Reperfusion In Mice Research Articles

GIV/Girdin Modulation of Microglial Activation in Ischemic Stroke: Impact of FTO-Mediated m6A Modification.

Ischemic stroke (IS) is one of the most common causes of death in the world. The lack of effective pharmacological treatments for IS was primarily due to a lack of understanding of its pathogenesis. Gα-Interacting vesicle-associated protein (GIV/Girdin) is a multi-modular signal transducer and guanine nucleotide exchange factor that controls important signaling downstream of multiple receptors. The purpose of this study was to investigate the role of GIV in IS. In the present study, we found that GIV is highly expressed in the central nervous system (CNS). GIV protein level was decreased, while GIV transcript level was increased in the middle cerebral artery occlusion reperfusion (MCAO/R) mice model. Additionally, GIV was insensitive lipopolysaccharide (LPS) exposure. Interestingly, we found that GIV overexpression dramatically restrained microglial activation, inflammatory response, and M1 polarization in BV-2 microglia induced by oxygen-glucose deprivation and reoxygenation (OGD/R). On the contrary, GIV knockdown had the opposite impact. Mechanistically, we found that GIV activated the Wnt/β-catenin signaling pathway by interacting with DVL2 (disheveled segment polarity protein 2). Notably, m6A demethylase fat mass and obesity-associated protein (FTO) decreased the N6-methyladenosine (m6A) modification-mediated increase of GIV expression and attenuated the inflammatory response in BV-2 stimulated by OGD/R. Taken together, our results demonstrate that GIV inhibited the inflammatory response via activating the Wnt/β-catenin signaling pathway which expression regulated in an FTO-mediated m6A modification in IS. These results broaden our understanding of the role of the FTO-GIV axis in IS development.

Lycium Barbarum Polysaccharide-Derived Nanoparticles Protect Visual Function by Inhibiting RGC Ferroptosis and Microglial Activation in Retinal Ischemia‒Reperfusion Mice.

Retinal ischemia‒reperfusion (IR) is a major contributor to vision impairment and irreversible vision loss due to retinal ganglion cell (RGC) injury or loss. Contemporary therapeutic approaches predominantly focus on the amelioration of symptoms rather than addressing the fundamental etiological factors. Oxidative stress is a notable feature and an important mediator of IR damage. Lycium barbarum polysaccharide (LBP), the main active ingredient of Lycium barbarum, has various pharmacological effects, including antioxidation, immunoregulation, and neuroprotective effects. In this study, the ROS-consumable moiety phenylboronic acid pinacol ester (PBA) is introduced to LBP molecules, which can self-assemble into nanoparticles in aqueous solution. This nanoparticle (termed PLBP) can reduce the cellular ROS levels and enhance the antioxidant capability of RGCs by activating the NRF2 pathway, thus protecting RGCs from ferroptosis and preserving visual function in response to IR injury. PLBP also reduces neuroinflammation by inhibiting the ability of microglia to phagocytose, migrate, secrete inflammatory cytokines, and activate the NF-κB pathway. In conclusion, this approach can be used as an inspiration for the future development of neuroprotective drugs.

Weisheng-tang protects against ischemic brain injury by modulating microglia activation through the P2Y12 receptor.

Background: Stroke, a leading cause of death and disability, lacks effective treatments. Post-stroke secondary damage worsens the brain microenvironment, further exacerbating brain injury. Microglia's role in responding to stroke-induced damage in peri-infarct regions is crucial. In this study, we explored Weisheng-tang's potential to enhance ischemic outcomes by targeting microglia. Methods: We induced middle cerebral artery occlusion and reperfusion in mice, followed by behavioral assessments and infarct volume analyses after 48h, and examined the changes in microglial morphology through skeleton analysis. Results: Weisheng-tang (300mg/kg) significantly reduced infarction volume and alleviated neurological and motor deficits. The number of activated microglia was markedly increased within the peri-infarct territory, which was significantly reversed by Weisheng-tang. Microglial morphology analysis revealed that microglial processes were retracted owing to ischemic damage but were restored in Weisheng-tang-treated mice. This restoration was accompanied by the expression of the purinergic P2Y12 receptor (P2Y12R), a key regulator of microglial process extension. Weisheng-tang increased neuronal Kv2.1 clusters while suppressing juxtaneuronal microglial activation. The P2Y12R inhibitor-ticagrelor-eliminated the tissue and functional recovery that had been observed with Weisheng-tang after ischemic damage. Discussion: Weisheng-tang improved experimental stroke outcomes by modulating microglial morphology through P2Y12R, shedding light on its neuroprotective potential in ischemic stroke.

