益气清热消癥方调控LAMP-1/CTSB/NLRP3轴抑制糖尿病肾病肾小管上皮细胞焦亡的机制研究

doi:10.3969/j.issn.1006-7795.2026.01.016

摘要/Abstract

摘要： 目的探索益气清热消癥方对高糖（high glucose, HG）联合脂多糖（lipopolysaccharides，LPS）诱导肾小管上皮细胞焦亡的影响，揭示益气清热消癥方改善糖尿病肾病（diabetic kidney disease, DKD）状态下肾小管上皮细胞焦亡效应机制。方法以体外培养的人肾近曲小管上皮细胞系HK-2为研究对象，以HG联合LPS刺激，CCK-8法检测HK-2细胞活性以筛选最佳造模浓度，将细胞分为正常对照组、模型组和益气清热消癥方组，通过鬼笔环肽染色检测HK-2细胞骨架形态，Annexin V/PI双染流式细胞术检测细胞凋亡情况。免疫荧光法共标溶酶体相关膜蛋白-1（lysosomal associated membrane protein-1，LAMP-1）和组织蛋白酶B（cathepsin B， CTSB），Western blotting法检测含吡啶结构域的 NOD 样受体家族成员 3（NOD-like receptor family, pyrin domain containing 3，NLRP3）和含CARD 结构域的凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）的蛋白表达水平。并以溶酶体膜透剂LLOMe为对照药，酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）法检测细胞上清中炎性细胞因子白细胞介素-18（interleukin-18，IL-18）、白细胞介素-1β（interleukin-1β，IL-1β）蛋白表达情况，观察各组溶酶体膜通透性与细胞焦亡情况。结果 30 mmol/L葡萄糖联合10 μg/mL LPS刺激24 h，HK-2细胞活性受到抑制，益气清热消癥方干预使HG联合LPS刺激的肾小管上皮细胞活性提升。与正常对照组相比，HG联合LPS刺激的肾小管上皮细胞骨架褶皱呈现多边形，应力纤维破坏，细胞凋亡水平显著增加，溶酶体膜透化，CTSB释放入胞质中，焦亡相关的NLRP3、ASC蛋白表达上调，促炎细胞因子IL-18、IL-1β表达上调。与HG联合LPS模型组相比，益气清热消癥方干预后可以稳定HK-2细胞骨架，抑制肾小管上皮细胞焦亡。益气清热消癥方能够保护溶酶体膜，抑制溶酶体膜透化造成的CTSB释放入胞质，从而抑制CTSB诱导的焦亡相关NLRP3及ASC的蛋白表达，减少细胞上清中IL-1β和IL-18的释放。结论益气清热消癥方可以减轻DKD高糖状态造成的肾小管上皮细胞损伤，其效应机制可能是通过调控LAMP-1/CTSB/NLRP3轴抑制细胞焦亡实现的。

关键词: 糖尿病肾病, 溶酶体细胞, 焦亡, 炎性细胞因子, 肾小管上皮细胞

Abstract: Objective To investigate the effect of Yiqi Qingre Xiaozheng Formula on pyroptotic injury in renal tubular epithelial cells induced by high glucose (HG) combined with lipopolysaccharide (LPS), and to elucidate the underlying mechanisms by which the formula ameliorates tubular cell pyroptosis in the context of diabetic kidney disease (DKD). Methods Human renal proximal tubular epithelial cells (HK-2) were used for in vitro experiments. Pyroptosis was induced by stimulation with high glucose and lipopolysaccharide, and cell viability was assessed by using the CCK-8 assay to determine optimal modeling conditions. HK-2 cells were divided into control, model, and treatment groups (treated with Yiqi Qingre Xiaozheng Formula). Phalloidin staining was used to observe cytoskeletal morphology. Cell apoptosis was evaluated by Annexin V/PI double staining and flow cytometry. Immunofluorescence staining was performed to detect lysosomalc-associated membrane protein-1 (LAMP-1) and cathepsin B (CTSB). Western blotting was performed to assess the protein expression levels of key NOD-like receptor family, pyrin domain containing 3（NLRP3) and apoptosis-associated speck-like protein containing a CARD（ASC). Lysosomal membrane permeabilizer LLOMe was used as a positive control. Levels of interleukin-18 (IL-18) and IL-1β in the cell supernatant were quantified by ELISA to assess lysosomal membrane permeability and pyroptosis. Results Stimulation with 30 mmol/L HG and 10 μg/mL LPS for 24 h significantly reduced HK-2 cell viability, while treatment with Yiqi Qingre Xiaozheng Formula restored cell viability. Compared with the control group, HG+LPS treatment led to disorganized cytoskeletal morphology, increased apoptosis, lysosomal membrane permeabilization, cytoplasmic release of CTSB, and upregulation of pyroptosis-related proteins NLRP3 and ASC, as well as increased pro-inflammatory cytokines of IL-1β and IL-18. In contrast, the formula stabilized cytoskeletal structure, inhibited apoptosis, protected lysosomal membrane integrity, and reduced CTSB release into the cytoplasm. Consequently, the expression of NLRP3 and ASC was downregulated, and the levels of IL-1β and IL-18 in the supernatant were significantly decreased. Conclusion Yiqi Qingre Xiaozheng Formula mitigates high glucose-induced tubular cell injury in DKD. Its protective mechanism may be associated with inhibition of pyroptosis via modulation of the LAMP-1/CTSB/NLRP3 signaling axis.

Key words: diabetic kidney disease, lysosomes, pyroptosis, inflammatory cytokines, renal tubular epithelial cells

中图分类号:

田蕾, 刘伟敬, 赵文景, 王耀献. 益气清热消癥方调控LAMP-1/CTSB/NLRP3轴抑制糖尿病肾病肾小管上皮细胞焦亡的机制研究[J]. 首都医科大学学报, 2026, 47(1): 126-134.

Tian Lei, Liu Weijing, Zhao Wenjing, Wang Yaoxian. Yiqi Qingre Xiaozheng Formula inhibits tubular cell pyroptosis in diabetic kidney disease by modulating the LAMP-1/CTSB/NLRP3 axis[J]. Journal of Capital Medical University, 2026, 47(1): 126-134.

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