The Role of the GRP78/PERK/ATF4 Pathway in the Ability of Gua Lou Gui Zhi Decoction to Attenuate Apoptosis by Inhibiting Endoplasmic Reticulum Stress after Ischemia-Reperfusion Injury.

BACKGROUND: Endoplasmic reticulum stress (ERS) is a key part of the apoptotic cascade that is initiated after cerebral ischemia-reperfusion injury and is very important for research on poststroke rehabilitation. In addition, the unfolded protein response (UPR) plays an important role in ERS because it activates downstream apoptotic signal transduction and induces apoptosis through the glucose-regulated protein 78 (GRP78)/protein kinase R (PKR)-like ER kinase (PERK)/activating transcription factor 4 (ATF4) pathway. The Gua Lou Gui Zhi Decoction (GLGZD) ameliorated neuronal apoptosis of ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO) had been proved in our previous study. The present study aims to underly the regulatory ability of GLGZD in ERS-induced apoptosis mediated by the GRP78/PERK/ATF4 pathway. METHODS: GLGZD was analyzed by HPLC. The effects of GLGZD were obversed on MCAO-induced ischemic rats. The cerebral infarct volume was detected by 2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) Staining. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling (TUNEL) were used to detect apoptosis. Transmission Electron Microscopy (TEM), Ca2+ levels and reactive oxygen species (ROS) detection were used to determine the function of endoplasmic reticulum. The GRP78/PERK/ATF4 signaling pathway was assessed by western blotting and immunohistochemistry. RESULTS: Our results showed that GLGZD exerted its effects on ischemia-reperfusion injury by significantly promoting the restoration of the quantity and morphology of the rough ER and reducing the neuronal apoptosis rate in the ischemic cortex. Moreover, both of the intracellular ROS and Ca2+ levels in ischemic cortical cells were found significantly reduced by GLGZD. The GLGZD-treated group showed increased levels of phosphorylation in both of PERK and eukaryotic translation initiation factor 2α (eIF2α), activation of cysteinyl aspartate-specific proteinase-3 (Caspase-3), upregulation of the total protein levels of sarcoplasmic/endoplasmic Ca2+ ATPase 2α (SERCA 2α) and B-cell leukemia/lymphoma gene 2 (Bcl-2). CONCLUSIONS: These findings suggest that GLGZD reduces oxidative stress-induced injury and promotes a dynamic calcium balance, thereby inhibiting ERS and exerting an antiapoptotic effect on neuronal ischemic injury, which are closely related to the activation of GRP78/PERK/ATF4 signaling pathway.

Cell membrane-coated biomimetic magnetic nanoparticles for the bio-specific extraction of components from Gualou Guizhi decoction exhibiting activities against oxygen-glucose deprivation/reperfusion injury.

Gua-Lou-Gui-Zhi decoction (GLGZD) is a classical multiherb traditional Chinese medicine formula that has ameliorative effects on oxygen-glucose deprivation/reperfusion (OGD/R) injury and has been applied for the treatment of stroke in clinical practice; however, its active ingredients remain unknown. The aim of this study was to develop an effective method for screening for components of GLGZD with potential therapeutic activity against OGD/R injury. Brain microvascular endothelial cell membrane-coated magnetic beads (CMs@rBMECs-MBs) were incubated with the GLGZD extract; the bound material was eluted and the constituents were identified using solid phase extraction and ultra-performance liquid chromatography-Orbitrap Fusion Tribrid mass spectrometry (UPLC-Orbitrap Fusion Tribrid MS). The biological activities of the identified GLGZD components were analyzed using OGD/R-exposed brain endothelial cells. Seven compounds bound to the CMs@rBMECs-MBs were identified as gallic acid, paeoniflorin, liquiritigenin, isoliquiritigenin, liquiritin, isoliquiritin, and formononetin. Among them, six (except formononetin) protected brain endothelial cells against OGD/R injury in a concentration-dependent manner (20-120 µM; P < 0.01-0.05) and downregulated the expression of hypoxia-inducible factor-1α (P < 0.01) involved in the pathogenic mechanisms triggered by stroke. Our findings suggest that the screening of bioactive compounds using cell membrane-coated magnetic beads combined with solid phase extraction and UPLC-Orbitrap Fusion Tribrid MS is an effective method for the bio-specific extraction and identification of ingredients responsible for the therapeutic activity of traditional Chinese medicines.

Preconditioning with Gua Lou Gui Zhi decoction enhances H2O2-induced Nrf2/HO-1 activation in PC12 cells.

