参芪糖肾安胶囊含药血清对脂多糖损伤的肾小管上皮细胞活性、超微结构及NLR蛋白3炎性小体的作用机制

张瑞方; 高峰; 杨薇; 李娟; 张轶欧; 姚吉太; 程刚

doi:10.7619/jcmp.20232072

参芪糖肾安胶囊含药血清对脂多糖损伤的肾小管上皮细胞活性、超微结构及NLR蛋白3炎性小体的作用机制

山西省中医院肾病三科, 山西太原, 030012

基金项目:

山西省中医药管理局科研课题计划 2022ZYYC004

详细信息

通讯作者:
高峰, E-mail: 13935118128@163.com

中图分类号: R692;R334
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- 文章访问数: 147
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出版历程
- 收稿日期: 2023-06-28
- 修回日期: 2023-08-30
- 网络出版日期: 2023-11-05

Mechanism of Shenqitang Shen'an Capsule containing serum on activity and ultrastructure of renal tubular epithelial cells as well as NLR family pyrin domain containing 3 inflammasome injured by lipopolysaccharide

the Third Department of Nephrology, Shanxi Provincial Hospital of Traditional Chinese Medicine, Taiyuan, Shanxi, 030012

摘要

摘要:
目的
探讨参芪糖肾安胶囊含药血清对脂多糖(LPS)损伤的肾小管上皮细胞活性、超微结构及NLR蛋白3(NLRP3)炎性小体的影响。
方法
随机将HK-2细胞分为空白组(HK-2+75 μL 7.5%空白血清)、LPS组(HK-2+LPS+75 μL 7.5%空白血清)、低浓度组(HK-2+LPS+75 μL 2.5%含药血清+75 μL 7.5%空白血清)、中浓度组(HK-2+LPS+75 μL 5%含药血清+75 μL 5%空白血清)、高浓度组(HK-2+LPS+150 μL 10%含药血清)。采用CCK-8法检测细胞存活率; 采用流式细胞仪检测细胞凋亡率; 采用酶联免疫吸附试验(ELISA)检测细胞中白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)水平; 采用电镜检测细胞的超微结构; 采用蛋白质印迹检测细胞中NLRP3和caspase-1蛋白表达。
结果
与空白组相比, LPS组细胞存活率降低，凋亡率及细胞中IL-6、IL-1β、TNF-α水平增高，差异有统计学意义(P < 0.05); 与LPS组相比，低浓度组、中浓度组和高浓度组细胞存活率均增高，凋亡率及细胞中IL-6、IL-1β、TNF-α水平均降低，差异有统计学意义(P < 0.05)。LPS组较空白组出现明显的程序性坏死，细胞体积变大，胞膜大量破裂，细胞大量坏死; 与LPS组相比，低浓度组、中浓度组和高浓度组细胞均有明显改善，坏死的细胞数量减少，仅可见少量细胞线粒体轻度肿胀和空泡样变，且呈剂量依赖性。与空白组相比，LPS组细胞中NLRP3和caspase-1蛋白表达升高，差异有统计学意义(P < 0.05); 与LPS组相比，低浓度组、中浓度组和高浓度组细胞中NLRP3和caspase-1蛋白表达降低，且高浓度组细胞中NLRP3和caspase-1蛋白表达低于低浓度组和中浓度组，差异有统计学意义(P < 0.05)。
结论
参芪糖肾安胶囊含药血清可抑制LPS损伤的肾小管上皮细胞凋亡，增加细胞存活率，改善超微结构，抑制NLRP3炎性小体介导的炎性反应，对急性肾损伤发挥保护作用。
- 参芪糖肾安胶囊含药血清 /
- 脂多糖 /
- 肾小管上皮细胞 /
- 超微结构 /
- NLRP3炎性小体
Abstract:
Objective
To investigate the effects of Shenqitang Shen'an Capsule containing serum on the activity, ultrastructure and NLR family pyrin domain containing 3 (NLRP3) inflammatory vesicles in lipopolysaccharide (LPS)-injured renal tubular epithelial cells.
Methods
HK-2 cells were randomly divided into blank group (HK-2+75 μL 7.5% blank serum), LPS group (HK-2+LPS+75 μL 7.5% blank serum), low concentration group (HK-2+LPS+75 μL 2.5% drug-containing serum+75 μL 7.5% blank serum), medium concentration group (HK-2+LPS+75 μL 5% drug-containing serum+75 μL 5% blank serum), and high concentration group (HK-2+LPS+150 μL 10% drug-containing serum). The cell survival rate was detected by CCK-8; the apoptosis rate was detected by flow cytometry; the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA); the ultrastructure of the cells was detected by electron microscopy; the expression of NLRP3 and caspase-1 protein was detected by protein blotting.
Results
Compared with the blank group, the cell survival rate was reduced, and apoptosis rate, the level of IL-6, IL-1β, and TNF-α in the cells of the LPS group were significantly decreased (P < 0.05). The cell survival rates in the low concentration group, medium concentration group, and high concentration group were higher than those in the LPS group, while the apoptosis rate and the levels of IL-6, IL-1β, and TNF-α in the cells were lower than those in the LPS group (P < 0.05). Compared with the blank group, LPS group showed obvious programmed necrosis, cell volume increased, cell membrane ruptured and cell necrosis. Compared with LPS group, the cells in the low concentration group, medium concentration group and high concentration group were significantly improved, the number of necrotic cells was reduced, and only a few cell mitochondria were slightly swollen and vacuole-like changes were observed in a dose-dependent manner. Compared with the blank group, the protein expression of NLRP3 and caspase-1 in the cells of the LPS group was significantly increased (P < 0.05). The protein expressions of NLRP3 and caspase-1 in the cells of the low concentration group, medium concentration group, and high concentration group were significantly lower than those in the LPS group (P < 0.05), and the protein expressions of NLRP3 and caspase-1 in the cells of the high concentration group were significantly lower than those in the low concentration group and medium concentration group (P < 0.05).
Conclusion
Shenqitang Shen'an Capsule containing serum can inhibit apoptosis of LPS-injured renal tubular epithelial cells, increase cell survival rate, improve ultrastructure, inhibit NLRP3 inflammatory vesicle-mediated inflammatory response, and exert protective effects on AKI.
- Shenqitang Shen'an Capsule containing serum /
- lipopolysaccharide /
- renal tubular epithelial cells /
- ultrastructure /
- NLRP3 inflammatory vesicles

