶(MAPK1)对牛磺胆酸钠诱导的胰腺腺泡细胞内胰蛋白酶原激活及自噬流的影响。方法：选择体外培养的AR42J细胞分为AR42J细胞+空白对照组，AR42J细胞+牛磺胆酸钠(TLC)(200 μM 的TLC作用40 min)，AR42J细胞+RNA1(MAPK1)。分别转染MAPK1 siRNA以及阴性对照，采用BZiPAR、流式细胞术及激光共聚焦显微镜检测胰蛋白酶原激活，western blot技术检测各组自噬相关蛋白LC3、Beclin1、Cathepsin L，组织蛋白酶L(Cathepsin L，CTSL，也称为CTSL1)和溶酶体膜蛋白(Lysosomal associated membrane protein 2，LAMP2)。结果：TLC处理AR42J细胞后，胰蛋白酶原激活显著增加(阳性细胞相对比：15.12 %±1.46% vs. 7.82 %±1.86 %, P<0.05，平均荧光强度：7.65±0.72 vs. 3.76±0.57, P<0.05)，MAPK1 siRNA转染后TLC处理AR42J细胞后细胞内胰蛋白酶原激活则较TLC组明显降低(阳性细胞相对比：9.25 %±1.16% vs. 15.12 %±1.46 %, P<0.05, 平均荧光强度:4.31±0.27 vs. 7.65±0.72, P<0.05)。TLC组Beclin1与LC3表达显著性高于对照组(Beclin1：2.237±0.097 vs. 1.103±0.057, P<0.05。LC3：1.908±0.039 vs. 0.973±0.081, P<0.05)，TLC＋MAPK1siRNA组Beclin1与LC3的表达显著低于TLC组(Beclin1：1.214±0.049 vs. 2.237±0.097, P<0.05。LC3：1.315±0.037 vs. 1.908±0.039, P<0.05)；而TLC组LAMP2及CTSL1表达较对照组显著下调(LAMP2：0.462±0.025 vs. 1.009±0.039, P<0.05。CTSL1：0.563±0.028 vs. 1.135±0.041, P<0.05)，TLC＋MAPK1siRNA组LAMP2及CTSL1表达显著高于TLC组(LAMP2：1.007±0.019 vs. 0.462±0.025, P<0.05。CTSL1：0.921±0.030 vs. 0.563±0.028, P<0.05)。结论：沉默MAPK1对TLC诱导的AR42J细胞中的胰蛋白酶原激活具有抑制作用，可能通过抑制MAPK1通路抑制自噬发生，同时使LAMP2及CTSL1的表达增强，自噬溶酶体的功能正常，自噬过程顺利进行，从而抑制胰蛋白酶原的激活，减轻急性胰腺炎。 |
| 英文摘要: |
| ABSTRACT Objective: To investigate silenced mitogen-activated protein kinase (MAPK1) has an effect on the intracellular activation of trypsinogen and autophagic flux in taurolithocholic-induced pancreatic acinar cells. Methods: AR42J cells cultured in vitro were divided into three groups: AR42J cells adding blank control group, AR42J cells adding taurolithocholic (TLC) (TLC effect of 200 μM for 40 minute), AR42J cells adding RNA1 (MAPK1). The above groups were transfected with MAPK1 siRNA respectively and negative control. There were many means to detect the activation of trypsinogen by BZiPAR, flow cytometry and laser confocal microscopy. Western blot was used to detect the expression of autophagy-related proteins LC3, Beclin1, Cathepsin L, (CTSL, also known as CTSL1) and Lysosomal associated membrane protein 2 (LAMP2) in each group. Results: After the treatment of AR42J cells with TLC, the activation of trypsin was significantly increased (relative proportion for positive cells: 15.12% ± 1.46 % vs. 7.82% ± 1.86 %, P<0.05, mean fluorescence intensity: 7.65 ± 0.72 vs. 3.76 ± 0.57, P<0.05). After MAPK1 siRNA transfection, the intracellular trypsinogen activation in TLC-treated AR42J cells was significantly lower than that in TLC group (relative proportion for positive cells: 9.25% ± 1.16 % vs. 15.12 % ± 1.46 %, P<0.05, mean fluorescence Intensity: 4.31 ± 0.27 vs. 7.65 ± 0.72, P<0.05). Beclin1 and LC3 expression in TLC group was significantly higher than that in control group (Beclin1: 2.237 ± 0.097 vs. 1.103 ± 0.057, P<0.05. LC3: 1.908 ± 0.039 vs. 0.973 ± 0.081, P<0.05). The expression of Beclin1 and LC3 in TLC adding MAPK1siRNA group was significantly lower than that in TLC group (Beclin1: 1.214 ± 0.049 vs. 2.237 ± 0.097, P<0.05. LC3: 1.315 ± 0.037 vs. 1.908 ± 0.039, P<0.05). But, The expression of LAMP2 and CTSL1 in TLC group was significantly lower than that in control group (LAMP2: 0.462 ± 0.025 vs. 1.009 ± 0.039, P<0.05. CTSL1: 0.563 ± 0.028 vs. 1.135 ± 0.041, P<0.05). The expression of LAMP2 and CTSL1 in TLC adding MAPK1siRNA group was significantly higher than that in TLC group (LAMP2: 1.007 ± 0.019 vs. 0.462 ± 0.025, P<0.05. CTSL1: 0.921 ± 0.030 vs. 0.563 ± 0.028, P<0.05). Conclusion: Silenced MAPK1 can inhibit the trypsinogen activation induced by TLC in AR42J cells, and it may inhibit autophagy by inhibiting the MAPK1 pathway. At the same time, the expression of LAMP2 and CTSL1 enhanced, the functions of autophagy lysosomes are normal and the process of autophagy proceeds smoothly, thereby inhibiting the activation of trypsinogen and reducing acute pancreatitis. |
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