活性氧簇/p38丝裂原活化蛋白激酶级联反应对大鼠肾结石形成的影响及机制

谢亚彬; 王飞

doi:10.16098/j.issn.0529-1356.2024.05.012

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解剖学报 ›› 2024, Vol. 55 ›› Issue (5) : 604-611. DOI: 10.16098/j.issn.0529-1356.2024.05.012

组织学胚胎学发育生物学

活性氧簇/p38丝裂原活化蛋白激酶级联反应对大鼠肾结石形成的影响及机制

谢亚彬王飞* 王康扬林师帅

作者信息 +

Effect and mechanism of reactive oxygen species/p38 mitogen-activated protein kinase cascade on renal stone formation in rats

XIE Ya-bin WANG Fei* WANG Kang-yang LIN Shi-shuai

Author information +

文章历史 +

摘要

目的探讨活性氧簇（ROS）/p38 MAPK级联反应对大鼠肾结石（KS）形成的影响及机制。方法 50只SD大鼠随机分为对照组（正常喂养）、N-乙酰半胱氨酸（NAC）组（腹腔注射200 mg/kg NAC）、KS组（构建草酸钙KS模型）、KS+NAC组（构建草酸钙KS模型后腹腔注射200 mg/kg NAC）、KS+NAC+衣霉素（TM）组（构建草酸钙KS模型后腹腔注射200 mg/kg NAC与1 mg/kg TM），每组10只。给药结束4周后，测量大鼠24 h尿量与尿草酸（Ox），全自动生化仪检测血清肌酐（Cr）、尿素氮（BUN）、尿酸（UA）水平，HE染色和Von Kossa染色观察肾组织病理学变化及晶体沉积情况，TUNEL染色检测肾组织细胞凋亡，试剂盒测定肾组织超氧化物歧化酶（SOD）活性与丙二醛（MDA）含量，二氢乙锭（DHE）荧光探针检测肾组织ROS水平，免疫组织化学染色检测肾组织微管相关蛋白1轻链3B（LC3B）与葡萄糖调节蛋白78（GRP78）的表达，Western blotting测定肾组织p-p38 MAPK/p38 MAPK与LC3Ⅱ/LC3Ⅰ、Beclin1、GRP78和CCAAT/增强子结合蛋白同源蛋白（CHOP）水平。结果与KS组比较，KS+NAC组Ox、血清BUN、Cr、UA水平降低(P<0.05），肾小管扩张程度减轻，草酸钙结晶减少，TUNEL阳性细胞率减少（P<0.05），SOD活性升高，MDA含量减少（P<0.05），ROS水平降低（P<0.05），LC3B与GRP78阳性染色水平降低（P<0.05），p-p38 MAPK/p38 MAPK、LC3Ⅱ/LC3Ⅰ、Beclin1、GRP78及CHOP蛋白相对表达量均下调（P<0.05）；与KS+NAC组比较，KS+NAC+TM组Ox、血清BUN、Cr、UA水平升高（P<0.05），肾小管扩张明显、草酸钙结晶增多，TUNEL阳性细胞率增加（P<0.05），SOD活性降低而MDA含量增加（P<0.05），ROS水平升高（P<0.05），LC3B与GRP78阳性染色水平升高（P<0.05），同时，p-p38 MAPK/p38 MAPK、LC3Ⅱ/LC3Ⅰ、Beclin1、GRP78、CHOP蛋白相对表达量均上调（P<0.05）。结论 ROS/p38 MAPK级联反应参与大鼠KS形成，其作用与其激活内质网应激介导的自噬途径有关。

Abstract

Objective To study the effect of reactive oxygen species (ROS)/p38 MAPK cascade reaction on the formation of kidney stones (KS) in rats and explore the mechanism. Methods Fifty SD rats were randomly divided into control group (normal feeding), N-acetylcysteine (NAC) group (intraperitoneally injected 200 mg/kg NAC), KS group (constructed calcium oxalate KS model), KS+NAC group (constructed calcium oxalate KS model, intraperitoneally injected 200 mg/kg NAC), KS+NAC+tunicamycin (TM) group(constructed calcium oxalate KS model, intraperitoneally injected 200 mg/kg NAC and 1 mg/kg TM), with 10 rats in each group. After 4 weeks of administration, 24 hours urine volume and oxalic acid (Ox) of each group were measured, serum creatinine (Cr), urea nitrogen (BUN) and uric acid (UA) levels were detected by automatic biochemical analyzer. HE staining and Von Kossa staining were used to observe the histopathological changes and crystal deposition of the kidney. TUNEL staining was used to detect apoptosis of renal tissue cells. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in renal tissue were measured by the kit. DHE fluorescent probes detected the levels of reactive oxygen species (ROS) in kidney tissue. Immunohistochemical staining was used to detect the expressions of microtubule-associated protein 1 light chain 3 B (LC3B) and glucose regulatory protein 78 (GRP78) in renal tissue, and the protein expression of LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78, CCAAT/enhancer binding protein homologous protein (CHOP) in renal tissue was determined by Western blotting. Results Compared with the KS group, Ox in KS+NAC group decreased (P<0.05), BUN, Cr and UA levels decreased (P<0.05), renal tubule dilatation and calcium oxalate crystallization decreased, TUNEL positive cell rate decreased (P<0.05), SOD activity increased and MDA content decreased (P<0.05), ROS levels decreased (P<0.05), LC3B and GRP78 positive staining levels decreased (P<0.05), the relative protein expressions of p-p38 MAPK/p38 MAPK, LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78 and CHOP were down-regulated(P<0.05). Compared with the KS+NAC group, Ox in KS+NAC+TM group increased (P<0.05), BUN, Cr and UA levels also increased (P<0.05), renal tubule dilated significantly, calcium oxalate crystals increased, TUNEL positive cell rate increased (P<0.05), SOD activity decreased and MDA content increased (P<0.05), ROS levels increased (P<0.05), LC3B and GRP78 positive staining levels increased (P<0.05), the relative protein expressions of p-p38 MAPK/p38 MAPK, LC3Ⅱ/LC3Ⅰ, Beclin1, GRP78 and CHOP were also up-regulated (P<0.05). Conclusion ROS/p38 MAPK cascade is involved in promoting KS formation in rats, which is related to the activation of endoplasmic reticulum stress-mediated autophagy pathway.

