STING高表达通过调控TLR4/NF-κB/NLRP3通路和影响炎症与凋亡水平促进小鼠肾脏缺血再灌注损伤

doi:10.12122/j.issn.1673-4254.2024.07.14

摘要/Abstract

摘要：

目的探讨STING在肾缺血再灌注损伤（IRI）中的表达水平以及相关作用机制。方法在体内水平，将24只C57BL/6小鼠分为假手术组（Sham）、IRI组、IRI+药物溶剂组（IRI+DMSO）、IRI+SN-011组，6只/组。通过肾动脉夹闭方法建立IRI模型，通过血清肌酐和尿素氮检测、PAS染色检测肾组织损伤变化，采用RT-qPCR、ELISA、Western blotting和IHC法检测肾组织中STING、KIM-1、Bcl-2、Bax、caspase-3、TLR4、P65、NLRP3、caspase-1、CD68、MPO、 IL-1β、IL-6、TNF-α的水平。在体外水平，将HK-2细胞分为对照组、缺氧复氧（H/R）组、H/R+药物溶剂组（H/R+DMSO）、H/R+SN-011组，用厌氧包模拟缺氧环境，RT-qPCR和Western blotting法检测STING表达水平，流式细胞术检测各组细胞凋亡率。结果在体内水平，与Sham组相比，IRI组的PAS染色显示组织损伤增加（P<0.05），小鼠血清肌酐、尿素氮含量以及组织KIM-1、STING、TLR4、P65、NLRP3、caspase-1、caspase-3、Bax、CD68、MPO、IL-1β、IL-6、TNF-α表达水平升高（P<0.05），Bcl-2水平降低（P<0.05），SN-011抑制STING表达后，逆转了上述结果（P<0.05）。在体外水平，与对照组相比，H/R组STING的mRNA与蛋白水平升高（P<0.05），流式细胞仪检测显示细胞凋亡率上升（P<0.05），SN-011抑制STING表达，细胞凋亡率下降（P<0.05）。结论 STING在肾脏IRI中表达水平上升，且可通过作用于TLR4/NF-κB/NLRP3通路以及影响炎症与凋亡水平促进肾损伤。

关键词: STING, TLR4, NF-κB, NLRP3, 肾缺血再灌注, 炎症, 凋亡

Abstract:

Objective To investigate renal expression level of STING in mice with renal ischemia-reperfusion injury (IRI) and its regulatory role in IRI. Methods C57BL/6 mice were divided into sham operation group, IRI (induced by clamping the renal artery) model group, IRI+DMSO treatment group, and IRI+SN-011 treatment group. Serum creatinine and blood urea nitrogen of the mice were analyzed, and pathological changes in the renal tissue were assessed with PAS staining. RT-qPCR, ELISA, Western blotting, and immunohistochemistry were used to detect the expression levels of STING, KIM-1, Bcl-2, Bax, caspase-3, TLR4, P65, NLRP3, caspase-1, CD68, MPO, IL-1β, IL-6, and TNF-α in the renal tissues. In the cell experiment, HK-2 cells exposed to hypoxia-reoxygenation (H/R) were treated with DMSO or SN-011, and cellular STING expression levels and cell apoptosis were analyzed using RT-qPCR, Western blotting or flow cytometry. Results In C57BL/6 mice, renal IRI induced obvious renal tissue damage, elevation of serum creatinine and blood urea nitrogen levels and renal expression levels of KIM-1, STING, TLR4, P65, NLRP3, caspase-1, caspase-3, Bax, CD68, MPO, IL-1β, IL-6, and TNF-α, and reduction of Bcl-2 expression level. Treatment of the mouse models with SN-011 for inhibiting STING expression significantly alleviated these changes. In HK-2 cells, H/R exposure caused significant elevation of cellular STING expression and obviously increased cell apoptosis rate, which was significantly lowered by treatment with SN-011. Conclusion Renal STING expression is elevated in mice with renal IRI to exacerbate renal injury by regulating the TLR4/NF-κB/NLRP3 pathway and promoting inflammation and apoptosis in the renal tissues.