Deficiency of flavin-containing monooxygenase 3 protects kidney function after ischemia–reperfusion in mice

The kidney is vulnerable to ischemia and reperfusion (I/R) injury that can be fatal after major surgery. Currently, there are no effective treatments for I/R-induced kidney injury. Trimethylamine N-oxide (TMAO) is a gut-derived metabolite linked to many diseases, but its role in I/R-induced kidney injury remains unclear. Here, our clinical data reveals an association between preoperative systemic TMAO levels and postoperative kidney injury in patients after post-cardiopulmonary bypass surgery. By genetic deletion of TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) and dietary supplementation of choline to modulate TMAO levels, we found that TMAO aggravated acute kidney injury through the triggering of endoplasmic reticulum (ER) stress and worsened subsequent renal fibrosis through TGFβ/Smad signaling activation. Together, our study underscores the negative role of TMAO in I/R-induced kidney injury and highlights the therapeutic potential through the modulation of TMAO levels by targeting FMO3, thereby mitigating acute kidney injury and preventing subsequent renal fibrosis.

Exogenous leptin alleviates glutamate-excitotoxic injury caused by cerebral ischemia–reperfusion in mice by affecting the expression of glutamate transporters

Ischemic stroke is characterized by high morbidity and mortality and a lack of effective therapeutic interventions. Leptin plays an important role in regulating oxidative stress, angiogenesis, hematopoiesis, etc. Although recent studies have found a neuroprotective effect of leptin, little is known about its role in cerebral ischemia. This study explores the possible roles and potential preventative mechanisms of leptin in cerebral ischemia–reperfusion injury (CIRI). An in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was used to replicate the CIRI model, low (0.5 mg/kg), medium (1 mg/kg) and high (2 mg/kg) concentrations of leptin were injected intraperitoneally immediately after inserting the embolic line. After 1.5 h of ischemia and 24 h of reperfusion, we examined the neural function of the mice, collected brain tissue for histological examination, and screened for the optimal concentrations of leptin intervention. On this basis, we observed the changes of cortical apoptosis injury, intracellular calcium fluorescence intensity and astrocyte glial fibrillary acidic protein (GFAP) expression and morphological changes. In addition, we also tested the expression of transporters and metabolism-related enzymes (VGLUT-1, VGLUT-2, GLAST, GLT-1, GS, ATP1α1), the expression of inflammatory factors and the content of glutamate (Glu). Compared with the I/R group, we found that leptin improved neurological deficits, reduced the area of infarcts, maintained the normal morphology of astrocytes (AST), downregulated the expression of VGLUT-1, and upregulates the expression of GLT-1 and GLAST, thereby reducing the content of Glu in the synaptic cleft. Our studies suggest that leptin may have a neuroprotective effect by decreasing the excitotoxicity of glutamate.

Obesity aggravates acute kidney injury resulting from ischemia and reperfusion in mice

In critically ill patients, overweight and obesity are associated with acute respiratory distress syndrome and acute kidney injury (AKI). However, the effect of obesity on ischemia–reperfusion injury (IRI)-induced AKI is unknown. We hypothesized that obesity would aggravate renal IRI in mice. We fed mice a standard or high-fat diet for eight weeks. The mice were divided into four groups and submitted to sham surgery or IRI: obese, normal, normal + IRI, obese, and obese + IRI. All studies were performed 48 h after the procedures. Serum glucose, cholesterol, and creatinine clearance did not differ among the groups. Survival and urinary osmolality were lower in the obese + IRI group than in the normal + IRI group, whereas urinary neutrophil gelatinase-associated lipocalin levels, tubular injury scores, and caspase 3 expression were higher. Proliferating cell nuclear antigen expression was highest in the obese + IRI group, as were the levels of oxidative stress (urinary levels of thiobarbituric acid-reactive substances and renal heme oxygenase-1 protein expression), whereas renal Klotho protein expression was lowest in that group. Expression of glutathione peroxidase 4 and peroxiredoxin 6, proteins that induce lipid peroxidation, a hallmark of ferroptosis, was lower in the obese + IRI group. Notably, among the mice not induced to AKI, macrophage infiltration was greater in the obese group. In conclusion, greater oxidative stress and ferroptosis might aggravate IRI in obese individuals, and Klotho could be a therapeutic target in those with AKI.