HTML全文

图 1 各组细胞存活率比较

A: 不同浓度含药血清细胞存活率比较，与LPS比较, *P < 0.05; B: 各组细胞存活率比较，与空白组比较, *P < 0.05; 与LPS组比较, #P < 0.05; 与低浓度组比较, △P < 0.05; 与中浓度组比较, ▲P < 0.05。

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图 2 各组细胞凋亡率比较

与空白组比较, *P < 0.05; 与LPS组比较, #P < 0.05; 与低浓度组比较, △P < 0.05; 与中浓度组比较, ▲P < 0.05。

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图 3 各组细胞中IL-6、IL-1β、TNF-α水平比较

A: 各组IL-6水平比较; B: 各组IL-1β水平比较; C: 各组TNF-α水平比较。与空白组比较, *P < 0.05; 与LPS组比较, #P < 0.05; 与低浓度组比较, △P < 0.05; 与中浓度组比较, ▲P < 0.05。

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图 4 各组细胞超微结构比较(透射电镜，放大20 000倍，标尺5 μm)

红色箭头为坏死细胞; 橙色箭头为空泡样变。

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图 5 各组细胞中NLRP3、caspase-1蛋白表达比较

A: 各组蛋白条带; B: 各组NLRP3蛋白表达; C: 各组caspase-1蛋白表达。与空白组比较, *P < 0.05; 与LPS组比较, #P < 0.05; 与低浓度组比较, △P < 0.05; 与中浓度组比较, ▲P < 0.05。

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