Key words: STING, TLR4, NF-κB, NLRP3, ischemia-reperfusion, inflammation, apotosis

陶怀祥, 骆金光, 闻志远, 虞亘明, 苏萧, 王鑫玮, 关翰, 陈志军. STING高表达通过调控TLR4/NF-κB/NLRP3通路和影响炎症与凋亡水平促进小鼠肾脏缺血再灌注损伤[J]. 南方医科大学学报, 2024, 44(7): 1345-1354.

Huaixiang TAO, Jinguang LUO, Zhiyuan WEN, Genming YU, Xiao SU, Xinwei WANG, Han GUAN, Zhijun CHEN. High STING expression exacerbates renal ischemia-reperfusion injury in mice by regulating the TLR4/NF-κB/NLRP3 pathway and promoting inflammation and apoptosis[J]. Journal of Southern Medical University, 2024, 44(7): 1345-1354.

图/表 5

表 1 RT-qPCR引物序列

Tab.1 Primer sequence for RT-qPCR

Gene	Primer sequence
STING	F:GTCCCTTGCACATGGTGTTG
STING	R:CAGGTCATGCTGTCGCCTAT
KIM-1	F:AGAAGACCCACAACTACAAGGC
KIM-1	R:TAGATGTTGGAGGAGTGGAGGT
IL-1β	F:GCCTGTGTTTTCCTCCTTGC
IL-1β	R:TGCTGCCTAATGTCCCCTTG
IL-6	F:GTGGCTAAGGACCAAGACCAT
IL-6	R:TCTGACCACAGTGAGGAATGTC
TNF-α	F:AGCCGATGGGTTGTACCTTG
TNF-α	R:ATAGCAAATCGGCTGACGGT
GAPDH	F:TGGAAAGCTGTGGCGTGAT
GAPDH	R:AGATCCACGACGGACACATT

图2 血清生化、Western blotting、RT-qPCR、PAS染色验证小鼠缺血再灌注模型构建效果

Fig.2 Serum biochemistry, Western blotting, RT-qPCR and PAS staining for assessing the effect of ischemia-reperfusion modeling. A, B: Serum BUN (A) and Cr (B) levels of the mice in sham and IRI group. C: RT-qPCR analysis of KIM-1 in Sham and IRI groups mice. D: Western blotting of KIM-1 in sham and IRI group. E: PAS staining of renal tissue from mice in sham and IRI groups (Original magnification: ×400). *P<0.05 vs Sham group.

图5 RT-qPCR、Western blotting、PAS染色检测肾组织损伤程度

Fig.5 RT-qPCR, Western blotting and PAS staining for detecting renal tissue injury. A: PAS staining of kidney tissues from mice in the sham, IRI, IRI+DMSO and IRI+SN-011 groups (×400). B, C: RT-qPCR and Western blotting of the expressions of KIM-1 in sham, IRI, IRI+DMSO and IRI+SN-011 groups. *P<0.05 vs Sham; #P<0.05 vs IRI.

图7 Western blotting、流式细胞仪技术检测凋亡水平

Fig.7 Cell apoptosis analyzed using Western blotting and flow cytometry. A: Western blotting of relative expression levels of caspase-3, Bcl-2 and Bax in mouse kidney tissue from sham, IRI, IRI+DMSO and IRI+SN-011 groups. B: Flow cytometric analysis of HK-2 cell apoptosis in control, H/R, H/R+DMSO, and H/R+SN-011 groups. *P<0.05 vs Sham; #P<0.05 vs IRI; ▲P<0.05 vs control; ☆P<0.05 vs H/R.

图8 Western blotting检测肾组织中TLR4、NF-κB P65、NLRP3、caspase-1蛋白表达水平

Fig.8 Western blotting for detecting TLR4, NF‑κB P65, NLRP3, and caspase-1 protein expressions in the renal tissue. A: Western blotting of TLR4 and NF-κB P65 in mouse kidney tissues from sham, IRI, IRI+DMSO and IRI+SN-011 groups. B: Western blotting of NLRP3 and caspase-1 in mice kidney tissue from the sham, IRI, IRI+DMSO and IRI+SN-011 groups. *P<0.05 vs Sham; #P<0.05 vs IRI.

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