Mechanism of microRNA regulating apoptosis after reperfusion in patients with mechanical thrombectomy.

Stroke is the second leading cause of death worldwide. Understanding of gene expression dynamics could bring new approaches in diagnostics and therapy of stroke. Small noncoding molecules termed 'microRNA' represent the most flexible network of gene expression regulators. To screen out miRNAs that are mainly regulated during reperfusion in mechanically embolized patients, and study their mechanisms of action in reperfusion injury after thrombectomy, in order to find new therapeutic targets for mechanically embolized patients. Serums from 30 patients with moderate to severe stroke after mechanical thrombectomy (MT) were collected to measure miRNA expressions. Clinical information of patients was analyze, and patients were divided into poor prognosis and good prognosis. Factors affecting prognosis was classified, and independent risk factors for poor prognosis were determined. Prognostic value of National Institutes of Health Stroke Scale (NIHSS) score on admission to patients with MT was assessed. ROC (receiver operating characteristic) curves were drawn, and Kaplan-Merier method determined whether different NIHSS scores at admission had any difference in the in-hospital survival rate of consistency index/random consistency index (CI/RI) patients treated with MT. An oxygen-glucose deprivation/reperfusion (OGD/R) cell model and an middle cerebral artery occlusion (MCAO)/reperfusion mouse model were established, in which miR-298 expression was tested. In OGD/R cells, proliferation, apoptosis, and autophagy were assessed after intervention with miR-298 and/or autophagy related gene 5 (ATG5). In MCAO mice, the infarct area was calculated, and neurological function was assessed. The relationship between miR-298 and ATG5 was explored and validated. Age, diabetes, hypertension, hemorrhage transformation, NIHSS score at admission, leukocyte, neutrophil count and neutrophil to lymphocyte ratio (NLR) level were associated with patient's prognosis. Diabetes, NIHSS score at admission, and hemorrhagic transformation were independent risk factors for predicting poor prognosis in patients treated with MT. NIHSS score on admission had a predictive value on patient's prognosis. miR-298 was upregulated in acute cerebral ischemia patients with MT (p<0.05), especially in those with poor prognosis. miR-298 was elevated in both cell and mouse models (p<0.05). Apoptosis and autophagy of cells were weakened after miR-298 knockdown, and infarction in the mouse brain tissues was reduced. ATG5 was a target of miR-298. Overexpressing ATG5 rescued miR-298-induced apoptosis and autophagy. In conclusion: regulation of miR-298 and ATG5 attenuates neuronal apoptosis and autophagy, providing a new strategy for brain injury after reperfusion in patients with MT.

Recombinant Human Heavy Chain Ferritin Nanoparticles Serve as ROS Scavengers for the Treatment of Ischemic Stroke.

