Triptolide inhibits ferroptosis and improves cerebral ischemia-reperfusion injury in a rat model of cerebral artery occlusion/reperfusion

doi:10.12307/2025.992

Abstract

Abstract: BACKGROUND: Triptolide, a bioactive component of the traditional Chinese medicine Tripterygium wilfordii, has a certain protective effect on neurons.
OBJECTIVE: To investigate the effect of triptolide on cerebral ischemia/reperfusion injury.
METHODS: (1) Cell experiment: Hippocampal neurons (HT22 cells) were randomly divided into control group, glucose oxygen deprivation/reoxygenation (OGD/R) group, OGD/R+triptolide group, OGD/R+triptolide+si-TIGAR group, OGD/R+si-TIGAR group, and OGD/R+triptolide+rapamycin group. HT22 cell viability was detected by cell counting kit 8. Tp53-induced glycolysis and apoptosis factors, glutathione peroxidase 4, 7 members of the solsolic vector family 11, sphingosine kinase 1 (SPHK1) and (mTOR) were detected by western blot assay. Glutathione, malondialdehyde and iron level were detected using the biochemical kit. (2) Animal experiment: Rats were randomly divided into sham surgery group, model group, and triptolide group. Cerebral artery occlusion/reperfusion rat models were prepared in the latter two groups. Rats in the triptolide group were orally administered 50 mg/kg triptolide for 7 days. Twenty-four hours after administration, LONGA method was used to evaluate the neurological impairment of rats, TTC method was used to observe the conditions of cerebral infarction, TUNEL staining was used to detect cell apoptosis, and western blot was performed to detect the expression level of related proteins.
RESULTS AND CONCLUSION: (1) At the cellular level, triptolide promoted cell viability and inhibited apoptosis in HT22 cells treated with OGD/R. Triptolide also increased the expression levels of Tp53-induced glycolysis and apoptosis factors, glutathione peroxidase 4, and 7 members of the solsolic vector family 11, activated the SPHK1/mTOR pathway, increased glutathione content, inhibited malondialdehyde content and iron levels. Rapamycin treatment counteracted the protective effect of triptolide on HT22 cells. (2) At the animal level, triptolide significantly reduced neurological deficits, infarct volume, and cell apoptosis, and inhibited neuronal ferroptosis in brain tissue of rats. To conclude, triptolide can inhibit ferroptosis by upregulating the expression level of Tp53-induced glycolysis and apoptosis factors and activating the SPHK1/mTOR signaling, and thereby reduced cerebral ischemia/reperfusion injury. These findings suggest that triptolide may be a candidate drug for the treatment of cerebral ischemia/reperfusion injury.

Key words: triptolide, ferroptosis, TIGAR, mammalian target of rapamycin, cerebral ischemia/reperfusion injury, engineered tissue construction

CLC Number:

Zou Rongji, Yu Fangfang, Wang Maolin, Jia Zhuopeng. Triptolide inhibits ferroptosis and improves cerebral ischemia-reperfusion injury in a rat model of cerebral artery occlusion/reperfusion[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(4): 873-881.

