组胺H3受体对尿道横纹肌控尿功能的作用与机制

Stress urinary incontinence (SUI) is a common disease, frequently occurring in women. The current treatment methods cannot completely solve this disease. The urethral striated muscle is the key position to treat SUI. The recent studies found histamine H3 receptor was involved in the smooth muscles and myocardium. Meanwhile, our previous study found the presence of H3 receptor in the adult urethral striated muscle and the folds’ increase expression of H3 receptor in the striated myogenesis of mouse muscle derived stem cells C2C12; additionally, activation of H3 receptor can reduce cytoplasmic calcium ion concentration in differentiated C2C12 cells under contraction evoked by electrical stimulation. Therefore, it is supposed that H3 receptor may influence the function of striated muscle cell by regulating cytoplasmic calcium ion concentration, thereby, to play a role in continence function through the urethral striated muscle. The present study would like to do the research from the new viewpoint of H3 receptor that it would set up the SUI mouse model to compare the expression of H3 receptor in the urethral striated muscle between normal and SUI mice, and to verify if the viral vector modulators of H3 receptor gene in the urethral striated muscle could improve urethral continence function on SUI mice ; additionally, we would verify how H3 receptor affects calcium channels on cell membrane and endoplasmic reticulum, and its functional signal pathway in the cells through differentiated C2C12 cells. Thus, these would help to uncover the role of H3 receptor in the striated muscle from animal’s and cellular molecular levels, which would provide new ideas for the clinical diagnosis and treatment of urethral diseases like SUI in the future.

压力性尿失禁（SUI）是女性的常见病、多发病，目前的治疗方法尚不能彻底解决该病。尿道横纹肌是治疗SUI的关键部位。近年发现组胺H3受体存在并参与平滑肌和心肌的收缩功能。对此，我们前期研究发现在成人尿道横纹肌中存在H3受体；小鼠肌源干细胞C2C12成横纹肌分化过程，H3受体表达呈倍增加；在分化后的细胞中激动该受体可以降低胞浆中钙离子浓度。由此推测，H3受体可能通过调节胞浆钙离子浓度影响横纹肌细胞功能，进而通过尿道横纹肌影响控尿功能。本研究拟从H3受体新视点，通过建立SUI小鼠模型来测量H3受体在SUI和正常小鼠尿道横纹肌的表达差异及病毒载体调控H3受体基因表达验证其对小鼠尿道控尿功能的作用；同时通过C2C12细胞阐明H3受体对胞膜和内质网上钙通道的影响及其在细胞中发挥功能的信号通路。从而在宏观动物和微观细胞分子层面揭示H3受体在横纹肌中的角色，为今后临床诊断治疗SUI等尿道疾病提供新思路。

项目背景目的：探讨成肌分化过程中组胺H3受体对TNFa诱导的C2C12成肌细胞炎症反应的作用,从分子角度阐明尿道括约肌发育和损伤修复过程对炎症刺激的抵抗。探讨佛司可林对人THP1巨噬细胞内NLRP3炎症小体活化及活化后IL1β炎症因子分泌的影响，为临床治疗炎症性疾病提供参考。主要研究内容 ：诱导C2C12细胞成肌分化，检测TNFa、H3受体激动剂Met和阻滞剂CXP对分化中细胞的生长活性和凋亡的影响；检测NLRP3以及早中晚细胞分化标志物MyoD1mRNA、MyogeninmRNA、Myosin2mRNA表达；检测炎性因子IL1β的分泌。诱导人单核细胞系THP1细胞分化成巨噬细胞，尼日利亚菌素活化巨噬细胞，佛司可林刺激活化的巨噬细胞；检测细胞炎症小体NLRP3、炎性因子IL1β和Caspase1mRNA和相应前体及功能性蛋白表达，以及cAMP蛋白量和IL1β的分泌。重要结果：C2C12细胞分化过程中，TNFa和TNFa+CXP降低细胞的生长活性,CXP导致D6细胞发生凋亡。TNFα诱导分化细胞NLRP3mRNA增加，Met降低TNFa作用，而CXP增加TNFa作用。TNFa使MyoD1mRNA、 MyogeninmRNA、Myosin2mRNA降低；Met加剧TNFa刺激下各标志物的降低，CXP增加TNFa刺激下的各分化标志物。TNFa诱导细胞分泌IL1β，Met降低TNFa诱导的IL1β分泌量，而CXP作用反之。尼日利亚菌素升高THP1巨噬细胞的NLRP3、IL1β和 Caspase1的mRNA，但佛司可林刺激其无影响。佛司可林对proIL1β和 procaspase1的蛋白量表达无影响，但明显降低NLRP3和 Caspase1蛋白量。尼日利亚菌素提高IL1β的分泌，而佛司可林可使分泌量降低。科学意义：TNFa能诱导C2C12细胞发生炎症反应并抑制其成肌分化过程；而组胺H3受体抑制TNFa诱导的炎症反应而有序调控分化进程减缓；从而说明尿道括约肌发育和手术后损失修复过程遭遇外源性炎症刺激时，组胺H3受体可抵抗外源性的炎症反应，维持细胞正常的发育修复。佛司可林不影响激活的THP1巨噬细胞炎症相关蛋白前体的基因转录和蛋白翻译表达，但降低其炎症因子IL1β翻译后的蛋白分泌量，从而抑制炎症反应，可用于治疗由IL1β引起的成人和小儿炎症性疾病。