Ischemic stroke is a high-incidence disease that threatens human well-being. The potent neuroprotective effects render reactive oxygen species (ROS) scavengers potential agents for acute ischemic stroke therapy. Challenges such as inadequate permeability across the blood-brain barrier (BBB), limited half-life, and adverse effects hinder the widespread utilization of small molecule and inorganic ROS scavengers. Thus, there is an urgent demand for efficacious neuroprotective agents targeting ischemic stroke. Our study discovered the superoxide dismutase (SOD)-mimetic activity of recombinant human heavy chain ferritin (rHF) nanoparticles expressed from Escherichia coli (E. coli). Subsequent investigations delved into the ROS-scavenging proficiency of rHF within neural cells, its therapeutic efficacy against ischemic stroke, and the elucidation of its neuroprotective mechanisms. rHF protein nanoparticles were expressed in E. coli and purified via size-exclusion chromatography. The superoxide anion (•O2-) scavenging SOD-mimetic activity of rHF nanoparticles was measured using a SOD detection kit. The ROS scavenging ability and protection effects against oxidative damage of rHF nanoparticles were studied in H2O2-induced PC12 cells. Therapeutic effects and neuroprotective mechanisms of rHF against ischemic stroke were investigated with transient middle cerebral artery occlusion (MCAO) reperfusion mice model. rHF nanoparticles can eliminate excessive ROS in nerve cells and alleviate oxidative damage. The results of animal experiments demonstrated that rHF nanoparticles passed across BBB, reduced infarct areas in brain tissue, and lowered the neurological deficit score of ischemia-reperfusion model mice. Additionally, rHF nanoparticles mitigated neuronal apoptosis and ferroptosis, suppressed microglial activation, maintained oxygen homeostasis, and exhibited negligible organ toxicity. rHF nanoparticle could be developed as a new ROS scavenger for nerve cells and has therapeutic potential as a drug for cerebral ischemia-reperfusion injury.

Downregulation of Nrp1 transcription promotes blood–brain barrier disruption following experimental cerebral ischemia–reperfusion

Disruption of the blood–brain barrier (BBB) following cerebral ischemia–reperfusion injury (CIRI) is a major factor in the pathophysiology of stroke. Endothelial cell–cell communication is essential for maintaining BBB integrity. By analyzing GSE227651 data, we found that a decrease in endothelial cell–cell communication mediated by Sema3/Nrp1 may be due to the downregulation of Nrp1 transcription, which could contribute to BBB breakdown after CIRI. We confirmed this hypothesis by using western blot analysis to show a reduction in Nrp1 protein levels in penumbra endothelial cells after CIRI in mice. We then overexpressed Nrp1 specifically in brain endothelial cells using adeno-associated virus in mice. Furthermore, Nrp1 overexpression had a protective effect on BBB integrity, as evidenced by a decrease in IgG and albumin leakage caused by CIRI in mice. Finally, we found that Nrp1 overexpression also reduced brain cell death and neurological deficits induced by cerebral ischemia–reperfusion in mice. Our findings suggest that Nrp1 downregulation may be a key factor in the breakdown of endothelial cell–cell communication and subsequent BBB disruption following CIRI. Targeting Nrp1-mediated pathways may be a promising approach for mitigating BBB damage and alleviating neurological consequences in stroke patients.

Hyperoxia Increases Kidney Injury During Renal Ischemia and Reperfusion in Mice.

Renal ischemia and reperfusion (IR) contribute to perioperative acute kidney injury, and oxygen is a key regulator of this process. We hypothesized that oxygen administration during surgery and renal IR would impact postoperative kidney function and injury in mice. Mice were anesthetized, intubated, and mechanically ventilated with a fraction of inspired oxygen (F io2 ) 0.10 (hypoxia), 0.21 (normoxia), 0.60 (moderate hyperoxia), or 1.00 (severe hyperoxia) during 67 minutes of renal IR or sham IR surgery. Additional mice were treated before IR or sham IR surgery with 50 mg/kg tempol, a superoxide scavenger. At 24 hours, mice were sacrificed, and blood and kidney collected. We assessed and compared kidney function and injury across groups by measuring blood urea nitrogen (BUN, primary end point), renal histological injury, renal expression of neutrophil gelatinase-associated lipocalin (NGAL), and renal heme oxygenase 1 ( Ho-1 ), peroxisome proliferator-activated receptor gamma coactivator 1-α ( Pgc1-α ), and glutathione peroxidase 4 ( Gpx-4 ) transcripts, to explore potential mechanisms of any effect of oxygen. Hyperoxia and hypoxia during renal IR surgery decreased renal function and increased kidney injury compared to normoxia. Baseline median (interquartile range) BUN was 22.2 mg/dL (18.4-26.0), and 24 hours after IR surgery, BUN was 17.5 mg/dL (95% confidence interval [CI], 1.3-38.4; P = .034) higher in moderate hyperoxia-treated animals, 51.8 mg/dL (95% CI, 24.9-74.8; P < .001) higher in severe hyperoxia-treated animals, and 64.9 mg/dL (95% CI, 41.2-80.3; P < .001) higher in hypoxia-treated animals compared to animals treated with normoxia ( P < .001, overall effect of hyperoxia). Hyperoxia-induced injury, but not hypoxia-induced injury, was attenuated by pretreatment with tempol. Histological injury scores, renal NGAL staining, and renal transcription of Ho-1 and suppression of Pgc1- α followed the same pattern as BUN, in relation to the effects of oxygen treatment. In this controlled preclinical study of oxygen treatment during renal IR surgery, hyperoxia and hypoxia impaired renal function, increased renal injury, and impacted expression of genes that affect mitochondrial biogenesis and antioxidant response. These results might have implications for patients during surgery when high concentrations of oxygen are frequently administered, especially in cases involving renal IR.

