TRPC6靶向miR-214负调控Caspase-1表达以改善肾缺血再灌注损伤的机制研究

doi:10.3877/cma.j.issn.1674-0785.2022.01.015

目的

探讨TRPC6对肾缺血再灌注损伤（RIRI）的缓解作用及潜在作用机制。

方法

建立氧糖剥离再灌注（OGD/R HK-2）模型细胞和缺血再灌注（I/R）模型小鼠后，采用ELISA方法检测白介素（IL）-1β和IL-18水平，通过RT-qPCR、Western blot和免疫组化检测caspase-1表达，并利用CCK-8和流式细胞仪检测细胞活力和焦亡情况。此外，通过双荧光素酶报告基因检测TRPC6与miR-214之间的相互作用。

结果

TRPC6在构建的OGD/R HK-2细胞和I/R模型小鼠中表达明显升高。同时，下调caspase-1可增强OGD/R HK-2细胞的活力，抑制炎症水平和细胞焦亡。TRPC6高表达能减轻I/R模型小鼠肾损伤并且下调caspase-1水平。从机制探究，我们发现TRPC6可通过调控miR-214下调caspase-1，并改善RIRI。

结论

TRPC6通过靶向调控miR-214下调caspase-1缓解RIRI，TRPC6/miR-214/caspase-1的作用通路可能为RIRI的治疗提供关键线索。

关键词: 肾缺血再灌注损伤, TRPC6, 微小RNA-214, caspase-1, 细胞焦亡

Objective

To investigate the alleviating effect and potential mechanism of TRPC6 on renal ischemic reperfusion injury (RIRI).

Methods

After establishing oxygen-glucose deprivation/reoxygenation, OGD/R HK-2 model cells and ischemia-reperfusion (I/R) model mice, the expression levels of IL-1β and IL-18 in model cells or mice were detected by using ELISA kits, whilst the expression of caspase-1 was examined by RT-qPCR, Western blot, and immunohistochemistry. And the cell viability and pyroptosis were determined by CCK-8 assay and flow cytometry. Moreover, the interaction between TRPC6 and miR-214 was identified by dual-luciferase reporter assay.

Results

The expression of TRPC6 was observably increased in OGD/R HK-2 cells and I/R model mice. Meanwhile, down-regulation of caspase-1 enhanced the vitality of OGD/R HK-2 cells and inhibited inflammation and pyroptosis. Overexpression of TRPC6 could alleviate renal injury and down-regulate caspase-1 in I/R model mice. TRPC6 could down-regulate caspase-1 partly by regulating miR-214, thereby improving RIRI.

Conclusion

TRPC6 alleviates RIRI by targeting miR-214 to down-regulate caspase-1 expression. The TRPC6/miR-214/caspase-1 regulatory pathway may provide key clues for the therapy of RIRI.

Key words: Renal ischemic reperfusion injury, TRPC6, MicroRNA-214, caspase-1, Pyroptosis

图1 caspase-1及其下游因子IL-1β和IL-18在OGD/R HK-2细胞中显著上调（n=3）。图a和b为利用酶联免疫吸附试验（ELISA）试剂盒检测IL-1β（图a）和IL-18（图b）在OGD/R HK-2模型细胞中的表达变化；图c为caspase-1在OGD/R HK-2细胞中的表达变化注：与假手术对照组（sham）比较，^*P＜0.05，^***P＜0.001；OGD/R为氧糖剥离再灌注组；IL为白介素

图2 缺血再灌注（I/R）模型小鼠的肾损伤及caspase-1表达。图a为I/R模型小鼠的肾脏损伤情况（HE，×200）；图b为I/R模型小鼠左右肾中caspase-1蛋白的表达变化（免疫组化，×200）注：与假手术对照组（sham）比较，^***P＜0.00

图3 caspase-1敲减对OGD/R HK-2细胞活力及焦亡的影响（n=3）。图a为实时荧光定量聚合酶链反应（RT-qPCR）检测caspase-1过表达或敲除后，OGD/R HK-2细胞中caspase-1 mRNA表达情况；图b为蛋白免疫印迹实验（WB）检测caspase-1过表达的OGD/R HK-2细胞中caspase-1蛋白表达情况；图c为WB检测caspase-1敲减的OGD/R HK-2细胞中caspase-1蛋白表达情况；图d、e为酶联免疫吸附试验（ELISA）试剂盒检测敲减caspase-1后，OGD/R HK-2细胞中白介素（IL）-1β和IL-18水平；图f为CCK-8法检测敲减caspase-1后OGD/R HK-2细胞的活力变化；图g为WB检测敲减caspase-1后OGD/R HK-2细胞中caspase-8和caspase-9表达情况注：组间比较，^*P＜0.05，^**P＜0.01，^***P＜0.001；OGD/R为氧糖剥离再灌注模型细胞组；OGD/R＋Vector为氧糖剥离再灌注模型细胞中转染过表达质粒的空载体组；OGD/R＋caspase-1为氧糖剥离再灌注模型细胞中转染caspase-1过表达质粒组；OGD/R＋sh-NC氧糖剥离再灌注模型细胞中转染干扰质粒对照组；OGD/R＋sh-caspase-1为氧糖剥离再灌注模型细胞中转染caspase-1干扰质粒组；Blank为空白对照组

