[Protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid on OGD/R-damaged HT22 cells via regulation of ASK1/MKK4/JNK signaling pathway].

To investigate the protective effect of Tongqiao Huoxue Decoction containing cerebrospinal fluid(TQHXD-CSF) on HT22 cells damaged by oxygen-glucose deprivation/reoxygenation(OGD/R) and whether the mechanism is related to the regulation of ASK1/MKK4/JNK signaling pathway. HT22 cells were subjected to OGD/R to simulate cerebral ischemia-reperfusion injury(CIRI). Then the cells were randomly divided into five groups: blank cerebrospinal fluid(control group), OGD/R group, TQHXD-CSF group, Z-VAD-FMK group(20 µmol·L~(-1)) and TQHXD-CSF+Z-VAD-FMK group. Except the control group, cells in the other groups were reoxygenated for 12 h after 6 h of oxygen and glucose deprivation for modeling OGD/R, and group administration was performed. Cell viability and cytotoxicity were detected by CCK8 and LDH assay kit, respectively and the morphology of HT22 cells was observed by inverted microscope. Western blot and qRT-PCR were employed to detect the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3, respectively. Then HT22 cells were assigned into the control group, OGD/R group, si-NC group, si-ASK1 group, TQHXD-CSF group and TQHXD-CSF+si-ASK1 group. Cell viability, proliferation and apoptosis were determined by CCK8, electric cell-substrate impedance sensing(ECIS), and Hoechst staining and flow cytometry, respectively. The protein expression of MKK4, p-MKK4, JNK, p-JNK, c-Jun, p-c-Jun, Cyt C, Bax, Bcl-2 and caspase-3 was tested by Western blot. The results showed that compared with OGD/R group, TQHXD-CSF significantly enhanced cell viability, improved cell morphology and reduced the protein and mRNA expression levels of Bax, Bcl-2 and caspase-3. In addition, when ASK1 was silenced, compared with OGD/R group, TQHXD-CSF remarkably improved cell viability, and decreased apoptosis rate and the protein expression levels of p-MKK4, p-JNK, p-c-Jun, Cyt C, Bax/Bcl-2 and caspase-3, but the effect was not as good as that of TQHXD-CSF+si-ASK1 group. In conclusion, TQHXD-CSF can inhibit apoptosis mediated by ASK1/MKK4/JNK signaling pathway in OGD/R-damaged HT22 cells, and has protective effect on ischemia-reperfusion injury.

Study on the mechanism of Tong-Qiao-Huo-Xue decoction regulating apoptosis via ASK1/MKK4/JNK pathway in MCAO/R rats.

BACKGROUND: Activation of blood stasis is a crucial aspect of stroke treatment, and the Tong-Qiao-Huo-Xue-Decoction (TQHXD) formula is commonly utilized for this purpose. However, the mechanism underlying the protective effects of TQHXD against cerebral ischemia-reperfusion (I/R) injury is unclear. PURPOSE: Identification of the TQHXD components responsible for its protective effects and determination of their mode of action against cerebral I/R injury. METHODS: Gas chromatography (GC) and high-performance liquid chromatography (HPLC) were carried out to determine the active aspects of TQHXD. The active components and targets of TQHXD were looked up in the TCMSP and HERB databases; the Genecards, OMIM, TTD, and DrugBank databases were used to identify targets related to cerebral infarction; and the intersecting targets were obtained. The drug-ingredient-target-disease network and PPI network were subsequently built using Cytoscape 3.7.1 and STRING websites. Autodock VINA was used to perform molecular docking between the core target ASK1 and the active components of TQHXD detected by HPLC and GC. After successfully creating a rat model of middle cerebral artery occlusion (MCAO), the therapeutic effect of TQHXD was observed using triphenyltetrazolium and hematoxylin-eosin staining. We used Tunel-NeuN staining and transmission electron microscopy (TEM) to quantify hippocampal apoptosis. RT-qPCR and western blotting were used to detect protein and mRNA expression, respectively. RESULTS: HPLC and GC identified six active ingredients. Network pharmacology analyses were performed to test 66 intersection targets, including ASK1, MKK4, and JNK. Ferulic acid, HSYA, ligustilide, paeoniflorin, and muscone all displayed high binding affinity with ASK1 in molecular docking studies. The neuroprotective effects of TQHXD in I/R rats were demonstrated in the experimental models. In comparison with the model group, TQHXD decreased the apoptosis rate and reduced the protein levels of p-ASK1, caspase 3, p-MKK4, CytC, p-c-Jun, Bax/Bcl-2, and p-JNK, while considerably increasing the mRNA levels of Bcl-2 and decreasing those of Bax. CONCLUSION: By controlling the ASK1/MKK4/JNK pathway, TQHXD protects neurons from I/R damage and prevents apoptosis. Thus, TQHXD may be effective for the treatment of ischemic stroke. And the mechanism behind these therapeutic actions of TQHXD is supported by this research.