Spasticity is common in various central neurological conditions, including after a stroke. Such spasticity may cause additional problems, and often becomes a primary concern for afflicted individuals. A number of studies have identified nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a key regulator in the adaptive survival response to oxidative stress. Elevated expression of Nrf2, combined with heme oxygenase 1 (HO-1) resistance, in the central nervous system is known to elicit key internal and external oxidation protection. Gua Lou Gui Zhi decoction (GLGZD) is a popular traditional Chinese formula with a long history of clinical use in China for the treatment of muscular spasticity following a stroke, epilepsy or a spinal cord injury. However, the mechanism underlying the efficacy of the medicine remains unclear. In the present study, the antioxidative effects of GLGZD were evaluated and the underlying molecular mechanisms were investigated, using hydrogen peroxide (H2O2)-induced rat pheochromocytoma cells (PC12 cells) as an in vitro oxidative stress model of neural cells. Upon application of different concentrations of GLGZD, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay and ATP measurement were conducted to assess the impact on PC12 cell proliferation. In addition, inverted microscopy observations, and the MTT and ATP assessments, revealed that GLGZD attenuated H2O2-induced oxidative damage and signaling repression in PC12 cells. Furthermore, the mRNA and protein expression levels of Nrf2 and HO-1, which are associated with oxidative stress, were analyzed using reverse transcription quantitative polymerase chain reaction (PCR) and confocal microscopy. Confocal microscopy observations, as well as the quantitative PCR assay, revealed that GLGZD exerted a neuroprotective function against H2O2-induced oxidative damage in PC12 cells. Therefore, the results demonstrated that GLGZD protected PC12 cells injured by H2O2, which may be associated with the upregulation of Nrf2 and HO-1 mRNA and protein expression levels in PC12 cells.

Chemical profiling and quantification of Gua-Lou-Gui-Zhi decoction by high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and ultra-performance liquid chromatography/triple quadrupole mass spectrometry.

Gua-Lou-Gui-Zhi decoction (GLGZD) is a classical formula of traditional Chinese medicine, which has been commonly used to treat dysfunction after stroke, epilepsy and spinal cord injury. In this study, a systematic method was established for chemical profiling and quantification analysis of the major constituents in GLGZD. For qualitative analysis, a method of high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (Q-TOF MS) was developed. 106 compounds, including monoterpene glycosides, galloyl glucoses, phenolic acids, flavonoids, gingerols and triterpene saponins were identified or tentatively presumed by comparison with reference standards or literature data. According to the qualitative results, a new quantitative analysis method of ultra-performance liquid chromatography/triple quadrupole mass spectrometry (QqQ-MS) was established. 24 representative compounds were simultaneously detected in 10 batches of GLGZD samples in 7.5 min. The calibration curves for all analytes showed good linearity (r>0.9959) within the test ranges. The LODs and the LOQs were less than 30.6 and 70.9 ng/mL, respectively. The RSDs of intra- and inter-day precision, repeatability and stability were below 3.64%, 4.85%, 4.84% and 3.87%, respectively. The overall recoveries ranged from 94.94% to 103.66%, with the RSDs within 5.12%. This study established a high sensitive and efficient method for the integrating quality control, including identification and quantification of Chinese medicinal preparation.

Study on Renal Protection of Gui-Zhi Decoction in Hyperuricemic Mice / 世界科学技术-中医药现代化

This study was aimed to investigate renal protective effects and mechanism of Gui-Zhi(GZ) decoction in hyperuricemic mice.Potassium oxonate was used to induce hyperuricemia mouse model.Mice were randomly divided into 6 groups,which were the blank control group,model group,allopurinol group (5 mg·kg-1) and GZ decoction group (900,1 799 and 3 598 mg·kg-1).Hematoxylin eosin staining was used to observe the histopathological changes of renal tissues in mice.Commercial assay kits were used to measure levels of uric acid (UA),creatinine (Cr) and blood urea nitrogen (BUN) in serum and urine,as well as the xanthine oxidase (XOD) activity in liver.Renal protein levels of urate transporter 1 (URAT1),glucose transporter 9 (GLUT9),ATP-binding cassette G member 2 (ABCG2),organic cation transporter 1 (OCT1),OCT2,organic cation/carnitine transporter 1 (OCTN1) and OCTN2 were detected by western blot.The results showed that compared with the model group,GZ decoction can obviously decrease serum levels of UA,Cr and BUN,increase urine levels of UA and Cr,resulting in the elevation of fractional excretion of UA in hyperuricemic mice.Additionally,GZ decoction obviously inhibited hepatic XOD activity in hyperuricemic mice.Furthermore,GZ decoction downregulated renal URAT1 and GLUT9 protein levels,upregulated renal ABCG2,as well as OCT1,OCT2,OCTN1 and OCTN2 protein levels in hyperuricemic mice.It was concluded that GZ decoction had hypouricemic and renal protective effects in hyperuricemic mice,which might be associated with the reduction of UA production via inhibiting hepatic XOD activity,promoting UA and other organic ion excretion via regulating renal organic ion transporter protein levels.