The effects of protein-rich extract from Rhizoma Gastrodiae against cerebral ischemia/reperfusion injury via regulating MAPK and PI3K/AKT signaling pathway

BackgroundRhizoma Gastrodiae is a highly valuable traditional Chinese medicine and functional health food that has been used in China to treat neurological disorders for thousands of years. Rhizoma Gastrodiae contains various of biological activities, such as antioxidative, neuroprotective, learning improvement, anxiolytic, and antidepressant effects. However, no studies have been conducted to explore the effects of the protein components in Rhizoma Gastrodiae (GEPS) and its potential protective effects against ischemic stroke.Our main goal was to investigate the effects of GEPS on ischemia/reperfusion (I/R) injury and its possible mechanisms. MethodsA middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia mouse model and an oxygen-glucose deprivation (OGD/R) injury model in HT22 cells were established. A neurobehavioral test was performed 24 h after MCAO, and brain infarction was measured. A Morris water maze experiment was conducted on Day 14 after reperfusion in mice. Hematoxylin and eosin (HE) and TUNEL staining were performed to assess apoptotic neuronal death. Immunohistochemical analysis was used to detect BDNF and GAP43 expression. The content of SOD, MDA, GSH-PX and ROS were detected. The protein expression was analyzed using Western blotting. Cell viability was determined by MTT assay. Cell apoptosis was examined by flow cytometry. ResultsGEPS reduced apoptosis, decreased cerebral infarction, improved neurological defects, and ameliorated oxidative stress in the ischemic penumbra. In addition, GEPS increased the expression of BDNF and GA43 in the penumbra. Mechanistically, GEPS counteracted MCAO-induced PI3K/AKT inhibition and activation of MAPK signaling pathways. ConclusionGEPS has a clear neuroprotective effect on I/R injury, and its mechanism may be linked to the PI3K/AKT and MAPK signaling pathways.

Dynamic Assessments of Coronary Flow Reserve after Myocardial Ischemia Reperfusion in Mice.

After cardiac ischemia, there is often insufficient myocardial perfusion, even if flow has been successfully and completely restored in an upstream artery. This phenomenon, known as the "no-reflow phenomenon," is attributed to coronary microvascular dysfunction and has been associated with poor clinical outcomes. In clinical practice, a reduction in coronary flow reserve (CFR) is frequently used as an indicator of coronary artery disease. CFR is defined as the ratio of the peak flow velocity induced by pharmacologic or metabolic factors to the resting flow velocity. This protocol focused on assessing the dynamic changes in CFR before and after ischemia-reperfusion (IR) using pulse wave Doppler measurements. In this study, normal mice exhibited the ability to increase the peak velocity of coronary blood flow up to two times higher than the resting values under isoflurane stimulation. However, after ischemia-reperfusion, the CFR at 1 h significantly decreased compared to the pre-operation baseline. Over time, the CFR showed gradual recovery, but it remained below the normal level. Despite the preservation of systolic function, early detection of microvascular dysfunction is crucial, and establishing a practical guide could aid doctors in this task, while also facilitating the study of cardiovascular disease progression over time.

