CAJ | 학술논문

目的：明确酪酸梭菌对人结肠腺癌细胞HT-29细胞水通道蛋白3（AQP3）mRNA和蛋白表达的影响以及c-Jun氨基末端激酶（JNK）和p38信号转导通路对其表达的调控作用。方法用酪酸梭菌上清液作用于HT-29细胞后，应用实时定量反转录聚合酶链反应（RT-PCR）和蛋白印迹方法检测AQP3 mRNA和蛋白表达水平。AQP3表达水平有差异后测定总JNK、p38以及磷酸化的JNK（P-JNK）和磷酸化的p38（P-p38）蛋白表达水平。后分别加用JNK抑制剂SP600125和p38抑制剂SB203580后测定AQP3蛋白表达量，观察JNK和p38对AQP3表达的调控作用。统计学处理采用单因素方差分析。结果实时定量RT-PCR和蛋白印迹法结果显示酪酸梭菌上清液不同浓度（1∶4和1∶8组）作用HT-29细胞12 h以及同一浓度1∶4作用不同时间（24、12、6 h组）的AQP3 mRNA和蛋白表达水平均较无酪酸梭菌上清液作用组明显上调（P均＜0．05）。蛋白印迹法结果显示酪酸梭菌上清液组的P-p38/p38和P-JNK/JNK蛋白相对量较空白对照组表达水平明显上调（F＝26．065和65．034，P＜0．05）。蛋白印迹法结果显示加入p38抑制剂SB20358010μg/ml组和20μg/ml的AQP3蛋白表达相对量为（0．7±0．4）和（0．6±0．6）、加入JNK抑制剂SP60012510μg/ml组和20μg/ml的AQP3蛋白表达相对量为（0．7±0．4）和（0．5±0．4）均较未加入抑制剂组（1．1±0．7）AQP3蛋白表达水平明显下调（F＝36．225，P＜0．05）。结论酪酸梭菌上清液能够上调HT-29细胞的AQP3表达进而调节肠道的水代谢。JNK和p38信号转导通路可能参与调控AQP3的表达。
목적：명학락산사균대인결장선암세포HT-29세포수통도단백3（AQP3）mRNA화단백표체적영향이급c-Jun안기말단격매（JNK）화p38신호전도통로대기표체적조공작용。방법용락산사균상청액작용우HT-29세포후，응용실시정량반전록취합매련반응（RT-PCR）화단백인적방법검측AQP3 mRNA화단백표체수평。AQP3표체수평유차이후측정총JNK、p38이급린산화적JNK（P-JNK）화린산화적p38（P-p38）단백표체수평。후분별가용JNK억제제SP600125화p38억제제SB203580후측정AQP3단백표체량，관찰JNK화p38대AQP3표체적조공작용。통계학처리채용단인소방차분석。결과실시정량RT-PCR화단백인적법결과현시락산사균상청액불동농도（1∶4화1∶8조）작용HT-29세포12 h이급동일농도1∶4작용불동시간（24、12、6 h조）적AQP3 mRNA화단백표체수평균교무락산사균상청액작용조명현상조（P균＜0．05）。단백인적법결과현시락산사균상청액조적P-p38/p38화P-JNK/JNK단백상대량교공백대조조표체수평명현상조（F＝26．065화65．034，P＜0．05）。단백인적법결과현시가입p38억제제SB20358010μg/ml조화20μg/ml적AQP3단백표체상대량위（0．7±0．4）화（0．6±0．6）、가입JNK억제제SP60012510μg/ml조화20μg/ml적AQP3단백표체상대량위（0．7±0．4）화（0．5±0．4）균교미가입억제제조（1．1±0．7）AQP3단백표체수평명현하조（F＝36．225，P＜0．05）。결론락산사균상청액능구상조HT-29세포적AQP3표체진이조절장도적수대사。JNK화p38신호전도통로가능삼여조공AQP3적표체。
Objective To investigate the effects of Clostridium butyricum and the regulation of c-Jun N-termi-nal Kinase (JNK)/p38 signaling transduction pathways on aquaporin3 (APQ3) mRNA and protein expression. Methods After HT-29 cells were treated with supernatant of Clostridium butyricum, the mRNA and protein expression of AQP3 were measured by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. After the altered expression of AQP3 was shown, protein expression levels of total JNK, p38, phosphorylated JNK (P-JNK), and phos-phorylated p38 (P-p38) were determined. Then, AQP3 protein expression was assayed after HT-29 cells were treated with a JNK inhibitor (SP600125) and a p38 inhibitor (SB203580), respectively. The regulation of JNK and p38 on AQP3 expression were analyzed. One-way analysis of variance was used for statistical processing. Results Real-time RT-PCR and Western blotting showed significant up-regulation of APQ3 mRNA and protein expression in HT-29 cells after 12 h treatment with different concentrations of Clostridium butyricum supernatant (1∶4 and 1∶8) or fixed concen-tration treatment (1∶4) for varied durations (24 h, 12 h and 6 h) compared with the group without treatment of Clostridium butyricum supernatant (all P<0.05). Western blotting showed that the relative levels of P-p38/p38 and P-JNK/JNK in the group treated with Clostridium butyricum supernatant was significantly up-regulated compared with the blank control group (F=26.065 and 65.034, P<0.05). Western blotting showed that the relative level of AQP3 pro-tein expression were (0.7 ±0.4) and (0.6 ±0.6), respectively, after addition of 10 μg/ml and 20 μg/ml p38 inhibitor SB203580; (0.7±0.4) and (0.5±0.4), respectively, after addition of 10 μg/ml and 20 μg/ml JNK inhibitor SP600125. The AQP3 protein expression levels were significantly down-regulated (F=36.225, P<0.05) compared with the group without inhibitors (1.1±0.7). Conclusion The supernatant of Clostridium butyricum can up-regulate AQP3 expres-sion in HT-29 cells and further regulate water metabolism in the bowels. JNK and p38 signaling transduction pathways may be involved in the regulation of AQP3 expression.