导出引用

谢亚彬王飞王康扬林师帅. 活性氧簇/p38丝裂原活化蛋白激酶级联反应对大鼠肾结石形成的影响及机制[J]. 解剖学报. 2024, 55(5): 604-611 https://doi.org/10.16098/j.issn.0529-1356.2024.05.012

XIE Ya-bin WANG Fei WANG Kang-yang LIN Shi-shuai. Effect and mechanism of reactive oxygen species/p38 mitogen-activated protein kinase cascade on renal stone formation in rats[J]. Acta Anatomica Sinica. 2024, 55(5): 604-611 https://doi.org/10.16098/j.issn.0529-1356.2024.05.012

中图分类号： R692.4

参考文献

［1］ Pavlakou P, Dounousi E, Roumeliotis S, et al. Oxidative stress and the kidney in the space environment［J］. Int J Mol Sci, 2018, 19(10):3176.

［2］ Tormos AM, Taléns-Visconti R, Nebreda AR, et al. p38 MAPK: a dual role in hepatocyte proliferation through reactive oxygen species［J］. Free Radic Res, 2013, 47(11):905-916.

［3］ Zhang ZhY, Zuo QJ, Song X, et al. Study on the effect and mechanism of p38 MAPK pathway mediated by pulmonium extract on calcium oxalate stone formation in rats ［J］. Journal of Sichuan of Traditional Chinese Medicine,2022,40(10):55-58. (in Chinese)

张智源,左庆军,宋旭,等.金钱草提取物介导P38MAPK通路对大鼠草酸钙结石形成的干预效果及机制研究［J］.四川中医,2022,40(10):55-58.

［4］ Liu Y, Chen S, Liu J, et al. Telmisartan inhibits oxalate and calcium oxalate crystal-induced epithelial-mesenchymal transformation via PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway ［J］. Life Sci, 2020, 241:117108.

［5］ Ge MY，Zhai XY. Study on effect and mechanism of Miniao Paishi Mixture on formation of calcium oxalate kidney stones in rats［J］. Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(S1):187-191. (in Chinese)

葛旻垚,翟新宇.泌尿排石合剂对大鼠草酸钙肾结石形成的影响及机制研究［J］.上海中医药大学学报,2022,36(S1):187-191.

［6］ Liang XF, Lu XG, Chen D, et al. Research progress of calcium oxalate stone formation induced by renal tubular epithelial cell injury ［J/OL］. Chinese Journal of Endourology (Electronic Edition),2018,12(4):281-283. (in Chinese)

梁雄发,卢小刚,陈东,等.肾小管上皮细胞损伤促进草酸钙结石形成的研究进展［J/OL］.中华腔镜泌尿外科杂志(电子版),2018,12(4):281-283.

［7］ Wu Y, Zhang J, Li C, et al. The activation of ROS/NF-κB/MMP-9 pathway promotes calcium-induced kidney crystal deposition［J］. Oxid Med Cell Longev, 2021, 2021:8836355.

［8］ Liu Y, Sun Y, Kang J, et al. Role of ROS-induced NLRP3 inflammasome activation in the formation of calcium oxalate nephrolithiasis［J］. Front Immunol, 2022, 13:818625.

［9］ Albert A, Tiwari V, Paul E, et al. Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease［J］. J Enzyme Inhib Med Chem, 2017, 32(1):426-433.

［10］ Halasi M, Wang M, Chavan TS, et al. ROS inhibitor N-acetyl-L-cysteine antagonizes the activity of proteasome inhibitors［J］. Biochem J, 2013, 454(2):201-208.

［11］ Liu J, Liu Q, Han J, et al. N-Acetylcysteine inhibits patulin-induced apoptosis by affecting ROS-mediated oxidative damage pathway［J］. Toxins (Basel), 2021, 13(9):595.

［12］ Xiong W, Chen H, Lu J, et al. IL-39 increases ROS production and promotes the phosphorylation of p38 MAPK in the apoptotic cardiomyocytes［J］. Folia Histochem Cytobiol, 2021, 59(3):195-202.

［13］ Li C, Chai X, Pan J, et al. β-Hydroxybutyrate alleviates low glucose-induced apoptosis via modulation of ROS-mediated p38 MAPK signaling［J］. J Mol Neurosci, 2022, 72(5):923-938.

［14］ Sharma M, Naura AS, Singla SK. A deleterious interplay between endoplasmic reticulum stress and its functional linkage to mitochondria in nephrolithiasis［J］. Free Radic Biol Med, 2021, 168:70-80.

［15］ Kang J, Sun Y, Deng Y, et al. Autophagy-endoplasmic reticulum stress inhibition mechanism of superoxide dismutase in the formation of calcium oxalate kidney stones［J］. Biomed Pharmacother, 2020, 121:109649.

［16］ Li H, Zhou Y, Xu W, et al. The role of autophagy in calcium oxalate kidney stone: a systematic review of the literature［J］. Front Physiol, 2022, 13:1008264.