[Muscone inhibits opening of mPTP to alleviate OGD/R-induced injury of HT22 cells].

This study aims to investigate the mechanism of muscone in inhibiting the opening of mitochondrial permeability transition pore(mPTP) to alleviate the oxygen and glucose deprivation/reoxygenation(OGD/R)-induced injury of mouse hippocampal neurons(HT22). An in vitro model of HT22 cells injured by OGD/R was established. CCK-8 assay was employed to examine the viability of HT22 cells, fluorescence microscopy to measure the mitochondrial membrane potential, the content of reactive oxygen species(ROS), and the opening of mPTP in HT22 cells. Enzyme-linked immunosorbent assay was employed to determine the level of ATP and the content of cytochrome C(Cyt C) in mitochondria of HT22 cells. Flow cytometry was employed to determine the Ca~(2+) content and apoptosis of HT22 cells. The expression of Bcl-2(B-cell lymphoma-2) and Bcl-2-associated X protein(Bax) was measured by Western blot. Molecular docking and Western blot were employed to examine the binding between muscone and methyl ethyl ketone(MEK) after pronase hydrolysis of HT22 cell proteins. After the HT22 cells were treated with U0126, an inhibitor of MEK, the expression levels of MEK, p-ERK, and CypD were measured by Western blot. The results showed that compared with the OGD/R model group, muscone significantly increased the viability, mitochondrial ATP activity, and mitochondrial membrane potential, lowered the levels of ROS, Cyt C, and Ca~(2+), and reduced mPTP opening to inhibit the apoptosis of HT22 cells. In addition, muscone up-regulated the expression of MEK, p-ERK, and down-regulated that of CypD. Molecular docking showed strong binding activity between muscone and MEK. In conclusion, muscone inhibits the opening of mPTP to inhibit apoptosis, thus exerting a protective effect on OGD/R-injured HT22 cells, which is associated with the activation of MEK/ERK/CypD signaling pathway.

Hydroxysafflor yellow A alleviates cerebral ischemia reperfusion injury by suppressing apoptosis via mitochondrial permeability transition pore.

BACKGROUND: Mitochondria are key cellular organelles that are essential for cell fate decisions. Hydroxysafflor yellow A (HSYA) has displayed an impressively essential role in protection of cerebral ischemia/reperfusion (I/R). However, the mitochondrial effect of HSYA on Brain Microvascular Endothelial Cells (BMECs) under I/R remains to be largely unclear. PURPOSE: To evaluate the protective effects of HSYA-mediated mitochondrial permeability transition pore (mPTP) on cerebral I/R injury and its mechanism. METHODS: Cerebral I/R injury was established by the model of Middle cerebral artery occlusion (MCAO) in rats. Furthermore, to further clarify the relevant mechanism of HSYA's effects on mPTP, inhibition of extracellular regulated protein kinases (ERK) with U0126 and transfect with Cyclophilin D (CypD) SiRNA to reversely verified whether the protective effects of HSYA were exerted by regulating the Mitogen-activated protein kinase kinase (MEK)/ERK/CypD pathway. RESULTS: HSYA treatment significantly increased BMECs viability, decreased the generation of ROS, opening of mPTP and translocation of cytochrome c after OGD/R. In addition to inhibited CypD, HSYA potentiated MEK and increased phosphorylation of ERK expression in BMECs, inhibited apoptosis mediated by mitochondrial. Notably, HSYA also significantly ameliorated neurological deficits and decreased the infarct volume in rats. CONCLUSION: HSYA reduced the CytC export from mitochondrial by inhibited the open of mPTP via MEK/ERK/CypD pathway, contributing to the protection of I/R. Thus, our study not only revealed novel mechanisms of HSYA for its anti-I/R function, but also provided a template for the design of novel mPTP inhibitor for the treatment of various mPTP-related diseases.

Naoluo Xintong capsule ameliorates apoptosis induced by endoplasmic reticulum stress in rats with cerebral ischemia/ reperfusion injury.

BACKGROUND: Naoluo Xintong (NLXT) capsuleis a newly developed drug recorded in the Chinese Pharmacopoeia. It is derived from traditional Chinese medicine (TCM) NLXT decoction, and has been widely used to treat cerebrovascular diseases in clinic. However, it is currently unknown whether it improve cerebral ischemia reperfusion (I/R) injury. METHODS: The effect of NLXT on regional cerebral blood flow (rCBF) was examined using Laser Doppler flower. The Terminal deoxyribonucleotide transferase-mediated Nick end labeling (Tunel) assay was performed to determine the effects of NLXT on apoptosis. Subsequently, cerebral water content and TTC staining were measured to assess cerebral edema and infarct volume, respectively. The protein expression levels were analyzed with Immunofluorescence and western blot assays. RESULTS: The results indicated that NLXT ameliorated MCAO-induced cerebral I/R injury by decreasing infract volume, inhibition of apoptosis, and upregulation rCBF. In addition, it decreased the expression of key protein involved endoplasmic reticulum (ER)-stress, including glucose-regulated protein 78 (GRP78), C/EBP-homologous protein (CHOP) and Caspase-12 at 24 h following reperfusion. This was accompanied reduced degradation level of TRPC6 and increased phosphorylation of cAMP/Ca2+ response elementbinding protein (p-CREB), and decreased calpain-specific αII-spectrin breakdown product (SBDP145) activity. Interestingly, inhibition of mitogen-activated protein kinase (MEK) activity abolished the effect of NLXT on CREB activity. CONCLUSIONS: Collectively, the results indicated that NLXT can improve I/R injury therapy by activating TRPC6/MEK/CREB signaling pathway to attenuate ER-stress related neuronal apoptosis.

