Effect of effective parts of Dracocephalum moldavica on the necroptosis of HBMECs after OGD/R injury
10.16438/j.0513-4870.2021-0855
- VernacularTitle:香青兰有效部位对OGD/R损伤诱导的人脑微血管内皮细胞程序性坏死的作用机制研究
- Author:
Zhi-hang YANG
1
;
Xue-meng WANG
1
;
Lei XU
2
;
Wen-ling SU
2
;
Kadeerye KADDER
2
;
Ming XU
3
;
Rui-fang ZHENG
2
;
Jian-guo XING
2
Author Information
1. College of Pharmacy, Shihezi University, Shihezi 832000, China
2. Xinjiang Institute of Materia Medica, Urumqi 830002, China; Xinjiang Uyghur Medicine Key Laboratory, Urumqi 830002, China
3. China Pharmaceutical University, Nanjing 210009, China
- Publication Type:Research Article
- Keywords:
effective parts of Dracocephalum moldavica;
ischemia-reperfusion injury;
oxygen-glucose deprivation/re-oxygenation;
necroptosis;
mixed lineage kinase domain like protein;
mitochondrial membrane permeability transformation pore
- From:
Acta Pharmaceutica Sinica
2022;57(2):409-418
- CountryChina
- Language:Chinese
-
Abstract:
We investigated the ability of Dracocephalum moldavica (EPDM) flavonoids to protect human brain microvascular endothelial cells (HBMECs) from necroptosis induced by ischemia-reperfusion injury. To mimic the process of cerebral ischemia-reperfusion injury, a necroptosis model was established by treatment with the pan-cysteine aspartic acid protease (caspase) inhibitor Z-VAD-FMK combined with oxygen-glucose deprivation/re-oxygenation (OGD/R) injury using HBMECs. Cell proliferation and cytotoxicity (cell counting kit-8, CCK-8) was used to measure cell viability. A Hoechst33342/PI fluorescent double-staining method was exploited to determine the rate of cell necroptosis. A commercial kit was used to detect lactate dehydrogenase in the cell culture supernate. DCFH-DA probes, calcein AM and JC-1 probes were used to measure changes in ROS production, mitochondrial membrane permeability transformation pore (MPTP) opening and mitochondrial membrane potential (MMP), respectively. Enzyme-linked immunosorbent assay (ELISA) kits were chosen to detect the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Western blotting was used to detect necroptosis-related proteins. The results show that relative to control group, Z-VAD-FMK combined with OGD/R injury reduced cell viability, increased the necroptosis rate and the levels of LDH and ROS in HBMECs. The MPTP of the model group cells opened and the MMP reduced. TNF-α, IL-1β, and IL-6 levels were significantly elevated. Furthermore, the expression of receptor-interacting protein kinase 3 (RIP3) and mitochondrial phosphoglycerate mutase 5 (PGAM5) was significantly increased, accompanied by an increase of phosphorylated mixed-lineage kinase domain-like protein (p-MLKL)/MLKL. EPDM partially reversed the changes of the above-mentioned factors in HBMECs induced by Z-VAD-FMK plus OGD/R injury. These results indicate that EPDM may protect HBMECs from cerebral ischemia-reperfusion injury by inhibiting the RIP3/MLKL/PGAM5 pathway and MPTP opening to maintain mitochondrial function, thereby providing a scientific basis for the use of EPDM in the treatment of cerebral ischemia-related diseases.
