Dl-3-N-butylphthalide alleviates renal ischemia-reperfusion injury by down-regulating NF-κB signaling pathway and inhibiting cell pyroptosis in rat models
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摘要:
目的 探讨丁苯酞对大鼠肾缺血-再灌注损伤（IRI）的作用机制。 方法 将40只SD大鼠随机分为假手术组（Sham组）、模型组（IRI组）、NF-κB抑制剂吡咯烷二硫代氨基甲酸酯（PDTC）组、丁苯酞低剂量组（NBP-L组）及丁苯酞高剂量组（NBP-H组），每组8只。检测各组大鼠血清肌酐（Scr）、血清胱抑素C（Cys-C）、血尿素氮（BUN）和血清白细胞介素（IL）-1β、IL-18水平，苏木素-伊红（HE）染色观察各组肾组织病理损伤情况，采用蛋白质印迹法和免疫组织化学法检测肾组织中炎症因子、核因子（NF）-κB信号通路及细胞焦亡相关蛋白表达水平。 结果 与Sham组比较，IRI组肾组织损伤较为严重，Scr、Cys-C、BUN和血清IL-1β、IL-18水平均升高，蛋白质印迹法结果显示NOD样受体蛋白（NLRP3）、Gasdermin D（GSDMD）、半胱氨酸天冬氨酸蛋白酶（Caspase）-1、IL-18、IL-1β、NF-κB p65、p-NF-κB p65蛋白相对表达量均增加，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均增多。与IRI组比较，PDTC组、NBP-L组和NBP-H组肾组织的损伤程度均减轻，Scr、Cys-C、BUN和血清IL-18、IL-1β水平均下降，蛋白质印迹法结果显示NLRP3、GSDMD、Caspase-1、IL-1β、IL-18、NF-κB p65、p-NF-κB p65蛋白表达均减少，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均下降。与NBP-L组比较，NBP-H组肾组织的损伤程度减轻，Scr、Cys-C、BUN和血清IL-18、IL-1β水平均下降，蛋白质印迹法结果显示NLRP3、GSDMD、Caspase-1、IL-1β、IL-18、NF-κB p65、p-NF-κB p65蛋白表达均减少，免疫组织化学染色结果显示NF-κB p65、p-NF-κB p65、IL-1β、IL-18和NLRP3蛋白表达均下降。 结论 丁苯酞可下调NF-κB/NLRP3信号通路的活性，降低肾IRI后焦亡相关蛋白的表达水平及炎症因子水平，进而抑制细胞焦亡，减轻肾IRI。 -
关键词:
- 丁苯酞 /
- 缺血-再灌注损伤 /
- 急性肾损伤 /
- 细胞焦亡 /
- 炎症 /
- NOD样受体蛋白3（NLRP3） /
- 血清肌酐 /
- 血尿素氮 /
- 核因子（NF）-κB信号通路 /
- 血清胱抑素C（Cys-C） /
- 白细胞介素-1β /
- 白细胞介素-18
Abstract:Objective To elucidate the mechanism of dl-3-N-butylphthalide (NBP) on renal ischemia-reperfusion injury (IRI) in rat models. Methods Forty SD rats were randomly divided into the sham operation group (Sham group), model group (IRI group), NF-κB inhibitor pyrrolidine dithiocarbamate group (PDTC group), low-dose NBP group (NBP-L group) and high-dose NBP group (NBP-H group), with 8 rats in each group. Serum creatinine (Scr), serum cystatin C(Cys-C), blood urea nitrogen (BUN) and serum interleukin (IL)-1β and IL-18 levels were detected in all groups. Pathological injury of renal tissues in each group was observed by Hematoxylin-eosin (HE) staining. The expression levels of inflammatory factors and nuclear factor (NF)-κB signaling pathway and cell pyroptosis-related proteins in renal tissues were measured by Western blot and immunohistochemical staining. Results Compared with the Sham group, renal tissue injury was more severe, and the levels of Scr, Cys-C, BUN and serum IL-1β and IL-18 were all up-regulated in the IRI group. Western blot showed that the relative expression levels of NOD-like receptor protein (NLRP3), Gasdermin D(GSDMD), cysteinyl aspartate specific proteinase (Caspase)-1, IL-18, IL-1β, NF-κB p65 and p-NF-κB p65 proteins were all up-regulated, and immunohistochemical staining revealed that the expression levels of NF-κB p65 and p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were all up-regulated in the IRI group. Compared with the IRI group, renal tissue injury was alleviated, and the levels of Scr, Cys-C, BUN and serum IL-18 and IL-1β were down-regulated in the PDTC, NBP-L and NBP-H groups. Western blot showed that the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated, and immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the PDTC, NBP-L and NBP-H groups, respectively. Compared with the NBP-L group, renal tissue injury was mitigated, and the levels of Scr, Cys-C, BUN, serum IL-18 and IL-1β were all down-regulated in the NBP-H group. Western