利用CRISPR-Cas9技术探究肠道ERβ对肠源性TPH1和5-HT水平的影响

Exploring the effect of intestinal ERβ on enterogenic TPH1 and 5-HT levels using CRISPR-Cas9 technology

摘要

目的 探索C57BL/6J小鼠结肠上皮雌激素受体β（estrogen receptor β，ERβ）缺失对结肠色氨酸羟化酶-1（TPH1，tryptophan hydroxylase-1）和5-羟色胺（5-hydroxytryptamine，5-HT）水平的影响。 方法 利用CRISPR-Cas9技术获得Pvillin-Cre^+/-小鼠、ERβflox^+/-小鼠，通过配种繁殖最终获得肠道ERβ敲除纯合子组（ERβflox^-/--Pvillin-Cre^+/+，ERβCKO，n=5）、杂合子组（ERβflox^+/--Pvillin-Cre^+/+，ERβCKO^+/-）（n=5）及同窝野生型（wild type，WT）小鼠。取6个月龄和12个月龄的各品系小鼠作为研究对象。结合苏木素-伊红染色（hematoxylin-eosin staining，HE）、酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）、免疫印迹（Western blot，WB）和免疫组化（immunohistochemistry，IHC）方法对各组小鼠肠道形态、肠道ERβ、5-HT和TPH1水平进行检测。 结果 基因鉴定验证肠道ERβCKO小鼠成功构建后，WB显示肠道ERβ水平明显下调（P<0.05）。HE显示肠道ERβ敲除后，肠道结构完整性与肠道黏膜上皮的绒毛发育均异常。ELISA结果显示，2个月龄段ERβCKO小鼠肠道5-HT水平较WT组小鼠下降明显（P<0.05），且呈月龄依赖方式。IHC结果显示，各组小鼠肠道均出现TPH1免疫阳性表达，与WT小鼠相比，ERβCKO小鼠肠道TPH1免疫阳性细胞数量减少（P<0.05），免疫阳性物表达量更低（P<0.05），但TPH1未呈现月龄依赖方式。 结论 结肠ERβ下调可引起结肠TPH1蛋白和5-HT水平含量降低，继而引发小鼠肠道功能异常，增加患肠道疾病的风险，这为以ERβ为靶点的肠道相关疾病的发病机制提供了实验室依据。

Abstract

Objective To investigate the effects of colonic epithelial estrogen receptor β（ERβ） deletion on colonic tryptophan hydroxylase-1（TPH1） and 5-hydroxytryptamine（5-HT） levels in C57BL/6J mice. Methods Pvillin-Cre^+/- mice and ERβflox^+/- mice were obtained using CRISPR-Cas9 technology，and intestinal ERβ knockout homozygous（ERβflox^-/--Pvillin-Cre^+/+，ERβCKO，n=5），heterozygous（ERβflox^+/--Pvillin-Cre^+/+，ERβCKO^+/-n=5），and littermate wild-type（WT） mice were ultimately obtained by mating and breeding. Mice of each strain at 6 and 12 months of age were used for the study. Hematoxylin-eosin staining（HE），enzyme-linked immunosorbent assay（ELISA），Western blotting（WB），and immunohistochemistry（IHC） were used for determining the intestinal morphology and measuring intestinal ERβ，5-HT，and TPH1 levels of mice in each group. Results After the successful construction of intestinal ERβCKO mice as verified by gene identification，WB showed that intestinal ERβ levels were significantly down-regulated（P<0.05）. HE showed abnormalities in intestinal structural integrity and intestinal mucosal epithelial villi development after intestinal ERβ knockdown. ELISA results showed that intestinal 5-HT levels of ERβCKO mice at 6 and 12 months of age were significantly decreased compared with those of WT mice（P<0.05），and the decrease was in a month-age-dependent manner. IHC results showed that TPH1 was expressed in the intestines of mice of all groups. However，ERβCKO mice showed a reduced number of TPH1 immunoreactive cells（P<0.05） and lower expression of immunoreactive substances（P<0.05） compared with WT mice，but the decline in TPH1 was not in a month-age-dependent manner. Conclusion Down-regulation of colonic ERβ induces a decrease in colonic TPH1 protein and 5-HT levels，which subsequently triggers abnormal intestinal function and increases the risk of developing intestinal diseases in mice. This study is expected to provide a laboratory basis for the pathogenesis of ERβ-targeted intestinal diseases.

关键词

雌激素受体β / 色氨酸羟化酶-1 / 5-羟色胺 / 肠道

Key words

estrogen receptor β / tryptophan hydroxylase-1 / 5-hydroxytryptamine / intestine

基金资助

国家自然科学基金资助项目(82371203)

重庆市基础与前沿研究计划资助项目(CSTB2023NSCQ-MSX0161)