METTL3-deficiency Suppresses Neural Apoptosis to Induce Protective Effects in Cerebral I/R Injury via Inhibiting RNA m6A Modifications: A Pre-clinical and Pilot Study.

N6-Methyladenosine (m6A) RNA methylation involves in regulating the initiation, progression and aggravation of cerebral ischemia-reperfusion (I/R) injury, however, the detailed functions and mechanisms by which m6A drives cerebral I/R injury are not fully understood. This study found that methyltransferase-like 3 (METTL3) m6A-dependently regulated cerebral I/R injury trough regulating a novel LncRNA ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α axis. Specifically, the middle cerebral artery occlusion (MCAO)/reperfusion mice models and glucose deprivation (OGD)/reoxygenation (RX) astrocyte cell models were respectively established, and we verified that METTL3, ABHD11-AS1 and HIF1AN were upregulated, whereas miR-1301-3p and HIF-1α were downregulated in both MCAO/reperfusion mice tissues and OGD/RX astrocytes. Mechanical experiments confirmed that METTL3 m6A dependently increased stability and expression levels of ABHD11-AS1, and elevated ABHD11-AS1 sponged miR-1301-3p to upregulate HIF1AN, resulting in the downregulation of HIF-1α. Moreover, silencing of METTL3 rescued MCAO/reperfusion and OGD/RX-induced oxidative stress-associated cell apoptosis and cell cycle arrest in both mice brain tissues in vivo and the mouse primary astrocytes in vitro, which were abrogated by overexpressing ABHD11-AS1 and downregulating miR-1301-3p. Taken together, our study firstly reported a novel METTL3/m6A/ ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α signaling cascade in regulating the progression of cerebral I/R injury, and future work will focus on investigating whether the above genes can be used as biomarkers for the treatment of cerebral I/R injury by performing clinical studies.

CCA repair or ECA ligation-Which middle cerebral artery occlusion is better in the reperfusion mouse model?

A reliable animal model is essential for ischemic stroke research. The implications of the external carotid artery (ECA) transection or common carotid artery (CCA) ligation have been described. Thus, a modified animal model, the CCA-repair model, has been established, and studies have shown that the CCA-repair model has potential advantages over the CCA-ligation model. However, whether the CCA-repair model is superior to the ECA-ligation model remains unclear. Sixty male C57BL/6 mice were randomly assigned to establish the CCA-repair (n = 34) or ECA-ligation (n = 26) models. Cerebral blood flow before middle cerebral artery occlusion (MCAO), immediately after MCAO and reperfusion were monitored and the operation duration, postoperative body weight, and food intake within 7 days, and the number of intraoperative and postoperative deaths within 7 days were recorded in the two models. Modified neurological severity scores and Bederson (0-5) scores were used to evaluate postoperative neurological function deficits on Days 1/3/5/7. 2,3,5-Triphenyltetrazolium chloride staining was used to quantify lesion volume on Day 7 after the operation. We found the establishment of the CCA-repair model required a longer total operation duration (p = 0.0175), especially the operation duration of reperfusion (p < 0.0001). However, there was no significant difference in body weight and food intake development, lesion volume and intragroup variability, neurological function deficits, mortality, and survival probability between the two groups. The CCA-repair model has no significant advantage over the ECA-ligation model. The ECA-ligation model is still a better choice for focal cerebral ischemia.

Elucidating the chemical interaction effects of herb pair Danshen-Chuanxiong and its anti-ischemic stroke activities evaluation