Tong-Qiao-Huo-Xue decoction activates PI3K/Akt/mTOR pathway to reduce BMECs autophagy after cerebral ischemia/reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong-Qiao-Huo-Xue Decoction (TQHXD) is a traditional classic Chinese Medicinal Formula (CMF) used for clinical treatment of ischemic stroke. TQHXD leads to improvement in the symptoms of the acute period of cerebral infarction and recovery period after stroke. Our previous studies also showed that TQHXD produced a significant protective effect on the brain after cerebral ischemia-reperfusion (I/R) injury. It is reported that autophagy is closely related to ischemic brain injury; however, the functional contribution of TQHXD to brain microvascular endothelial cell (BMEC) autophagy and its underlying mechanism remains unclear. AIM OF THE STUDY: The purpose of this study was to investigate the effects and mechanism of TQHXD in inhibiting cerebral ischemia-induced endothelial autophagy. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint of the chemical constituents from TQHXD was established for the quality control, and the Longa method was used to evaluate the efficacy of TQHXD in rats with middle cerebral artery occlusion (MCAO). The expression of LC3 was determined by immunofluorescence double staining. To evaluate the protective effects of TQHXD-containing cerebrospinal fluid (CSF) on BMECs injured by oxygen-glucose deprivation and reperfusion, cell survival rate was determined using the CCK-8 assay and cell apoptosis was determined by fluorescein isothiocyanate (FITC)-Annexin V/PI. Autophagy was detected using transmission electron microscopy. RESULTS: The results showed that TQHXD-CSF significantly ameliorated oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in BMECs. Confocal microscopy and Western blot results showed that TQHXD-CSF reduced autophagy-related protein expression and autophagosome number. The results of the western blotting indicated that TQHXD-CSF caused a marked increase in the phosphorylation of protein kinase B and phosphoinsotide-3 kinase (Akt/p-Akt and PI3K/p-PI3K, respectively) and their expression levels were down-regulated after treatment with pathway inhibitor, ZSTK474. Furthermore, in a MCAO model in rats, TQHXD markedly increased p-PI3K, p-Akt and p-mTOR, whereas the autophagy related proteins decreased. CONCLUSIONS: Taken together, these findings demonstrate that TQHXD protects against ischemic insult by inhibiting autophagy through the regulation of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway and that TQHXD may have therapeutic value for protecting BMECs from cerebral ischemia.

Ligustilide ameliorates hippocampal neuronal injury after cerebral ischemia reperfusion through activating PINK1/Parkin-dependent mitophagy.

BACKGROUND: Mitophagy plays a critical role in cerebral ischemia/reperfusion by timely removal of dysfunctional mitochondria. In mammals, PINK1/Parkin is the most classic pathway mediating mitophagy. And the activation of PINK1/Parkin mediated mitophagy exerts neuroprotective effects during cerebral ischemia reperfusion injury (CIRI). Ligustilide (LIG) is a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis (Oliv.) diels that exerts neuroprotective activity after cerebral ischemia reperfusion injury (CIRI). However, it still remains unclear whether LIG could attenuates cerebral ischemia reperfusion injury (CIRI) through regulating mitophagy mediated by PINK1/Parkin. PURPOSE: To explore the underlying mechanism of LIG on PINK1/Parkin mediated mitophagy in the hippocampus induced by ischemia reperfusion. METHODS: This research used the middle cerebral artery occlusion and reperfusion (MCAO/R) animal model and oxygen-glucose deprivation and reperfusion (OGD/R) as in vitro model. Neurological behavior score, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Hematoxylin and Eosin (HE) Staining were used to detect the neuroprotection of LIG in MCAO/R rats. Also, the levels of ROS, mitochondrial membrane potential (MMP) and activities of Na+-K+-ATPase were detected to reflect mitochondrial function. Moreover, transmission electron microscope (TEM) and fluorescence microscope were used to observe mitophagy and the western blot was performed to explore the changes in protein expression in PINK1/Parkin mediated mitophagy. Finally, exact mechanism between neuroprotection of LIG and mitophagy mediated by PINK1/Parkin was explored by cell transfection. RESULTS: The results show that LIG improved mitochondrial functions by mitophagy enhancement in vivo and vitro to alleviate CIRI. Whereas, mitophagy enhanced by LIG under CIRI is abolished by PINK1 deficiency and midivi-1, a mitochondrial division inhibitor which has been reported to have the function of mitophagy, which could further aggravate the ischemia-induced brain damage, mitochondrial dysfunction and neuronal injury. CONCLUSION: LIG could ameliorate the neuronal injury against ischemia stroke by promoting mitophagy via PINK1/Parkin. Targeting PINK1/Parkin mediated mitophagy with LIG treatment might be a promising therapeutic strategy for ischemia stroke.

