基于成骨细胞Na/K-ATPase探讨质子泵抑制剂引起骨质疏松的机制

Proton pump inhibitors (PPIs), binding with gastric hydrogen-potassium adenosine triphosphatase (H/K-ATPase, proton pump), are one of the most popular and prevalent used medications. Long-term using of PPIs increase the risk of osteoporosis, but the mechanism is still not understandable. Our preliminary experiments show the results that: (1) Proton pump inhibitors can bind with Na/K-ATPase by using the method of computer-aided drug design. (2) Intracellular calcium concentration of mouse osteoblasts decreases when treating with PPIs by using the methods of laser confocal scanning, flow cytometry, and fluorescence microscope, and the decline extent is correlated with dosage. (3) Cell viability of mouse osteoblasts decreases when treating with PPIs by the experiment of cell counting kit-8. The mechanism deserves further investigation. In order to explore the possible mechanism of action, we propose our hypothesis that proton pump inhibitors distribute to bone tissue and bind with osteoblasts Na/K-ATPase, which displays a high sequence homology with gastric H/K-ATPase. The activation of Na/K-ATPase aggrandizes sodium-potassium exchange and leads to an abnormal increase of extracellular sodium, then activates sodium-calcium exchange and decreases intracellular calcium, as well as weakens the cell viability of osteoblasts, finally leads to high risk of osteoporosis. In order to validate the hypothesis, experiments are in planning from three levels including ossification cell lines (MC3T3-E1), mouse treated by PPIs and patients with osteoporsis. Then a series of techniques, such as radioisotopic labeling, Micro-PET/CT, patch clamp, flow cytometry, laser confocal scanning, siRNA transfection, real-time PCR, and western blot, and so on, will be used to explore the binding site of Na/K-ATPase in osteoblasts with PPIs after distributed to the bone tissue, sodium-potassium exchange and sodium-calcium exchange current on osteoblast membrane, intracellular calcium concentration, and cell viability, etc. Thus we could explore and confirm the molecular ion mechanism of osteoporosis induced by proton pump inhibitors. It will provide theoretical reference for clinical rational use of PPIs, and provide new targets for pharmaceutical in drug-induced osteoporosis.

质子泵抑制剂（PPIs）与胃H/K-ATP酶结合抑制胃酸分泌，是临床应用最广泛的药物之一，长期使用增加骨质疏松风险，机制不明。预实验发现：成骨细胞在PPIs干预后胞内Ca浓度及增殖活性均下降；计算机模拟显示PPIs可以与Na/K-ATP酶结合。我们提出假说，PPIs通过与胃H/K-ATP酶同源性较高的成骨细胞Na/K-ATP酶结合，增加Na/K交换，使胞外Na异常升高，进而促进Na/Ca交换，降低胞内Ca浓度，减弱成骨细胞活性，致骨质疏松发生。为了验证假说，拟以成骨细胞、小鼠和患者为研究对象，采用放射性同位素标记、Micro-PET/CT、膜片钳、流式细胞术、激光共聚焦扫描和siRNA转染等方法，探讨和确证PPIs在骨组织分布与结合位点，细胞膜Na/K和Na/Ca交换电流增加，胞内钙及细胞活性下降等致骨质疏松的分子离子机制。为PPIs合理用药提供理论依据，为药源性骨质疏松的治疗提供新靶点。

背景： 兰索拉唑是临床中常用的抑酸药，其良好的治疗效果和安全性得到了广泛的认可。但是根据相关报道，临床患者长期服用 PPIs 后骨折风险增加，患者以老年患者和绝经后妇女为主，但其损伤的机制尚不明确。. 目的： 探讨兰索拉唑致骨质疏松的相关机制，为预防及治疗兰索拉唑引起的药源性骨质疏松提供新的依据和方法。. 方法： 本研究从体内、体外两方面探讨兰索拉唑导致肝内胆管损伤的机制。ICR小鼠长期予兰索拉唑灌胃给药 6 个月后，使用血生化检查、H&E 染色和免疫组化等判断小鼠骨组织损伤机制以及程度。 使用 EDU细胞增殖试剂盒研究兰索拉唑对成骨细胞功能的影响，使用激光共聚焦和流式细胞仪研究经兰索拉唑处理后细胞内钙离子的变化情况，利用成骨细胞进行 Western blot和凋亡实验等。. 结果：小鼠血清生化结果显示随着小鼠给药剂量增加，与空白溶媒对照组相比，对于S-IP，LPZ低剂量组（250 mg/kg）与空白对照组相比有一定程度下降但无显著性差异（P>0.05），高剂量组（1000 mg/kg）小鼠与空白对照组相比S-IP水平均显著下降（P<0.05）；对于S-Ca，低剂量组和高剂量组小鼠与空白对照组相比S-Ca水平均显著下降，且药物剂量越大降低程度越高。小鼠股骨组织 HE 染色显示高剂量组出现骨小梁变薄变少。小鼠 CHOP 免疫组化显示空白溶媒对照组几乎不表达， 低剂量组表达略有增加， 高剂量组某些个体存在高表达。 小鼠组织 Western blot 显示与空白溶媒对照组相比Caspase-12,Caspase-3,CHOP,Grp78 均随着给药量增加而增加 （P＜0.05） 。 EDU 细胞增殖实验显示当在培养基中加入兰索拉唑后，成骨细胞的增殖能力减弱（P＜0.05）。激光共聚焦实验结果显示当加入兰索拉唑后，会出现异常的[Ca2+]i 信号升高。流式检测提示我们预先孵育钙离子螯合剂可以缓解给药组的细胞凋亡百分比。.结论 长期使用兰索拉唑后会导致成骨细胞损伤而致骨丢失。其作用机制为兰索拉唑抑制了细胞钙泵调节，再进一步激活了 IP3/Ca2+介导的钙释放，进而使成骨细胞钙超载而凋亡，导致慢性损伤。