图4 TRPC6过表达对缺血再灌注损伤（I/R）模型小鼠肾组织损伤及caspase-1和促炎细胞因子的影响（n=3）。图a为TRPC6过表达的I/R模型小鼠肾组织损伤情况（HE染色，×200）；图b为TRPC6过表达的I/R模型小鼠肾组织caspase-1表达情况（免疫组化，×200）；图c为蛋白免疫印迹实验（WB）检测TRPC6过表达对I/R模型小鼠caspase-1蛋白表达的影响；图d为WB检测敲减TRPC6对I/R模型小鼠caspase-1蛋白表达的影响；图e为酶联免疫吸附试验（ELISA）试剂盒检测TRPC6过表达或敲减的I/R模型小鼠血清中白介素（IL）-1β和IL-18水平注：组间比较，^*P＜0.05，^**P＜0.01，^***P＜0.001；sham为假手术对照组；sham＋I/R为缺血再灌注模型小鼠组；I/R＋Vector为缺血再灌注模型小鼠中过表达空载体组；I/R＋TRPC6为缺血再灌注模型小鼠中过表达TRPC6组；I/R＋sh-NC为缺血再灌注模型小鼠中干扰TRPC6对照组；I/R＋sh-TRPC6为缺血再灌注模型小鼠中干扰TRPC6组

图5 TRPC6介导miR-214对氧糖剥离模型细胞中caspase-1的表达调控（n=3）。图a、b为在用转染sh-TRPC6或TRPC6过表达质粒后，通过实时荧光定量聚合酶链反应（RT-qPCR）评估OGD/R HK-2细胞中miR-181和miR-214的表达；图c为通过双荧光素酶报告基因实验检测miR-181和miR-214对TRPC6的转录调控作用；图d、e分别为应用RT-qPCR（图d）和蛋白免疫印迹实验（WB）（图e）检测miR-214对OGD/R HK-2细胞中caspase-1表达的负调控作用注：组间比较，^*P＜0.05，^**P＜0.01，^***P＜0.001；OGD/R为氧糖剥离再灌注模型细胞组；OGD/R＋sh-TRPC6为氧糖剥离再灌注模型细胞中干扰TRPC6组；OGD/R＋TRPC6为氧糖剥离再灌注模型细胞中过表达TPRC6组；Blank为空白对照组；miR-214 mimics为miR-214模拟物处理组；miR-214 inhibitor为miR-214抑制物处理组；NC mimics为miRNA模拟物对照组；NC inhibitor为miRNA抑制物对照组；miR-181 mimics为miR-181模拟物处理组；miR-181 inhibitor为miR-181抑制物处理组；OGD/R＋NC为氧糖剥离再灌注模型细胞中转染miR-214阴性对照组；OGD/R＋miR-214为氧糖剥离再灌注模型细胞中转染miR-214组

图6 TRPC6通过靶向miR-214有效地诱导了OGD/R HK-2细胞的活力并抑制了炎症和细胞焦亡（n=3）。图a为酶联免疫吸附试验（ELISA）试剂盒检测TRPC6和miR214对OGD/R HK-2细胞中白介素（IL）-1β和IL-18水平的影响；图b为CCK-8法检测TRPC6和miR214对OGD/R HK-2细胞活力的影响；图c为流式细胞仪测定TRPC6和miR214对OGD/R HK-2细胞焦亡的影响注：组间比较，^*P＜0.05，^**P＜0.01，^***P＜0.001；sham为假手术对照组；OGD/R为氧糖剥离再灌注模型细胞组；OGD/R＋sh-TRPC6为氧糖剥离再灌注组模型细胞中干扰TPRC6组；OGD/R＋sh-TRPC6+mimics为氧糖剥离再灌注组模型细胞中干扰TPRC6后miR-214模拟物处理组；OGD/R＋TRPC6为氧糖剥离再灌注组模型细胞中过表达TPRC6组；OGD/R＋TRPC6＋inhibitor为氧糖剥离再灌注组模型细胞中过表达TPRC6后miR-214抑制物处理组

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TRPC6 ameliorates renal ischemic reperfusion injury by targeting miR-214 to prevent caspase-1 expression

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TRPC6 ameliorates renal ischemic reperfusion injury by targeting miR-214 to prevent caspase-1 expression