blot showed the expression levels of NLRP3, GSDMD, Caspase-1, IL-1β, IL-18, NF-κB p65 and p-NF-κB p65 proteins were down-regulated in the NBP-H group. Immunohistochemical staining indicated that the expression levels of NF-κB p65, p-NF-κB p65, IL-1β, IL-18 and NLRP3 proteins were down-regulated in the NBP-H group. Conclusions NBP may down-regulate the activity of NF-κB/NLRP3 signaling pathway and reduce the expression levels of cell pyroptosis-related proteins and inflammatory factors after renal IRI, thereby suppressing cell pyroptosis and alleviating renal IRI. -
Key words:
- Dl-3-N-butylphthalide /
- Ischemia-reperfusion injury /
- Acute renal injury /
- Cell pyroptosis /
- Inflammation /
- NOD-like receptor protein 3 (NLRP 3) /
- Serum creatinine /
- Blood urea nitrogen /
- Nuclear factor (NF)-κB signaling pathway /
- Serum cystatin C (Cys-C) /
- Interleukin-1β /
- Interleukin-18
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图 1 各组大鼠肾组织病理学和肾功能指标变化
注：A图为各组大鼠肾组织病理学改变（HE，×400）；B图为各组BUN、Scr和Cys-C水平，与Sham组比较，aP < 0.05，与IRI组比较，bP < 0.05，与NBP-L组比较，cP < 0.05。
Figure 1. Changes of renal histopathology and renal function indexes of rats in each group
图 2 各组大鼠血清及肾组织炎症因子水平
注：A图为各组大鼠血清IL-1β和IL-18水平，与Sham组比较，aP < 0.05，与IRI组比较，bP < 0.05，与NBP-L组比较，cP < 0.05；B图为蛋白质印迹法检测各组大鼠肾组织IL-1β和IL-18蛋白表达；C图为各组大鼠肾组织IL-1β和IL-18蛋白表达情况（免疫组化，×400）。
Figure 2. Levels of inflammatory factors in serum and kidney tissue of rats in each group
图 3 各组大鼠NF-κB信号通路及细胞焦亡相关蛋白的蛋白质印迹法结果
Figure 3. Results of Western blotting of NF-κB signaling pathway and pyroptosis related protein of rats in each group
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[1] ABDEL-RAZEK HA, RIZK MS, AMER GS, et al. Impact of combined ischemic preconditioning and melatonin on renal ischemia-reperfusion injury in rats[J]. Iran J Basic Med Sci, 2023, 26(2): 235-240. DOI: 10.22038/IJBMS.2022.67127.14722. [2] HUANG D, CHEN D, HU T, et al. GATA2 promotes oxidative stress to aggravate renal ischemia-reperfusion injury by up-regulating Redd1[J]. Mol Immunol, 2023, 153: 75-84. DOI: 10.1016/j.molimm.2022.09.012. [3] ZHANG B, WAN S, LIU H, et al. Naringenin alleviates renal ischemia reperfusion injury by suppressing ER stress-induced pyroptosis and apoptosis through activating Nrf2/HO-1 signaling pathway[J]. Oxid Med Cell Longev, 2022: 5992436. DOI: 10.1155/2022/5992436. [4] LAU A, RAHN JJ, CHAPPELLAZ M, et al. Dipeptidase-1 governs renal inflammation during ischemia reperfusion injury[J]. Sci Adv, 2022, 8(5): eabm0142. DOI: 10.1126/sciadv.abm0142. [5] LI T, SUN H, LI Y, et al. Downregulation of macrophage migration inhibitory factor attenuates NLRP3 inflammasome mediated pyroptosis in sepsis-induced AKI[J]. Cell Death Discov, 2022, 8(1): 61. DOI: 10.1038/s41420-022-00859-z. [6] LAN TY, DUN RL, YAO DS, et al. Effects of resveratrol on renal ischemia-reperfusion injury: a systematic review and meta-analysis[J]. Front Nutr, 2023, 9: 1064507. DOI: 10.3389/fnut.2022.1064507. [7] LUO Y, ZHU J, ZHAO F, et al. PIM1 attenuates renal ischemia-reperfusion injury by inhibiting ASK1-JNK/P38[J]. Int Immunopharmacol, 2023, 114: 109563. DOI: 10.1016/j.intimp.2022.109563. [8] GUO R, WANG H, CUI N. Autophagy regulation on pyroptosis: mechanism and medical implication in sepsis[J]. Mediators Inflamm, 2021: 9925059. DOI: 10.1155/2021/9925059. [9] 韩明伟, 马瑞雪, 刘梓谕, 等. 炎性小体在炎症性疾病中的研究进展[J]. 