Ligustilide Ameliorates the Permeability of the Blood-Brain Barrier Model In Vitro During Oxygen-Glucose Deprivation Injury Through HIF/VEGF Pathway.

Chuanxiong rhizome has been widely used for the treatment of cerebral vascular disease in traditional Chinese medicine. The integrity of blood-brain barrier (BBB) is closely linked to the cerebral vascular disease. The protective effects of ligustilide, the major bioactive component in Chuanxiong rhizome, on cerebral blood vessels have been reported previously, but its effects and potential mechanism on BBB have not been entirely clarified. In the current work, the effects of ligustilide on BBB permeability and the underlying molecular mechanisms had been investigated using the model of BBB established by coculturing astrocytes and brain microvascular endothelial cells isolated from the rat brain. The ischemia-damaged model of BBB has been established with oxygen and glucose deprivation (OGD). Our results indicated that OGD significantly increased the permeability in the coculture BBB model. This OGD-induced increase in permeability could suppress by ligustilide in a concentration-dependent manner. Also, ligustilide promoted both gene and protein expression of tight junction proteins. Ligustilide suppressed the upregulation of HIF-1α, vascular endothelial growth factor, and AQP-4 in the BBB model induced by OGD. Collectively, all results have demonstrated that ligustilide is capable of reducing the permeability of BBB in vitro model induced by OGD through HIF-1α/vascular endothelial growth factor pathway and AQP-4, which provide a new target for the clinical application of ligustilide on BBB after stroke in future.

YiQi Tongluo Granule against Cerebral Ischemia/Reperfusion Injury in Rats by Freezing GluN2B and CaMK II through NMDAR/ERK1/2 Signaling.

Yiqi Tongluo Granule (YQTL) is a kind of proprietary Chinese medicine, manufactured by China Shineway Pharmaceutical Group Ltd., under the authority of China Food and Drug Administration (CFDA) treating cardiovascular and cerebrovascular diseases such as ischemic stroke in China, however the underlying mechanism of YQTL on treating ischemic stroke has not been revealed. This study is aimed to evaluate the protective effect of YQTL on cerebral ischemia/reperfusion (I/R) injury and inquire into its underlying mechanisms. Cerebral I/R injury was induced by occluding the middle cerebral artery for 2 h followed by 24 h reperfusion. And regional cerebral flow was monitored by Laser Doppler flow during ischemia phase. The infarct volume was evaluated by Triphenyte-trazolium chloride staining. The protective effects of YQTL were assessed by a number of parameters, including neurological scores, regional cerebral blood flow, pathological changes of neuron in hippocampuses and hippocampus calcium level. The proteins of extracellular signal-regulated kinase (ERK), N-methyl D-aspartate receptor subtype 2B (GluN2B) and p-calcium-dependent protein kinaseII (CaMKII) response were assayed by Western blotting. I/R caused significant change in neurological deficit scores, regional cerebral flow and infarct volume. However results in YQTL groups and Nimodipine Tablets (NMDP) group were reversed. Subsequently YQTL reduced I/R-induced calcium influx. Results of hematoxylin-eosin staining manifested that YQTL significantly improved neuronal injury after I/R in rats. Meanwhile, microdialysis data demonstrated that extracellular glutamate was increased in the striatum during ischemia reperfusion, which was reduced by YQTL. YQTL and mitogen-activated protein extracellular kinase (MEK) inhibitor suppressed the I/R-mediated over-expression of GluN2B, p-ERK, ERK and p-CaMKII proteins expression. Putting these together, our results suggest that YQTL played a neuroprotective role in cerebral I/R injury, which might be exerted by inhibiting the excitotoxicity and expression of GluN2B, p-CaMKII and MEK/ERK signal pathway.

The Establishment of the Method of Cell Biochromatograpy and Analysis of the Active Ingredients from TongQiaoHuoXue Decoction Acting on the Neurocytes.

A novel strategy for screening active components in traditional Chinese medicines (TCM) using living cells and HPLC and GC analysis are proposed. The hypothesis is that when cells are incubated with the extract of Tongqiao Huoxue Decoction (TQHXD), a famous ancient prescription in TCM, the potential active components in the TQHXD should selectively combine with the cells, and the cell-combining components would be detectable in the extract of denatured cells. The identities of the cell-combining components could be determined by HPLC and GC analysis. Using the proposed approach, two characteristic active ingredients binding to the membrane of the PC12 cells are indicated. In the fingerprint of HPLC, there are two characteristic peaks. One active ingredient with its retention time was at around 70 min had been identified as muscone by HPLC, GC, which came from Moschus herb, the other active ingredient may come from the Allium fistulosum, its structure needs further research. Also, the protective effect of muscone on PC12 cells induced by Oxygen and glucose deprivation (OGD) had been explored. These results show that the pretreatment with muscone on PC12 cells observably increased cell viability, reduced the release of lactate dehydrogenase (LDH) and cell apoptosis. Combined with the pharmacodynamic study of muscone on neuroprotective effect, it could be identified as one of the effective components in TQHXD.