RESUMO
This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 µg·mL~(-1)) group, and TFR(30 µg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Assuntos
Isquemia Encefálica , Flavonoides , Traumatismo por Reperfusão , Animais , Masculino , Ratos , Apoptose , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Caspase 3 , Interleucina-1 , Interleucina-6 , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Fator de Necrose Tumoral alfa/genética , Flavonoides/farmacologia , Rhododendron/químicaRESUMO
The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.