转化医学杂志, 2021, 10(2): 116-120. DOI: 10.3969/j.issn.2095-3097.2021.02.012.HAN MW, MA RX, LIU ZY, et al. Research progress of inflammasomes in inflammatory diseases[J]. Transl Med J, 2021, 10(2): 116-120. DOI: 10.3969/j.issn.2095-3097.2021.02.012. [10] HAN B, XU J, SHI X, et al. Dl-3-N-butylphthalide attenuates myocardial hypertrophy by targeting Gasdermin D and inhibiting Gasdermin D mediated inflammation[J]. Front Pharmacol, 2021, 12: 688140. DOI: 10.3389/fphar.2021.688140. [11] 肖海燕, 杨淑贤, 韩雯, 等. 丁苯酞及其衍生物药理学研究进展[J]. 中国中药杂志, 2022, 47(13): 3425-3431. DOI: 10.19540/j.cnki.cjcmm.20220117.701.XIAO HY, YANG SX, HAN W, et al. Research progress on pharmacology of butylphthalide and its derivatives[J]. China J Chin Mater Med, 2022, 47(13): 3425-3431. DOI: 10.19540/j.cnki.cjcmm.20220117.701. [12] DONG Y, YIN J, CHEN T, et al. Dl-3-N-butylphthalide pretreatment attenuates renal ischemia/reperfusion injury[J]. Biochem Biophys Res Commun, 2021, 557: 166-173. DOI: 10.1016/j.bbrc.2021.04.006. [13] HUANG Y, PAN L, WU T. Improvement of cerebral ischemia-reperfusion injury by L-3-N-butylphthalide through promoting angiogenesis[J]. Exp Brain Res, 2021, 239(1): 341-350. DOI: 10.1007/s00221-020-05978-6. [14] SHAN HUANG S, ZHOU B, XIAN ZENG G, et al. Neuroprotective effect and mechanism of butylphthalide after cerebral ischemia-reperfusion injury in rats[J]. Folia Neuropathol, 2021, 59(2): 131-142. DOI: 10.5114/fn.2021.107667. [15] ZHANG D, ZHENG N, FU X, et al. Dl-3-N-butylphthalide attenuates myocardial ischemia reperfusion injury by suppressing oxidative stress and regulating cardiac mitophagy via the PINK1/Parkin pathway in rats[J]. J Thorac Dis, 2022, 14(5): 1651-1662. DOI: 10.21037/jtd-22-585. [16] XIA W, LI Y, WU M, et al. Gasdermin E deficiency attenuates acute kidney injury by inhibiting pyroptosis and inflammation[J]. Cell Death Dis, 2021, 12(2): 139. DOI: 10.1038/s41419-021-03431-2. [17] NA SW, JANG YJ, HONG MH, et al. Protective effect of joa-gui em through the improvement of the NLRP3 and TLR4/NF-κB signaling by ischemia/reperfusion-induced acute renal failure rats[J]. Evid Based Complement Alternat Med, 2021: 7178868. DOI: 10.1155/2021/7178868. [18] 马卓奇, 敖娜, 杨楠, 等. NLRP3炎性小体与非酒精性脂肪性肝病的相关性及利拉鲁肽对其的影响[J]. 中华肝脏病杂志, 2022, 30(6): 624-630. DOI: 10.3760/cma.j.cn501113-20200326-00142.MA ZQ, AO N, YANG N, et al. Study of the effect of liraglutide on the correlation between NLRP3 inflammasome and non-alcoholic fatty liver disease[J]. Chin J Hepatol, 2022, 30(6): 624-630. DOI: 10.3760/cma.j.cn501113-20200326-00142. [19] CAI Q, SUN Z, XU S, et al. Disulfiram ameliorates ischemia/reperfusion-induced acute kidney injury by suppressing the Caspase-11-GSDMD pathway[J]. Ren Fail, 2022, 44(1): 1169-1181. DOI: 10.1080/0886022X.2022.2098764. [20] YIN F, ZHENG PQ, ZHAO LQ, et al. Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of pannexin1 in acute kidney disease[J]. Acta Pharmacol Sin, 2022, 43(1): 86-95. DOI: 10.1038/s41401-021-00619-2. [21] 张瑞波, 申开文, 袁强, 等. NF-κB相关信号通路在肾缺血-再灌注损伤中作用的研究进展[J]. 器官移植, 2022, 13(3): 349-355. DOI: 10.3969/j.issn.1674-7445.2022.03.011.ZHANG RB, SHEN KW, YUAN Q, et al. Research progress of NF-κB signaling pathway in kidney ischemia-reperfusion injury[J]. Organ Transplant, 2022, 13(3): 349-355. DOI: 10.3969/j.issn.1674-7445.2022.03.011. [22] SU X, LIU B, WANG S, et al. NLRP3 inflammasome: a potential therapeutic target to minimize renal ischemia/reperfusion injury during transplantation[J]. Transpl Immunol, 2022, 75: 101718. DOI: 10.1016/j.trim.2022.101718. [23] 袁强, 申开文, 张瑞波, 等. NLRP3炎症小体与相关炎症信号通路在肾缺血-再灌注损伤中的作用[J]. 