Department of Microbiology, School of Biological Sciences, Central University of Punjab, Bathinda, Punjab, India.
Department of Zoology, School of Biological Sciences, Central University of Punjab, Bathinda, Punjab, India.
J Biochem Mol Toxicol. 2022 Aug;36(8):e23096. doi: 10.1002/jbt.23096. Epub 2022 May 9.
The present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H O , and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease.
本研究探讨了小麦蛋白麦胶蛋白诱导的氧化应激和硝化应激及其在人肠 HCT-116 和 HT-29 细胞中的下游反应。在体外模型研究中，评估了膳食植物化学姜黄素的有益作用和多功能酶脱嘌呤/脱嘧啶内切核酸酶 1（APE1）在不耐受麦胶蛋白的 HCT-116 和 HT-29 细胞系中碱基切除修复（BER）途径中的主要作用。培养的细胞暴露于麦胶蛋白、H 2 O 2 和姜黄素后，通过分光光度法、PCR、流式细胞仪、Western 印迹、共聚焦显微镜等方法评估氧化应激及其后果。结果表明，3 h 预处理姜黄素后，再用麦胶蛋白处理 24 h，可通过以下方式保护 HCT-116 和 HT-29 细胞：（i）减少 ROS/RNS，恢复线粒体跨膜电位；（ii）重建细胞抗氧化防御系统（超氧化物歧化酶、过氧化氢酶和 GSH）；（iii）增强 APE1 的功能，即核酸内切酶活性和转录因子 Nrf-2 的氧化还原激活，后者与抗氧化反应元件（ARE）结合并激活参与细胞存活的下游靶标。还通过免疫荧光成像和共免疫沉淀测定建立了 APE1 和 Nrf-2 之间的串扰。总之，麦胶蛋白诱导氧化/硝化应激、线粒体功能障碍，并损害肠细胞中的细胞生物分子。因此，它可能与小麦不耐受相关的肠病中的组织损伤和疾病发病机制有关。通过 BER 途径和 ARE 途径，用姜黄素预处理可显著减少麦胶蛋白诱导的应激及其后果；这通过这两种必需蛋白之间的相互作用得到证明。因此，建议对姜黄素和其他天然膳食植物化学物质进行干预，以管理和治疗与麦胶蛋白不耐受相关的肠道疾病，如乳糜泻。
