靶向治疗贲门失弛缓症：基于硫化氢减少导致食管运动功能障碍的研究

The etiology of achalasia is unclear, with a difficulty of treatment. It is proven that surgical treatment is invasive and easy to relapse, while the efficacy of pharmacological therapy with NO as the target is poor. So, it is urgent to find new targets of pharmacological therapy. We found that normal human esophagus expressed hydrogen sulfide synthesis enzymes, and the enzymes decreased significantly in patients with achalasia. We also found that hydrogen sulfide could relax animal esophageal smooth muscle, and symptoms of achalasia patients relieved after application of donor of hydrogen sulfide, with relaxing of LES. Therefore, we hypothesized that decreased hydrogen sulfide synthesis leads to defeated LES relaxation, which will cause achalasia. Hydrogen sulfide is a new therapeutic target of achalasia. Firstly, we studied the expression of hydrogen sulfide synthesis enzymes in esophagus of achalasia and the synthesis of hydrogen sulfide by PCR, deprotein method, and so on. Secondly, we explored the effects of hydrogen sulfide and combined with NO to esophagus, esophageal muscle strips and smooth muscle cells by esophageal manometry, electrophysiology and patch clamp technique. Lastly, we investigated the therapeutic effect of hydrogen sulfide donor and combined with NO donor on achalasia animal model. Also, we conducted dynamic comparison of influence of the two treatment protocols to esophageal motor function of achalasia patients. The aim of the study is to lay the foundation of.combined pharmacological therapy with multi-target for achalasia.

贲门失弛缓症病因未明，治疗困难。手术治疗有创且存在复发等问题，以NO为靶点的药物治疗效果欠佳，故迫切需要寻找新的药物治疗靶点。我们发现正常人食管组织中存在一种消化道抑制性递质硫化氢的合成酶，且该酶在贲门失弛缓症患者食管组织中明显减少；还发现硫化氢可松弛动物食管平滑肌；给予贲门失弛缓症患者硫化氢供体可减低LES压力缓解症状。因此，我们推测硫化氢合成减少导致LES松弛障碍可引起贲门失弛缓症，硫化氢是新的治疗靶点。本研究首先通过RT-PCR、去蛋白法等分别分析硫化氢合成酶在食管组织中的表达及硫化氢的合成；进而通过食管测压、电生理学、膜片钳等技术探讨硫化氢及联合NO对贲门失弛缓症动物食管、肌条及平滑肌细胞的调节作用及机制；再应用食管测压、食管排空等技术观察硫化氢供体及联合NO供体对贲门失弛缓症动物模型的治疗作用及对贲门失弛缓症患者食管运动功能的作用。旨在为多靶点药物联合治疗贲门失弛缓症奠定基础。

贲门失弛缓症病因未明，手术治疗有创且存在复发等问题，以NO为靶点的药物治疗效果欠佳，故迫切需要寻找新的药物治疗靶点。硫化氢是一种重要的抑制性神经递质，多项研究表明其可松弛胃肠道平滑肌。且我们的前期研究表明，贲门失弛缓患者食管组织较正常对照硫化氢合成酶减少。因此，本研究致力于阐明硫化氢减少对食管下括约肌（LES）松弛的影响及在贲门失弛缓症发病过程中的作用及可能机制，探讨硫化氢供体对贲门失弛缓症的治疗作用，以期为药物治疗贲门失弛缓症提供新方法。本研究表明贲门失弛缓症患者食管组织和正常对照均可表达硫化氢合成酶，且贲门失弛缓症患者食管各部位硫化氢合成酶表达均减少，以LES部位减少明显；外源性硫化氢供体NaHS和内源性硫化氢供体L-半胱氨酸均可抑制LES肌条收缩幅度和频率，且呈浓度依赖性，NaHS还可抑制乙酰胆碱介导的食管收缩活动，硫化氢合成酶抑制剂可增加LES肌条自发性收缩幅度和频率，其作用也呈浓度相关性，为硫化氢松弛食管平滑肌提供了部分证据；同时发现NaHS对食管的作用可能是通过L-型钙通道实现的，而不是通过介导其它神经递质的释放或通过KATP+通道、Kca+通道实现；通过在家兔食管下括约肌注射BAC建立贲门失弛缓症动物模型，NaHS和乙酰半胱氨酸均可降低正常动物和贲门失弛缓症模型动物的LES压力，初步临床试验证实，服用乙酰半胱氨酸后，贲门失弛缓症患者Eckardt评分和4sIRP、LES压力均降低，乙酰半胱氨酸可缓解贲门失弛缓症症状，该作用与其减轻LES压力有关。因此，本研究通过在体及离体实验、动物及人体实验等表明贲门失弛缓症的发生可能与硫化氢减少有关，硫化氢供体有望成为治疗贲门失弛缓症的新方法。