器官移植, 2021, 12(2): 177-183. DOI: 10.3969/j.issn.1674-7445.2021.02.007.YUAN Q, SHEN KW, ZHANG RB, et al. Role of NLRP3 inflammasome and related inflammatory signaling pathways in renal ischemia-reperfusion injury[J]. Organ Transplant, 2021, 12(2): 177-183. DOI: 10.3969/j.issn.1674-7445.2021.02.007. [24] PANG Y, ZHANG PC, LU RR, et al. Andrade-oliveira salvianolic acid B modulates caspase-1-mediated pyroptosis in renal ischemia-reperfusion injury via Nrf2 pathway[J]. Front Pharmacol, 2020, 11: 541426. DOI: 10.3389/fphar.2020.541426. [25] LIU JJ, LU L, HU FQ, et al. Methylene blue attenuates renal ischemia-reperfusion injury by negative regulation of NLRP3 signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2018, 22(9): 2847-2853. DOI: 10.26355/eurrev_201805_14986. [26] SHIN JH, KIM SS, SEO SR. Pyrrolidine dithiocarbamate suppresses cutibacterium acnes-induced skin inflammation[J]. Int J Mol Sci, 2023, 24(5): 4444. DOI: 10.3390/ijms24054444. [27] XU J, LI S, YANG X, et al. Mechanism of nonylphenol induced gastric inflammation through NF-κB/NLRP3 signaling pathway[J]. Toxicology, 2022, 479: 153294. DOI: 10.1016/j.tox.2022.153294. [28] YU Y, ZHU Y, SUN X, et al. Dl-3-N-butylphthalide protects H9c2 cardiomyoblasts from ischemia/reperfusion injury by regulating HSP70 expression via PI3K/Akt pathway activation[J]. Exp Ther Med, 2021, 22(3): 1008. DOI: 10.3892/etm.2021.10441. [29] LIU CY, ZHAO ZH, CHEN ZT, et al. Dl-3-N-butylphthalide protects endothelial cells against advanced glycation end product-induced injury by attenuating oxidative stress and inflammation responses[J]. Exp Ther Med, 2017, 14(3): 2241-2248. DOI: 10.3892/etm.2017.4784. [30] LIAO D, XIANG D, DANG R, et al. Neuroprotective effects of dl-3-N-butylphthalide against doxorubicin-induced neuroinflammation, oxidative stress, endoplasmic reticulum stress, and behavioral changes[J]. Oxid Med Cell Longev, 2018: 9125601. DOI: 10.1155/2018/9125601. [31] HAN J, SHI X, XU J, et al. Dl-3-N-butylphthalide prevents oxidative stress and atherosclerosis by targeting Keap-1 and inhibiting Keap-1/Nrf-2 interaction[J]. Eur J Pharm Sci, 2022, 172: 106164. DOI: 10.1016/j.ejps.2022.106164. [32] ZHAO S, LIU F, SHI W, et al. Dl-3-N-butylphthalide promotes hippocampal neurogenesis and reduces mossy fiber sprouting in chronic temporal lobe epilepsy rats[J]. BMC Neurol, 2022, 22(1): 3. DOI: 10.1186/s12883-021-02516-x. [33] GAO L, GUO X, LIU S, et al. Dl-3-N-butylphthalide imparts neuroprotection via Nrf2/SIRT3 pathway in a mouse model of vascular dementia[J]. Brain Res, 2022, 1779: 147785. DOI: 10.1016/j.brainres.2022.147785. [34] WANG J, LI Y, YU H, et al. Dl-3-N-butylphthalide promotes angiogenesis in an optimized model of transient ischemic attack in C57BL/6 mice[J]. Front Pharmacol, 2021, 12: 751397. DOI: 10.3389/fphar.2021.751397. [35] SUN H, WANG J, BI W, et al. Dl-3-N-butylphthalide (NBP) mitigates muscular injury induced by limb ischemia/reperfusion in mice through the HMGB1/TLR4/NF-κB pathway[J]. Evid Based Complement Alternat Med, 2022: 5556067. DOI: 10.1155/2022/5556067. -
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