[Molecular mechanism of ligustilide attenuating OGD/R injury in PC12 cells by inhibiting ferroptosis].

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.

[Mechanism of total flavonoids of Rhododendra simsii in alleviating ischemic brain injury].

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.

[Protective effect of total flavonoids of Rhododendron simsii on focal cerebral ischemia-reperfusion injury through SOCE pathway in rats].

This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg~(-1)) group, TFR(60 mg·kg~(-1))+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg~(-1)) group, and 2-APB(2.5 mg·kg~(-1)) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg~(-1)) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg~(-1)) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.

Total Flavone of Rhododendron Improves Cerebral Ischemia Injury by Activating Vascular TRPV4 to Induce Endothelium-Derived Hyperpolarizing Factor-Mediated Responses.

BACKGROUND: Total flavonoids of Rhododendron (TFR) is extracted from Rhododendron, a herbal medicine widely used in China. The main components are flavone compounds such as warfarin, rutin, quercetin, and hyperoside. We investigated the role of TRPV4 channel in the TFR induced endothelium-dependent hyperpolarizing factor- (EDHF-) mediated responses against ischemia/reperfusion injury (IR) in cerebral IR (CIR) rats. METHODS: The morphological changes of cerebral cortex, the relaxation of cerebral basal artery (CBA), and cell membrane potential recording were studied in CIR rats. The outward potassium current in smooth muscle cell was recorded by whole-cell patch clamp recording. The protein expression of TRPV4, SKca, and IKca was determined. Confocal laser was used to measure the Ca2+ fluorescence intensity. RESULTS: After treatment with TFR, the number of pyramidal cells in brain tissue increased and the number of empty or lightly stained cells decreased and these effects were eliminated by using HC-067047, Apamin, or TRAM-34. TFR induced and EDHF-mediated dilatation and hyperpolarization in CBA were also attenuated by using these inhibitors. The increased outward current density elicited by TFR in acutely isolated CBA smooth muscle cells was abolished by using TRAM-34 and Apamin. TFR upregulated the protein expression of TRPV4, SKca, and IKca that was also eliminated by these inhibitors. Laser scanning showed that the increased mean fluorescence intensity of Ca2+ by CIR was decreased by using TFR and that this effect was again eliminated by the above inhibitors. CONCLUSIONS: We conclude that in the CBA of the CIR rats the protective effect of TFR on ischemic cerebrovascular injury may be related to the activation of the TRPV4 in both endothelium and smooth muscle by increasing its expression and activity. The activation of TRPV4 channel in the endothelium may be linked to the opening of endothelial IKca/SKca channels that induces EDHF-mediated relaxation and hyperpolarization in the smooth muscle cell. In addition, the activation of TRPV4 in the smooth muscle cell in CBA may be linked with the activation of BKCa channel through a TRPV4-dependent pathway, reduce Ca2+ concentration in the cell, and relaxes the vessel. These findings may form a new therapeutic target for protection of ischemic brain injury and facilitate the use of Chinese medicine in brain protection.

[Protective effect against myocardial ischemia reperfusion injuries induced by hyperoside preconditioning and its relationship with PI3K/Akt signaling pathway in rats].

To investigate the protective effect of preconditioning with hyperoside ( Hyp) against myocardial ischemia-reperfusion injury (MIRI) in rats and the role of PI3K/Akt signaling pathway. MIRI was established by ligation of left anterior descending coronary artery for 30 min followed by reperfusion for 120 min in rats. Male SD rats were randomly divided into five groups: sham group,model group (MIRI),Hyp preconditioning group(Hyp), Hyp preconditioning + LY294002 (a PI3K/Akt signaling pathway inhibitor) group (Hyp + LY), and LY294002 group (LY). At the end of reperfusion, hemodynamic parameters were recorded as left ventricular systolic pressure (LVSP) , left ventricular end-diastolic pressure ( LVEDP) and maximal rate of increase and decrease of left ventricular pressure (± dP/dt(max)). Myocardial infaret size, the oxidative stress markers, myocardial enzymes indicators and inflammatory factors were also analyzed. The expressions of Akt, p-Akt, Bax and Bcl-2 proteins was detected by using Western blot method. The results showed that Hyp preconditioning remarkably improved cardiac constriction and relaxation function, reduced myocardial infarct size and enhanced the activities of oxidative stress markers about correlated to MIRI, such as superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px) and decreased the contents of malondialdehyde (MDA) as compared with MIRI group. Simultaneouly, the levels of myocardial enzymes, i. e. creatine kinase ( CK) and creatine kinase MB isoenzyme (CK-MB), and inflammatory factors, for instance tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were decreased. Hyp pretreatment apparently restrained myocardial apoptosis as evidenced by decreasing the level of Bax expression, increasing the levels of phosphorylation of Akt and Bcl-2 expression. These effects were inhibited by LY294002, a blocker of PI3K/Akt signaling pathway. These findings indicated that the cardioprotection of Hyp preconditioning against MIRI may be related to activating PI3K/Akt signaling pathway, upregulating the expression of BCL-2 protein and down-regulating the expression of Bax protein.

[Effects and mechanisms of hyperoside on vascular endothelium function in middle cerebral arteries of rats ex vivo].

To investigate the effects and potential mechanisms of hyperoside (Hyp) on the vascular endothelium function in middle cerebral artery (MCA) ex vivo in rats. Isolated arterial segments from MCAs of rats were used for surveying vasomotoricity in a pressurized chamber. Transmembrane potential was recorded by using glass microelectrodes to evaluate hyperpolarization. Hyp (1 x 10(-6)-1 x 10(-4) mol . L-1) was utilized to observe the effect on 1 x 10(-7) mol . L-1 U46619-preconstricted MCA in rats. The results showed that 1 x 10(-6)-1 x 10(-4) mol . L-1 Hyp significantly induced concentration-dependent vasodilatation and hyperpolarization, leading to the maximal diastolic ratio of (73. 2 ± 6. 1)% and maximal changes in membrane potentials of (-13. 2 ± 2. 2) mV. Hyp still elicited vasorelaxation and hyperpolarization by removal of endothelium in MCA of rat, which was notably attenuated as compared with vascular endothelium-intact group (P <0. 01). In the MCAs preconstricted by U46619 (1 x 10(-7) mol . L-1), Hyp (1 x 10(-6)-1 x 10(-4) mol . L-1) produced concentration-dependent vasorelaxation and hyperpolarizition that were partially attenuated by 3 x 10(-5) mol . L-1 L-NAME(a NOS inhibitor) plus 1 x 10(-5) mol . L-1 PGI2 ,(a synthetase inhibitor). The residual effects were further decreased by 1 x 10(-3) mol . L-1 TEA (an inhibitor of Ca2+-activated potassium channel) or 1 x 10(-5) mol . L-1 PPG (a blocker of endogenous H2S synthese-CSE). Similarly, 1 x 10(-5)-1 x 10(-3) mol . L-1 NaHS (a donor of exogenous H2S) or 1 x 10(-5)-1 x 10(-3) mol . L-1 L-Cys (the substrate of endogenous H2S synthesis) obviously evoked dose-dependent vasodilatation and hyperpolarization of MCA in rats. These findings indicated that Hyp may induce endothelium-dependent and endothelium-independent responses. And the endothelium-dependent vasodilatation may be related to the increases of endogenous H2S that has been promoted Hyp in the endotheliocyte of MCAs, and activated Kca and opening of Kca channels, resulting in the hyperpolarization of vascular smooth muscle cell membrane and subsequent reduction of Ca2+ influx and vasodilation.