DataSheet4_Pharmacological basis of bergapten in gastrointestinal diseases focusing on H+/K+ ATPase and voltage-gated calcium channel inhibition: A toxicological evaluation on vital organs.docx

Aim and objectives: This study aimed to establish a pharmacological basis for evaluating the effects of bergapten (5-methoxypsoralen) in gastrointestinal diseases and assessment of its toxicological profile.
Methods: The pharmacokinetic profile was evaluated using the SwissADME tool. AUTODOCK and PyRx were used for evaluating the binding affinities. The obtained results were further investigated for a post-dock analysis using Discovery Studio Visualizer 2016. The Desmond software package was used to conduct molecular dynamic simulations of best bound poses. Bergapten was further investigated for antidiarrheal, anti-secretory, charcoal meal transit time, anti-ulcer, anti-H. pylori activity.
Results: Bergapten at a dose of 50, 100, and 200 mg/kg was proved effective in reducing diarrheal secretions, intestinal secretions, and distance moved by charcoal meal. Bergapten at the aforementioned doses acts as a gastroprotective agent in the ethanol-induced ulcer model that can be attributed to its effectiveness against H. pylori. Bergapten shows concentration-dependent relaxation of both spontaneous and K+ (80 mM)-induced contractions in the isolated rabbit jejunum model; the Ca2+ concentration–response curves (CRCs) were shifted to the right showing potentiating effect similar to papaverine. For molecular investigation, the H+/K+ ATPase inhibitory assay indicated inhibition of the pump comparable to omeprazole. Oxidative stress markers GST, GSH, and catalase showed increased expression, whereas the expression of LPO (lipid peroxidation) was reduced. Histopathological examination indicated marked improvement in cellular morphology. ELISA and western blot confirmed the reduction in inflammatory mediator expression. RT-PCR reduced the mRNA expression level of H+/K+ ATPase, confirming inhibition of the pump. The toxicological profile of bergapten was evaluated by an acute toxicity assay and evaluated for behavioral analysis, and the vital organs were used to analyze biochemical, hematological, and histopathological examination.
Conclusion: Bergapten at the tested doses proved to be an antioxidant, anti-inflammatory, anti-ulcer, and antidiarrheal agent and relatively safe in acute toxicity assay.

研究目的：本研究旨在为评价佛手柑内酯（bergapten，5-甲氧基补骨脂素）在胃肠道疾病中的作用及其毒理学特征建立药理学基础。
方法：采用SwissADME工具评估佛手柑内酯的药代动力学特征；使用AUTODOCK与PyRx评估其与靶点的结合亲和力；所得结果进一步通过Discovery Studio Visualizer 2016开展对接后分析；利用Desmond软件包对最佳结合构象开展分子动力学模拟。此外，本研究还对佛手柑内酯的止泻、抗分泌、炭末推进时间、抗溃疡及抗幽门螺杆菌（H. pylori）活性进行了考察。
结果：研究显示，剂量为50、100及200 mg/kg的佛手柑内酯可有效减少腹泻分泌量、肠道分泌量以及炭末推进距离。在乙醇诱导的胃溃疡模型中，上述剂量的佛手柑内酯可发挥胃黏膜保护作用，这一效应可归因于其抗幽门螺杆菌活性。在离体家兔空肠模型中，佛手柑内酯可呈浓度依赖性舒张自发收缩与K+（80 mM）诱导的肠道收缩；钙浓度-反应曲线（CRCs）发生右移，显示出与罂粟碱相似的增强效应。分子层面的研究显示，H+/K+ ATP酶抑制实验表明，其对该酶泵的抑制效果可与奥美拉唑（omeprazole）相媲美。氧化应激标志物谷胱甘肽S转移酶（glutathione S-transferase，GST）、谷胱甘肽（glutathione，GSH）及过氧化氢酶的表达水平显著上调，而脂质过氧化（lipid peroxidation，LPO）产物的表达水平则显著下调。组织病理学检查显示细胞形态得到明显改善。酶联免疫吸附实验（enzyme-linked immunosorbent assay，ELISA）与蛋白质印迹（western blot）结果证实，炎症介质的表达水平有所降低。实时荧光定量聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）检测显示，H+/K+ ATP酶的mRNA表达水平下调，进一步证实了其对该酶泵的抑制作用。本研究通过急性毒性实验评估了佛手柑内酯的毒理学特征，同时开展行为学分析，并采集主要脏器进行生化指标、血液学指标及组织病理学检查。
结论：受试剂量下的佛手柑内酯具有抗氧化、抗炎、抗溃疡及止泻活性，且在急性毒性实验中表现出相对安全性。