Ligustilide attenuates ischemic stroke injury by promoting Drp1-mediated mitochondrial fission via activation of AMPK.

BACKGROUND: Ischemic stroke is a major global cause of death and permanent disability. Studies have suggested that mitochondria play a critical role in maintaining cellular energy homeostasis and inevitably involved in neuronal damage during cerebral ischemic. Ligustilide is the main active ingredient of Angelica sinensis and Ligusticum chuanxiongs with neuroprotective activity. PURPOSE: These study sought to exlopre the role of LIG in improving mitochondrial function and the relationship between LIG induced mitochondrial fission and mitophagy in ischemic stroke. METHODS: Cerebral I/R injury was established by the model of Oxygen-glucose deprivation/reperfusion (OGD/R) in HT22 cells and middle cerebral artery occlusion (MCAO) in rats. Mitochondrial functions of were detected by flow cytometry and immunofluorescence, and mitochondrial fission were detected by western blots. Furthermore, we studied the role of AMPK pathway in the neuroprotective effect of LIG. RESULTS: LIG treatment significantly increased the MMP and ATP production, decreased the reactive oxygen species (ROS) generation and Ca2+ overload, and further induced mitochondrial fission and mitophagy. Moreover, we found that blocking mitochondrial fission by mdivi-1 resulted in accumulation of damaged mitochondria mainly through selectively blocking mitophagy, thereby inhibiting viability of HT-22 cells after OGD/R. Also, Drp-1 inhibitor mdivi-1 increased the infarct volume and aggravated the neurological deficits after MCAO operation in vivo. Additionally, LIG triggered AMP-activated protein kinase (AMPK) pathway. AMPKα2 knockdown attenuated LIG-induced mitochondrial fission through inhibiting the expression of Drp1 and Fis1, and led to nerve cell apoptosis. CONCLUSION: Our study indicate that LIG attenuated the injury of ischemic stroke by improving mitochondrial function and highlight the critical role of LIG in the regulation of LIG-induced mitochondrial fission and mitophagy via an AMPK-dependent manner. These findings indicate that LIG protects nerve damage against ischemic stroke by inducing Drp1-mediated mitochondrial fission via activation of AMPK signaling pathway in vivo and in vitro.

Chrysophanol exerts neuroprotective effects via interfering with endoplasmic reticulum stress apoptotic pathways in cell and animal models of Alzheimer's disease.

OBJECTIVES: Chrysophanol (CHR), also well-known as Rhei radix et rhizome, is a crucial component in traditional Chinese medicine. It has been widely studied as a potential treatment for many diseases due to its anti-inflammatory effects. However, there are very few studies to establish the potential therapeutic effect of CHR in cell and animal models of Alzheimer's disease (AD). Therefore, we aim to investigate whether CHR could be used as a potential therapeutic approach to patients with AD and further disclose the underlying mechanism. Increasing studies have shown that endoplasmic reticulum (ER) calcium (Ca2+) homeostasis emerges as a central player in AD pathogenesis. Moreover, augmentation of ER stress (ERS) promotes neuronal apoptosis, and excessive oxidative stress is an inducer of ERS. Therefore, we believe that ERS-mediated apoptosis may be one of the causes of AD. METHODS: This study examined the neuroprotective effects of CHR on AD rats and AD cell models and explored its potential mechanism. KEY FINDINGS: CHR could reduce the damage of neurons. In AD cell models, CHR significantly inhibited Aß 25-35-induced neuronal damage, reduced the number of apoptotic cells and improved cell survival rate. Western blot showed that the expression of caspases 3, 9 and 12 was decreased after CHR treatment, and CHR also affected the ERS signalling pathway. In addition, the higher expression of pro-apoptotic proteins in the AD cell model was reduced after CHR treatment by inhibiting GRP78 signalling. Further studies have shown that overexpressed protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) inhibited the regulatory effect of CHR on PERK and weakened the neuroprotective effect of CHR on the AD cell model. CONCLUSIONS: This study revealed a novel mechanism through which CHR plays a neuroprotective role by regulating ERS when it comes to the therapy of AD.

