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.

参考文献 45

1	Hoste EAJ, Kellum JA, Selby NM, et al. Global epidemiology and outcomes of acute kidney injury[J]. Nat Rev Nephrol, 2018, 14(10): 607-25.
2	Zuk A, Bonventre JV. Acute kidney injury[J]. Annu Rev Med, 2016, 67: 293-307.
3	Arai S, Kitada K, Yamazaki T, et al. Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice[J]. Nat Med, 2016, 22(2): 183-93.
4	Inagi R, Ishimoto Y, Nangaku M. Proteostasis in endoplasmic reticulum: new mechanisms in kidney disease[J]. Nat Rev Nephrol, 2014, 10(7): 369-78.
5	Yan MJ, Tang CY, Ma ZW, et al. DNA damage response in nephrotoxic and ischemic kidney injury[J]. Toxicol Appl Pharmacol, 2016, 313: 104-8.
6	Malek M, Nematbakhsh M. Renal ischemia/reperfusion injury; from pathophysiology to treatment[J]. J Renal Inj Prev, 2015, 4(2): 20-7.
7	Fang R, Wang CG, Jiang QF, et al. NEMO-IKKβ are essential for IRF3 and NF-κB activation in the cGAS-STING pathway[J]. J Immunol, 2017, 199(9): 3222-33.
8	Fang R, Jiang QF, Guan YK, et al. Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING[J]. Immunity, 2021, 54(5): 962-75.e8.
9	Ren P, Cao JL, Lin PL, et al. Molecular mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion injury in H9c2 cardiomyocytes based on molecular docking[J]. Zhongguo Zhong Yao Za Zhi, 2021, 46(21): 5665-73.
10	Bi R, Yang YL, Liao HW, et al. Porphyromonas gingivalis induces an inflammatory response via the cGAS-STING signaling pathway in a periodontitis mouse model[J]. Front Microbiol, 2023, 14: 1183415.
11	Pressly JD, Park F. DNA repair in ischemic acute kidney injury[J]. Am J Physiol Renal Physiol, 2017, 312(4): F551-5.
12	Hu HL, Zou C. Mesenchymal stem cells in renal ischemia-reperfusion injury: biological and therapeutic perspectives[J]. Curr Stem Cell Res Ther, 2017, 12(3): 183-7.
13	Inagi R. Endoplasmic reticulum stress in the kidney as a novel mediator of kidney injury[J]. Nephron Exp Nephrol, 2009, 112(1): e1-9.
14	Cao Q, Wang YP, Niu ZG, et al. Potentiating tissue-resident type 2 innate lymphoid cells by IL-33 to prevent renal ischemia-reperfusion injury[J]. J Am Soc Nephrol, 2018, 29(3): 961-76.
15	Havasi A, Borkan SC. Apoptosis and acute kidney injury[J]. Kidney Int, 2011, 80(1): 29-40.
16	Yang DH, Tang M, Zhang MM, et al. Downregulation of G protein-coupled receptor kinase 4 protects against kidney ischemia-reperfusion injury[J]. Kidney Int, 2023, 103(4): 719-34.
17	Li XR, Liao J, Su XJ, et al. Human urine-derived stem cells protect against renal ischemia/reperfusion injury in a rat model via exosomal miR-146a-5p which targets IRAK1 [J]. Theranostics, 2020, 10(21): 9561-78.
18	Wang J, Xiong MR, Fan Y, et al. Mecp2 protects kidney from ischemia-reperfusion injury through transcriptional repressing IL-6/STAT3 signaling[J]. Theranostics, 2022, 12(8): 3896-910.
19	van Timmeren MM, van den Heuvel MC, Bailly V, et al. Tubular kidney injury molecule-1 (KIM-1) in human renal disease[J]. J Pathol, 2007, 212(2): 209-17.
20	Gkirtzimanaki K, Kabrani E, Nikoleri D, et al. IFNα impairs autophagic degradation of mtDNA promoting autoreactivity of SLE monocytes in a STING-dependent fashion[J]. Cell Rep, 2018, 25(4): 921-33.e5.
21	Gao YP, Zhang NN, Zeng ZH, et al. LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signalling in non-small cell lung cancer[J]. Clin Transl Med, 2022, 12(4): e792.
22	Li X, Liu YJ, Wang Y, et al. Epoxy triglyceride enhances intestinal permeability via caspase-1/NLRP3/GSDMD and cGAS-STING pathways in dextran sulfate sodium-induced colitis mice[J]. J Agric Food Chem, 2023, 71(10): 4371-81.
23	Wu JJ, Zhao L, Hu HG, et al. Agonists and inhibitors of the STING pathway: potential agents for immunotherapy[J]. Med Res Rev, 2020, 40(3): 1117-41.
24	Barber GN. STING: infection, inflammation and cancer[J]. Nat Rev Immunol, 2015, 15(12): 760-70.
