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肠胶质细胞系TRPV4依赖性Ca2+内流、ATP释放与炎症因子表达
Transient receptor potential vanilloid 4-dependent calcium influx, ATP release and expression of inflammatory factors in enteric glial cells
- 陆军军医大学学报 2023年45卷第5期 页码：417-425
- 作者机构：
1.400037 重庆，陆军军医大学(第三军医大学)第二附属医院普通外科
2.400037 重庆，陆军军医大学(第三军医大学)第二附属医院消化科
- 作者简介：
[肖卫东，E-mail：xiaoweidong@tmmu.edu.cn XIAO Weidong, E-mail: xiaoweidong@tmmu.edu.cn
- 基金信息：国家自然科学基金面上项目(82270585);国家自然科学基金面上项目(81770524);重庆英才计划(15014);军事医学高新技术培育项目(CX2019JS212)
DOI：10.16016/j.2097-0927.202211062
中图分类号： R322.45;R329.27;R392.3收稿：2022-11-09，
修回：2022-12-22，
纸质出版：2023-03-15
- 稿件说明：
移动端阅览
谭千山, 陈君, 谢会超, 等. 肠胶质细胞系TRPV4依赖性Ca2+内流、ATP释放与炎症因子表达[J]. 陆军军医大学学报, 2023,45(5):417-425.
Qianshan TAN, Jun CHEN, Huichao XIE, et al. Transient receptor potential vanilloid 4-dependent calcium influx, ATP release and expression of inflammatory factors in enteric glial cells[J]. Journal of Army Medical University, 2023, 45(5): 417-425.
谭千山, 陈君, 谢会超, 等. 肠胶质细胞系TRPV4依赖性Ca2+内流、ATP释放与炎症因子表达[J]. 陆军军医大学学报, 2023,45(5):417-425. DOI： 10.16016/j.2097-0927.202211062.
Qianshan TAN, Jun CHEN, Huichao XIE, et al. Transient receptor potential vanilloid 4-dependent calcium influx, ATP release and expression of inflammatory factors in enteric glial cells[J]. Journal of Army Medical University, 2023, 45(5): 417-425. DOI： 10.16016/j.2097-0927.202211062.
摘要
目的
2
探讨瞬时感受器电位离子通道香草素受体4(transient receptor potential vanilloid 4，TRPV4)在调节肠胶质细胞外Ca
2+
内流中的作用及其意义。
方法
2
收集大鼠肠胶质细胞系CRL-2690、大鼠肠上皮细胞系IEC6和人胚肾细胞系HEK293T样本，采用RT-qPCR、Western blot和免疫荧光方法检测TRPV4的mRNA、蛋白表达与定位；将CRL-2690细胞依次分为GSK组与GSK+HC组，低渗组与低渗+HC组，GdCl
3
组与GdCl
3
+HC组以及CaCl
2
组与CaCl
2
+HC组，采用Fura-2荧光探针和单细胞荧光Ca
2+
检测仪评估CRL-2690细胞中TRPV4通道活性。将CRL-2690细胞分为对照组、GSK组与GSK+HC组，通过荧光素-荧光素酶实验测量细胞ATP的释放水平；采用RT-qPCR检测炎症相关基因GFAP、IL-1β、IL-6、IL-10的mRNA表达。
结果
2
在大鼠肠胶质细胞系CRL-2690中检测到TRPV4 mRNA和蛋白表达；TRPV4特异性激动剂GSK1016790A和低渗透压刺激明显增强了CRL-2690细胞内钙荧光强度，而TRPV4特异性抑制剂HC067047可抑制此作用(0.430 38±0.063 65
vs
0.004 23±0.005 78，
P
<
0.000 1)；钙敏感受体(calcium-sensing receptor，CaSR)激活诱导的外Ca
2+
内流也可被HC067047所抑制(
P
<
0.05)；功能上，激活TRPV4可促ATP释放、EGC反应性增生标志物GFAP和炎症因子IL-1β、IL-6的表达，并抑制抗炎因子IL-10的表达，该作用可被HC067047所抑制(
P
<
0.05)。
结论
2
激活TRPV4可引起肠胶质细胞外Ca
2+
内流，并促进ATP释放与炎症因子表达，可能参与肠道炎症的发生。
Abstract
Objective
2
To investigate the role and physiological significance of transient receptor potential vanilloid receptor 4 (TRPV4) in regulation of calcium influx in rat intestinal glial cells.
Methods
2
The expression of TRPV4 at mRNA and protein levels and its localization were detected by RT-qPCR
Western blotting and immunofluorescence assay in rat enteric glial cell line CRL-2690
rat intestinal epithelial cell line IEC6 and human embryonic kidney cell line HEK293T. The CRL-2690 cells were divided into GSK1016790A (GSK
TRPV4-specific agonist) group and GSK+HC067047 (HC
TRPV4-specific inhibitor) group
hypotonic group and hypotonic+HC group
GdCl
3
group and GdCl
3
+HC group
and CaCl
2
group and
CaCl
2
+HC group
while the activity of TRPV4 channel was assessed by Fura-2 fluorescent probe and single-cell fluorescent calcium ion assay. CRL-2690 cells were also divided into control group
GSK group and GSK+HC group
and the level of cellular ATP release was measured by luciferin-luciferase assay
and the mRNA levels of inflammatory genes GFAP
IL-1β
IL-6 and IL-10 were detected by RT-qPCR.
Results
2
TRPV4 was expressed at mRNA and protein levels in rat intestinal glial cell line CRL-2690. GSK and low osmolarity stimulation significantly enhanced intracellular calcium fluorescence intensity in CRL-2690 cells
which was inhibited by HC (0.430 38±0.063 65
vs
0.004 23±0.005 78
P
<
0.000 1). Calcium-sensing receptor (CaSR) activation-induced Ca
2+
influx was also inhibited by HC (
P
<
0.05). Functionally
activation of TRPV4 promoted ATP release
expression of EGC reactive proliferation marker
GFAP
and inflammatory factors IL-1 β as well as IL-6
and inhibited the expression of anti-inflammatory factor IL-10 respectively. But all these effects were inhibited by HC treatment (
P
<
0.05).
Conclusion
2
Activation of TRPV4 induces extracellular Ca
2+
influx and promotes ATP release and expression of inflammatory cytokines in enteric glial cell
which may be closely involved in the underlying mechanisms of bowel inflammation.
关键词
Keywords
references
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