Figures/Tables 6

2.1 实验动物数量分析实验最终选用造模成功及假手术组共60只SD大鼠，均无脱失，以每组20只进入结果分析。 2.2 雷公藤内酯酮提高了体外神经元细胞活力并抑制凋亡 CCK-8法检测雷公藤内酯酮作用于HT22细胞的最佳浓度，与未添加雷公藤内酯酮组相比，10 nmol/L和20 nmol/L的雷公藤内酯酮均能显著抑制HT22细胞活力(P < 0.01)，而2.5 nmol/L和5 nmol/L雷公藤内酯酮对HT22细胞活力没有影响，见图1A。故为了排除药物毒性影响，后续研究使用5 nmol/L雷公藤内酯酮处理HT22细胞。与对照组比较，糖氧剥夺/复氧模型组中HT22细胞活力显著降低(P < 0.01)；而相较于糖氧剥夺/复氧组，5 nmol/L雷公藤内酯酮可显著增加HT22细胞活力(P < 0.05)，见图1B。流式细胞术分析结果显示糖氧剥夺/复氧模型组比对照组凋亡细胞比例显著升高(P < 0.01)，而在应用雷公藤内酯酮后凋亡细胞比例较糖氧剥夺/复氧组显著下降(P < 0.01)，见图1C，D。结果表明，雷公藤内酯酮能恢复HT22细胞活力，抑制糖氧剥夺/复氧诱导的HT22细胞凋亡。 2.3 雷公藤内酯酮激活TIGAR抑制HT22细胞铁死亡 CCK-8结果显示，与单独雷公藤内酯酮组比较，TIGAR敲低后HT22细胞活力显著降低(P < 0.01)，见图2A。如图2B-I所示，Western blotting检测结果显示，与对照组相比，糖氧剥夺/复氧处理组TIGAR、GPX4和xCT表达水平降低(P < 0.01)，谷胱甘肽表达水平降低，丙二醛以及亚铁离子水平升高(P < 0.01)。与糖氧剥夺/复氧处理组比较，糖氧剥夺/复氧+雷公藤内酯酮组的TIGAR、GPX4和xCT表达水平升高(P < 0.01)，谷胱甘肽表达水平上升，丙二醛及亚铁离子表达水平下降(P < 0.01)。与糖氧剥夺/复氧+雷公藤内酯酮组相比，加入TIGAR-siRNA组GPX4和xCT的表达水平显著降低(P < 0.01)，谷胱甘肽的表达降低(P < 0.01)，丙二醛及亚铁离子水平升高(P < 0.01)。结果提示，雷公藤内酯酮能有效抑制糖氧剥夺/复氧条件下的HT22细胞铁死亡，其抑制效果依赖其对TIGAR的激活。 2.4 雷公藤内酯酮通过TIGAR激活HT22细胞中SPHK1/mTOR通路如图3所示，相较于对照组，糖氧剥夺/复氧处理后SPHK1蛋白表达显著降低(P < 0.01)，mTOR磷酸化水平显著降低(P < 0.01)；糖氧剥夺/复氧+雷公藤内酯酮组与糖氧剥夺/复氧模型组对比，SPHK1蛋白表达水平及mTOR磷酸化水平均显著升高(P < 0.01)；而敲低TIGAR后，与糖氧剥夺/复氧+雷公藤内酯酮比较，糖氧剥夺/复氧+雷公藤内酯酮+si-TIGAR组中SPHK1蛋白表达水平和mTOR磷酸化水平也均显著降低；与糖氧剥夺/复氧+雷公藤内酯酮+si-TIGAR组比较，糖氧剥夺/复氧+si-TIGAR组中SPHK1蛋白表达再次降低，mTOR磷酸化水平也显著降低(P < 0.05)。以上结果提示，雷公藤内酯酮能够通过上调TIGAR表达激活SPHK1/mTOR通路。 2.5 雷公藤内酯酮通过TIGAR/mTOR抑制HT22细胞铁死亡如图4所示，应用雷公藤内酯酮和mTOR抑制剂雷帕霉素，共同处理糖氧剥夺/复氧条件下的HT22细胞。Western blotting检测结果显示，相较于糖氧剥夺/复氧组，糖氧剥夺/复氧+雷公藤内酯酮组中SPHK1的表达和mTOR磷酸化水平显著升高，而当雷帕霉素和雷公藤内酯酮共同处理HT22细胞后与单独雷公藤内酯酮组比较，SPHK1表达显著降低(P < 0.01)，mTOR磷酸化水平也显著下降(P < 0.01)。此外，Western blotting检测结果显示相较于糖氧剥夺/复氧组，糖氧剥夺/复氧+雷公藤内酯酮组中GPX4和xCT表达水平均显著升高；而与糖氧剥夺/复氧+雷公藤内酯酮组相比，雷帕霉素和雷公藤内酯酮共同处理能够显著降低细胞中GPX4和xCT的表达水平(P < 0.01)。ELISA检测雷帕霉素和雷公藤内酯酮处理的HT22细胞上清液中丙二醛和谷胱甘肽的含量，结果显示，相较于糖氧剥夺/复氧组，糖氧剥夺/复氧+雷公藤内酯酮组升高了谷胱甘肽含量，降低了丙二醛含量和亚铁离子水平，然而与糖氧剥夺/复氧+雷公藤内酯酮组比较，与雷帕霉素联合使用降低了谷胱甘肽的含量(P < 0.01)，升高丙二醛含量(P < 0.01)和亚铁离子水平(P < 0.05)。结果提示，雷公藤内酯酮激活mTOR磷酸化，这与其抑制糖氧剥夺/复氧条件下HT22细胞铁死亡有关。 2.6 雷公藤内酯酮对缺血再灌注大鼠的神经保护作用假手术组大鼠未出现任何神经功能缺损。与假手术组相比，模型组表现出明显的神经功能缺损，提示有严重的神经损伤。雷公藤内酯酮对模型大鼠神经功能缺损的治疗作用显著(P < 0.01)，见图5A。此外，与假手术组大鼠相比，模型组大鼠脑梗死体积显著增加，雷公藤内酯酮治疗有效降低了模型组大鼠的脑梗死体积(P < 0.01)，见图5B，C。如图5C所示，模型组手术完成即刻，缺血侧血流量明显下降，再灌注后血流有所恢复，表明大鼠脑缺血再灌注模型建立成功。此外，TUNEL染色显示，与假手术组大鼠相比，模型组大鼠的脑组织中细胞凋亡加重(P < 0.01)；雷公藤内酯酮治疗后，模型组大鼠凋亡细胞数量明显减少(P < 0.01)，见图5D，E。上述结果表明，雷公藤内酯酮治疗可减轻大鼠脑缺血再灌注损伤。Western blotting检测脑组织中SPHK1、p-mTOR、mTOR、GPX4蛋白表达水平，结果显示，模型组大鼠中SPHK1、p-mTOR/mTOR和GPX4水平显著降低；在雷公藤内酯酮处理后，模型组大鼠中SHKP1表达水平和mTOR的磷酸化水平升高(P < 0.01)，铁死亡相关蛋白GPX4表达水平上升(P < 0.01)，见图5F-I。这些数据表明，雷公藤内酯酮治疗可减轻大鼠脑缺血再灌注损伤。"

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