| 熊小春.散瘀止痛方含药血清对椎间盘退行性变大鼠髓核细胞焦亡的影响[J].浙江中西医结合杂志,2025,35(3): |
| 散瘀止痛方含药血清对椎间盘退行性变大鼠髓核细胞焦亡的影响 |
| Effect of Sanyu Zhitong decoction containing serum on pyroptosis of nucleus pulposus cells in rats with intervertebral disc degeneration |
| 投稿时间：2024-05-17 修订日期：2025-01-24 |
| DOI： |
| 中文关键词: 椎间盘退变 散瘀止痛方 含药血清 髓核细胞 细胞焦亡 |
| 英文关键词:Intervertebral disc degeneration, Sanyu Zhitong Decoction medicated serum nucleus pulposus cells pyroptosis. |
| 基金项目:浙江省中医药科技计划项目(2022ZA181)；舟山市科学技术局,编号2024C31018,神经干细胞联合血旺氏细胞移植治疗脊髓损伤的实验研究。 |
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| 中文摘要: |
| 目的：研究散瘀止痛方含药血清对过氧化氢诱导大鼠髓核细胞焦亡的影响。
方法：12周龄SD大鼠以散瘀止痛方灌胃，提取含药血清。另从SD大鼠分离提取髓核细胞，采用甲苯胺蓝染色和II型胶原免疫组化染色进行鉴定。将髓核细胞随机分为空白组、模型组及含药血清低/中/高剂量组。空白组使用正常完全培养基；模型组使用200μM过氧化氢培养24小时；含药血清低、中、高剂量组分别用5%，10%，20%散瘀止痛方含药血清预孵育24h，随后200μM过氧化氢处理。采用细胞计数试剂盒8（CCK-8）测定细胞活力；使用免疫荧光法检测细胞中GSDMD表达情况；使用WB检测GSDMD、Caspase-1、IL-1β、NLRP3、MMP-3/13、ADAMTS-4/5、Aggrecan蛋白的表达情况；使用酶联免疫反应（ELISA）检测细胞中IL-1β表达水平；使用相应试剂盒测定细胞中LDH、ROS、MDA、GSH-Px、SOD表达情况。结果：与对照组相比，模型组髓核细胞活性显著降低，焦亡水平显著上升，IL-1β和LDH表达显著上升；炎性小体活化相关蛋白蛋白表达显著上升；ROS和MDA水平显著上升，GSH-Px和SOD水平显著下降；细胞外基质降解相关蛋白表达显著上升，Collagen Ⅱ和Aggrecan蛋白显著下降。与模型组相比，散瘀止痛方含药血清低/中/高剂量组髓核细胞活性显著上升，焦亡水平显著下降，IL-1β和LDH表达显著下降；炎性小体活化相关蛋白表达被显著抑制；ROS和MDA水平显著下降，GSH-Px和SOD水平显著上升；细胞外基质降解相关蛋白表达显著抑制，Collagen Ⅱ和Aggrecan蛋白显著上升，且呈剂量依赖。结论：散瘀止痛方含药血清在体外条件下能显著抑制过氧化氢诱导大鼠髓核细胞的焦亡、炎症小体活化、氧化应激以及细胞外基质降解。 |
| 英文摘要: |
| Objective:? To study the effect of Sanyu Zhitong decoction on pyroptosis of nucleus pulposus cells induced by hydrogen peroxide in rats.Methods: SD rats at 12 weeks of age were given Sanyu Zhitong decoction by intragastric administration to extract drug-containing serum. Nucleus pulposus cells were isolated from SD rats and identified by toluidine blue staining and type II collagen immunohistochemical staining. Nucleus pulposus cells were randomly divided into blank group, model group and drug-containing serum low/medium/high dose group. Normal complete medium was used in blank group. The model group was cultured with 200μM hydrogen peroxide for 24 hours. The low, medium and high dose groups were preincubated with 5%, 10% and 20% Sanyu Zhitong decoction drug-containing serum for 24h, respectively, and then treated with 200μM hydrogen peroxide. Cell viability was measured with cell counting kit 8 (CCK-8). The expression of GSDMD was detected by immunofluorescence assay. WB was used to detect the expression of GSDMD, Caspase-1, IL-1β, NLRP3, MMP-3/13, ADAMTS-4/5 and Aggrecan. The expression level of IL-1β was detected by enzyme-linked immunoassay (ELISA). The expressions of LDH, ROS, MDA, GSH-Px and SOD in the cells were determined by the corresponding kit. Results: Compared with the control group, the activity of nucleus pulposus cells in model group was significantly decreased, the level of pyrodeath was significantly increased, and the expressions of IL-1β and LDH were significantly increased. The expression of inflammasome activation-related proteins increased significantly. The levels of ROS and MDA increased significantly, while the levels of GSH-Px and SOD decreased significantly. The expressions of extracellular matrix degradation related proteins were significantly increased, while the proteins of Collagen II and Aggrecan were significantly decreased. Compared with the model group, the activity of nucleus pulposus cells and the expression of IL-1β and LDH were significantly decreased in the low/medium/high dose of serum containing SBUzhitong prescription. The expression of proteins related to inflammasome activation was significantly inhibited. The levels of ROS and MDA decreased significantly, while the levels of GSH-Px and SOD increased significantly. Extracellular matrix degradation related protein expression was significantly inhibited, Collagen II and Aggrecan proteins were significantly increased in a dose-dependent manner. Conclusion: The serum containing Sanyu Zhitong decoction can significantly inhibit the pyroptosis, the activation of inflammatory bodies, oxidative stress and extracellular matrix degradation of nucleus pulposus cells induced by hydrogen peroxide in vitro. |
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