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Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

ABSTRACT: Guhong injection (GHI) is a drug for ischemic stroke created by combining safflower, a traditional Chinese medicine, and aceglutamide, a Western medicine. In this study, we investigated the curative effect of GHI on cerebral ischemia-reperfusion (I/R) injury via the PKC/HIF-1α pathway in rats. Adult male Sprague Dawley rats were randomly divided into seven groups: sham-operated, middle cerebral artery occlusion (MCAO), GHI, nimodipine injection (NMDP), MCAO + LY317615 (PKC inhibitor), GHI + LY317615, and NMDP + LY317615. After establishing an MCAO rat model, we performed neurological deficit testing, 2,3,5-triphenyltetrazolium chloride staining, hematoxylin and eosin (HE) staining, enzyme-linked immunosorbent assay, Western blotting, and q-PCR to detect the brain damage in rats. Compared with the MCAO group, the GHI and GHI + LY317615 group showed neurological damage amelioration as well as decreases in serum hypoxia-inducible factor-1α (HIF-1α), protein kinase C (PKC), and erythropoietin levels; brain HIF-1α and inducible nitric oxide synthase protein expression; and brain HIF-1α and NOX-4 mRNA expression. These effects were similar to those in the positive control groups NMDP and NMDP + LY317615. Thus, our results confirmed GHI can ameliorate cerebral I/R injury in MCAO rats possibly via the PKC/HIF-1α pathway.

SUBMITTER: Yu L

PROVIDER: S-EPMC8443782 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

Yu Li L Zhang Yangyang Y Zhao Xixi X Wan Haitong H He Yu Y Jin Weifeng W

Frontiers in pharmacology 20210902

Guhong injection (GHI) is a drug for ischemic stroke created by combining safflower, a traditional Chinese medicine, and aceglutamide, a Western medicine. In this study, we investigated the curative effect of GHI on cerebral ischemia-reperfusion (I/R) injury via the PKC/HIF-1α pathway in rats. Adult male Sprague Dawley rats were randomly divided into seven groups: sham-operated, middle cerebral artery occlusion (MCAO), GHI, nimodipine injection (NMDP), MCAO + LY317615 (PKC inhibitor), GHI + LY31 ...[more]

PMID: 34539402

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Project description:Ischemic stroke is an acute and severe neurological disease with high mortality and disability rates worldwide. Polymerase I and transcript release factor (PTRF) plays a pivotal role in regulating cellular senescence, glucose intolerance, lipid metabolism, and mitochondrial bioenergetics, but its mechanism, characteristics, and functions in neuronal cells following the cerebral ischemia-reperfusion (I/R) injury remain to be determined. Methods: Transcription factor motif analysis, chromatin immunoprecipitation (ChIP), luciferase and co-Immunoprecipitation (co-IP) assays were performed to investigate the mechanisms of PTRF in neuronal cells after I/R injury. Lentiviral-sgRNA against PTRF gene was introduced to HT22 cells, and adeno-associated virus (AAV) encoding a human synapsin (hSyn) promoter-driven construct was transduced a short hairpin RNA (shRNA) against PTRF mRNA in primary neuronal cells and the cortex of the cerebral I/R mice for investigating the role of PTRF in neuronal damage and PLA2G4A change induced by the cerebral I/R injury. Results: Here, we reported that neuronal PTRF was remarkably increased in the cerebral penumbra after I/R injury, and HIF-1α and STAT3 regulated the I/R-dependent expression of PTRF via binding to its promoter in neuronal cells. Moreover, overexpression of neuronal PTRF enhanced the activity and stability of PLA2G4A by decreasing its proteasome-mediated degradation pathway. Subsequently, PTRF promoted reprogramming of lipid metabolism and altered mitochondrial bioenergetics, which could lead to oxidative damage, involving autophagy, lipid peroxidation, and ferroptosis via PLA2G4A in neuronal cells. Furthermore, inhibition of neuronal PTRF/PLA2G4A-axis markedly reduced the neurological deficits, cerebral infarct volumes, and mortality rates in the mice following cerebral I/R injury. Conclusion: Our results thus identify that the STAT3/HIF-1α/PTRF-axis in neurons, aggravating cerebral I/R injury by regulating the activity and stability of PLA2G4A, might be a novel therapeutic target for ischemic stroke.