YiQiFuMai lyophilized injection attenuates cerebral ischemic injury with inhibition of neuronal autophagy through intervention in the NMMHC IIA-actin-ATG9A interaction.

BACKGROUND: YiQiFuMai lyophilized injection (YQFM) is derived from a traditional Chinese medicine prescription termed Shengmai San.YQFM is clinically applied to the treatment of cardiovascular and cerebrovascular diseases. It has been found that critical components of YQFM affect non-muscle myosin heavy chain IIA (NMMHC IIA), but its regulation in the excessive autophagy and the underlying mechanism has yet to be clarified. PURPOSE: To evaluate whether YQFM has neuroprotective effects on cerebral ischemia/reperfusion-induced injury by inhibiting NMMHC IIA-actin-ATG9A interaction for autophagosome formation. METHODS: The neuroprotective effects of YQFM were investigated in vivo in mice with middle cerebral artery occlusion/reperfusion (MCAO/R) (n = 6) by detecting neurological deficits, infarct volume, and histopathological changes. The NMMHC IIA-actin-ATG9A interaction was determined using immunofluorescence co-localization, co-immunoprecipitation, and proximity ligation assay. Rat pheochromocytoma (PC12) cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic neurons in in vitro experiments. RESULTS: In MCAO/R model mice, YQFM (1.342 g/kg) attenuated brain ischemia/reperfusion-induced injury by regulating NMMHC IIA-actin-mediated ATG9A trafficking. YQFM (400 µg/ml) also exerted similar effects on OGD/R-induced PC12 cells. Furthermore, RNAi of NMMHC IIA weakened the NMMHC IIA-F-actin-dependent ATG9A trafficking and, therefore, attenuated the neuroprotective activities of YQFM in vitro. CONCLUSION: These findings demonstrated that YQFM exerted neuroprotective effects by regulating the NMMHC IIA-actin-ATG9A interaction for autophagosome formation. This evidence sheds new light on the potential mechanism of YQFM in the treatment of cerebral ischemia/reperfusion.

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.

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway.

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood-brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.

YiQiFuMai Powder Injection Protects against Ischemic Stroke via Inhibiting Neuronal Apoptosis and PKCδ/Drp1-Mediated Excessive Mitochondrial Fission.

YiQiFuMai (YQFM) powder injection has been reported to be used in cardiovascular and nervous system diseases with marked efficacy. However, as a treatment against diseases characterized by hypoxia, lassitude, and asthenia, the effects and underlying mechanisms of YQFM in neuronal mitochondrial function and dynamics have not been fully elucidated. Here, we demonstrated that YQFM inhibited mitochondrial apoptosis and activation of dynamin-related protein 1 (Drp1) in cerebral ischemia-injured rats, producing a significant improvement in cerebral infarction and neurological score. YQFM also attenuated oxidative stress-induced mitochondrial dysfunction and apoptosis through increasing ATP level and mitochondria membrane potential (Δψm), inhibiting ROS production, and regulating Bcl-2 family protein levels in primary cultured neurons. Moreover, YQFM inhibited excessive mitochondrial fission, Drp1 phosphorylation, and translocation from cytoplasm to mitochondria induced by oxidative stress. We provided the first evidence that YQFM inhibited the activation, association, and translocation of PKCδ and Drp1 upon oxidative stress. Taken together, we demonstrate that YQFM ameliorates ischemic stroke-induced neuronal apoptosis through inhibiting mitochondrial dysfunction and PKCδ/Drp1-mediated excessive mitochondrial fission. These findings not only put new insights into the unique neuroprotective properties of YQFM associated with the regulation of mitochondrial function but also expand our understanding of the underlying mechanisms of ischemic stroke.