25	Lu L, Zhou HM, Ni M, et al. Innate immune regulations and liver ischemia-reperfusion injury[J]. Transplantation, 2016, 100(12): 2601-10.
26	DeWolf SE, Kasimsetty SG, Hawkes AA, et al. DAMPs released from injured renal tubular epithelial cells activate innate immune signals in healthy renal tubular epithelial cells[J]. Transplantation, 2022, 106(8): 1589-99.
27	Raup-Konsavage WM, Wang YM, Wang WW, et al. Neutrophil peptidyl arginine deiminase-4 has a pivotal role in ischemia/reperfusion-induced acute kidney injury[J]. Kidney Int, 2018, 93(2): 365-74.
28	Salvadori M, Rosso G, Bertoni E. Update on ischemia-reperfusion injury in kidney transplantation: Pathogenesis and treatment[J]. World J Transplant, 2015, 5(2): 52-67.
29	Liu CH, Wang QD, Niu L. Sufentanil inhibits Pin1 to attenuate renal tubular epithelial cell ischemia-reperfusion injury by activating the PI3K/AKT/FOXO1 pathway[J]. Int Urol Nephrol, 2023, 55(8): 1903-16.
30	Wu B, Xu MM, Fan C, et al. STING inhibitor ameliorates LPS-induced ALI by preventing vascular endothelial cells-mediated immune cells chemotaxis and adhesion[J]. Acta Pharmacol Sin, 2022, 43(8): 2055-66.
31	Liu R, Li JY, Shao JC, et al. Innate immune response orchestrates phosphoribosyl pyrophosphate synthetases to support DNA repair[J]. Cell Metab, 2021, 33(10): 2076-89.e9.
32	Yang B, Li X, Fu Y, et al. MEK inhibition remodels the immune landscape of mutant KRAS tumors to overcome resistance to PARP and immune checkpoint inhibitors[J]. Cancer Res, 2021, 81(10): 2714-29.
33	Zhang YN, Dong YL, Hao WP, et al. Increased cGAS/STING signaling components in patients with Mooren's ulcer[J]. Int J Ophthalmol, 2021, 14(11): 1660-5.
34	Hong Z, Mei JH, Li CH, et al. STING inhibitors target the cyclic dinucleotide binding pocket[J]. Proc Natl Acad Sci U S A, 2021, 118(24): e2105465118.
35	Diao FF, Bai J, Jiang CL, et al. The papain-like protease of porcine reproductive and respiratory syndrome virus impedes STING translocation from the endoplasmic reticulum to the Golgi apparatus by deubiquitinating STIM1[J]. J Virol, 2023, 97(4): e0018823.
36	Yang BX, Xie XR, Wu ZY, et al. DNA damage-mediated cellular senescence promotes hand-foot syndrome that can be relieved by thymidine prodrug[J]. Genes Dis, 2022, 10(6): 2557-71.
37	Ablasser A, Chen ZJ. cGAS in action: expanding roles in immunity and inflammation[J]. Science, 2019, 363(6431): eaat8657.
38	Gulen MF, Koch U, Haag SM, et al. Signalling strength determines proapoptotic functions of STING[J]. Nat Commun, 2017, 8(1): 427.
39	Lehnardt S, Massillon L, Follett P, et al. Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway[J]. Proc Natl Acad Sci USA, 2003, 100(14): 8514-9.
40	Wang L, Yang JW, Lin LT, et al. Acupuncture attenuates inflammation in microglia of vascular dementia rats by inhibiting miR-93-mediated TLR4/MyD88/NF‑κB signaling pathway[J]. Oxid Med Cell Longev, 2020, 2020: 8253904.
41	Zhang NX, Guan C, Liu ZY, et al. Calycosin attenuates renal ischemia/reperfusion injury by suppressing NF‑κB mediated inflammation via PPARγ/EGR1 pathway[J]. Front Pharmacol, 2022, 13: 970616.
42	Alaaeldin R, Bakkar SM, Mohyeldin RH, et al. Azilsartan modulates HMGB1/NF-κB/p38/ERK1/2/JNK and apoptosis pathways during renal ischemia reperfusion injury[J]. Cells, 2023, 12(1): 185.
43	Ding HS, Huang Y, Qu JF, et al. Panaxynol ameliorates cardiac ischemia/reperfusion injury by suppressing NLRP3-induced pyroptosis and apoptosis via HMGB1/TLR4/NF-κB axis[J]. Int Immunopharmacol, 2023, 121: 110222.
44	Liu YY, Lei ZL, Chai H, et al. Salidroside alleviates hepatic ischemia-reperfusion injury during liver transplant in rat through regulating TLR-4/NF‑κB/NLRP3 inflammatory pathway[J]. Sci Rep, 2022, 12(1): 13973.
45	Li N, Zhou H, Wu HM, et al. STING-IRF3 contributes to lipopolysaccharide-induced cardiac dysfunction, inflammation, apoptosis and pyroptosis by activating NLRP3[J]. Redox Biol, 2019, 24: 101215.

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