Project description:AimReprogramming of glycometabolism plays a crucial role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). Sestrin2 (SESN2), a sensor upstream of the mTORC1, is closely related to glycometabolism. However, the effect and mechanism of SESN2 in CIRI are unclear. The goal of this research was to explore the effect of SESN2 on CIRI and its potential mechanisms related to glycometabolism.MethodsLentiviral vectors carrying SESN2 shRNA (Lenti-SESN2) or negative NC virus (Lenti-GFP) or rapamycin (mTOR inhibitor) were employed in the oxygen-glucose deprivation/reoxygenation (OGD/R) model and in the middle cerebral artery occlusion (MCAO) mice. In all, 3 days after I/R, neurological deficit scores and infarct size were assessed. The glycolysis and SESN2 levels were determined by RT-qPCR, Western blots, and immunofluorescence staining. Lactate levels were detected by a lactate assay kit, and the expression of the p-mTOR/HIF-1α signaling pathway was measured by immunofluorescence staining and protein blotting.ResultsLocal SESN2 deficiency in brain tissue increased the infarct size and reduced neurological scores 3 days after I/R. Moreover, the results showed that local SESN2 deficiency in brain tissue increased the expression of glycolysis-related proteins, including HK2, PFKM, PKM1, PKM2, and GLUT1. The lactate assay kit showed that local SESN2 deficiency in brain tissue increased lactate levels. In addition, local SESN2 deficiency in brain tissue improved the expression of the p-mTOR/HIF-1α pathway. However, rapamycin (RAP) treatment reversed these results, suggesting that SESN2 may influence IS injury by regulating glycometabolism via p-mTOR/HIF-1α pathway regulation. SESN2 knockdown in BV2 cells improved the glycolysis levels and the expression of the mTOR/HIF-1α pathway in the OGD/R model in vitro, but RAP treatment can also reverse these results.ConclusionsKnockdown of SESN2 in MCAO mice increased the expression of the p-mTOR/HIF-1α pathway, which increased glycolysis and lactate levels and, in turn, affected IS injury.

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Project description:Guhong injection (GHI) has been applied in the therapy of cardio-cerebrovascular disease in clinic, but there is no report about the pharmacokinetic/pharmacodynamic (PK/PD) research on GHI treating myocardial ischemia/reperfusion (MI/R) injury in rats. In this study, eight compounds of GHI in plasma, including N-acetyl-L-glutamine (NAG), chlorogenic acid (CGA), hydroxysafflor yellow A (HSYA), p-coumaric acid ( pCA), rutin, hyperoside, kaempferol-3-O-rutinoside, and kaempferol-3-O-glucoside, were quantified by LC-MS/MS. We discovered that the values of t1/2β, k12, V2, and CL2 were larger than those of t1/2α, k21, V1, and CL1 for all compounds. The levels of four biomarkers, creatine kinase-MB (CK-MB), cardiac troponin I (cTn I), ischemia-modified albumin (IMA), and alpha-hydroxybutyrate dehydrogenase (α-HBDH) in plasma were determined by ELISA. The elevated level of these biomarkers induced by MI/R was declined to different degrees via administrating GHI and verapamil hydrochloride (positive control). The weighted regression coefficients of NAG, HSYA, CGA, and pCA in PLSR equations generated from The Unscrambler X software (version 11) were mostly minus, suggesting these four ingredients were positively correlated to the diminution of the level of four biomarkers. Emax and ED50, two parameters in PK/PD equations that were obtained by adopting Drug and Statistics software (version 3.2.6), were almost enlarged with the rise of GHI dosage. Obviously, all analytes were dominantly distributed and eliminated in the peripheral compartment with features of rapid distribution and slow elimination. With the enhancement of GHI dosage, the ingredients only filled in the central compartment if the peripheral compartment was replete. Meanwhile, high-dose of GHI generated the optimum intrinsic activity, but the affinity of compounds with receptors was the worst, which may be caused by the saturation of receptors. Among the eight analytes, NAG, HSYA, CGA, and pCA exhibited superior cardioprotection, which probably served as the pharmacodynamic substance basis of GHI in treating MI/R injury.

Dataset Information

Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

Publications

Guhong Injection Alleviates Cerebral Ischemia-Reperfusion Injury via the PKC/HIF-1α Pathway in Rats.

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