[Mechanism of Huangqi Chongteng Yin in improving OGD/R-induced injury and mitochondrial dysfunction in HT22 neurons via necroptosis mediated by RIPK1/RIPK3/MLKL signaling axis].

This study aimed to investigate whether Huangqi Chongteng Yin(HQCTY) alleviates oxygen-glucose deprivation/reoxygenation(OGD/R)-induced injury and mitochondrial dysfunction in mouse hippocampal HT22 neurons by modulating the receptor-interacting protein kinase 1(RIPK1)/receptor-interacting protein kinase 3(RIPK3)/mixed lineage kinase domain-like protein(MLKL)-mediated necroptosis signaling pathway. After preparing HQCTY-containing serum and verifying its safety and efficacy, HT22 cells were divided into the control group, OGD/R group, HQCTY-containing serum low-, medium-, and high-dose groups(5%, 10%, and 20%), and an edaravone(Eda) group. Cell viability and membrane damage were assessed using the cell counting kit-8(CCK-8) assay and lactate dehydrogenase(LDH) release assays, respectively, while nuclear morphology was evaluated by DAPI staining. The expression and phosphorylation levels of RIPK1, RIPK3, and MLKL were detected by Western blot, and the expression and subcellular localization of MLKL and phosphorylated(p)-MLKL were examined by immunofluorescence assay. Mitochondrial membrane potential, energy metabolism, mitochondrial network morphology, and ultrastructural alterations were evaluated using JC-1 staining, ATP assay kit, Mito-Tracker Green staining, and transmission electron microscopy, respectively. In addition, fluorescence staining was performed to assess the morphology of translocase of outer mitochondrial membrane 20(TOM20) and the cytoskeleton(phalloidin staining). The levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, and IL-18 were determined by ELISA. The results showed that, compared with the OGD/R group, HQCTY significantly increased HT22 cell viability, reduced LDH release and nuclear condensation, markedly downregulated the protein expression of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL, while ameliorating the abnormal localization of p-MLKL. Furthermore, HQCTY significantly improved mitochondrial membrane potential, increased ATP production, and promoted the restoration of mitochondrial network and ultrastructure. It also significantly reduced the levels of TNF-α, IL-1β, IL-6, and IL-18. These findings indicate that HQCTY may directly protect neurons from OGD/R injury by inhibiting RIPK1/RIPK3/MLKL-mediated necroptosis signaling pathway and improving mitochondrial structure and energy metabolism, thereby providing new insights and intervention strategies for the application of traditional Chinese medicine formulas in neuroprotection after cerebral ischemia-reperfusion injury.