Ethnopharmacological relevanceSalvia miltiorrhiza Bunge (Danshen) and Ligusticum chuanxiong Hort. (Chuanxiong) is the core herb pair in traditional Chinese medicines (TCMs) formulae for treating ischemic stroke. However, the synergistic effect of Danshen-Chuanxiong against anti-ischemic stroke and its compatibility mechanism remains unclear. Aim of the studyThis study aimed to uncover the compatibility mechanism of Danshen-Chuanxiong against ischemic stroke through chemical profiling, pharmacodynamics evaluation, network pharmacology and experimental validation. Materials and methodsUltra-high performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS) and UHPLC connected with tandem triple quadrupole mass spectrometry (QQQ-MS) were utilized to conduct the chemical interaction analysis. Then the synergistic effects of Danshen-Chuanxiong against ischemic stroke were comprehensively evaluated by the middle cerebral artery occlusion reperfusion (MCAO/R) mice model, zebrafish ischemic stroke model and glutamic acid-induced PC12 cells injury model. Afterwards, network pharmacology and molecular docking were applied to dissect the significant active compounds and potential mechanisms. Finally, the key target proteins were experimentally validated by Western blot. Results83 compounds were characterized in Danshen-Chuanxiong by UHPLC-QTOF-MS analysis, and 4 compounds were tentatively identified for the first time. The quantification results (24 accurately identified compounds) in 13 proportions of Danshen-Chuanxiong revealed that Danshen significantly increased the dissolution of most phthalides (from Chuanxiong), while Chuanxiong facilitated the dissolution of most phenolic acids (from Danshen) in solution. The anti-ischemic stroke effects of Danshen-Chuanxiong were significantly better than Danshen or Chuanxiong in attenuating infarct size, reducing brain edema and neurological scores in MCAO/R mice. Also, compared with single herbs, this herb pair exerted better effects of suppressing the incidence of cerebral thrombosis in zebrafish, and increasing the cell viability of glutamic acid-induced PC12 cells. In network pharmacology, 7 effective compounds (rosmarinic acid, chlorogenic acid, salvianolic acid B, (Z)-ligustilide, ferulic acid, caffeic acid, tanshinone IIA) and 5 hub targets (AKT, TNF−α, IL-1β, CASP3 and BCL2) as well as 4 key pathways were predicted. Western blot results showed that Danshen-Chuanxiong exert therapeutic effects mainly through decreasing the protein expressions of TNF-α, IL-1β and Cleaved-CASP3, elevating the levels of p-AKT and BCL2. ConclusionsThis work provided an integration strategy for uncovering the synergistic effects and compatibility mechanism of Danshen-Chuanxiong herb pair for treating ischemic stroke, and laid foundation for the further development and utilization of this herb pair.

Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice

The protective effects of remote ischemic conditioning (RIC) on acute ischemic stroke have been reported. However, the protective mechanisms of RIC have not been fully elucidated. This study aimed to investigate whether RIC could reduce oxidative stress and inflammatory responses in middle cerebral artery occlusion (MCAO)-reperfusion mice via the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. C57BL/6 mice were subjected to MCAO and underwent RIC twice daily at 1, 3, and 7 days after MCAO. ML385 was used to specifically inhibit Nrf2 in MCAO mice. Neurological deficit scores, infarct volume, and hematoxylin-eosin (HE) staining were assessed. Oxidative stress levels were assessed based on total antioxidant capacity (TAC), malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione/glutathione disulfide (GSH/GSSG). mRNA levels were detected using real-time polymerase chain reaction (PCR), and protein levels were detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Protein localization was investigated using immunofluorescence staining. RIC significantly reduced infarct volume and improved neurological function and histological changes after MCAO. RIC significantly increased TAC, SOD, and GSH/GSSG levels and decreased MDA levels. RIC significantly increased Nrf2 and HO-1 mRNA levels and decreased Keap1, NLRP3, and Cleaved Caspase-1 mRNA levels. RIC significantly increased Nrf2, HO-1, and NQO1 protein expression and decreased Keap1, NLRP3, Cleaved Caspase-1, Cleaved IL-1β, IL-6, and TNF-α protein expression. RIC promoted the activation and translocation of Nrf2 into the nucleus. The protective effects of RIC were abolished by ML385 treatment. In conclusion, our findings suggest that RIC alleviates oxidative stress and inflammatory responses via the Nrf2/HO-1 pathway, which in turn improves neurobehavioral function. RIC may provide novel therapeutic options for acute ischemic stroke.

Mannose binding lectin-associated serine protease-1 is a novel contributor to myocardial ischemia/reperfusion injury

BackgroundThe lectin pathway has been demonstrated to play a critical role in the pathological process of myocardial ischemia/reperfusion injury (IRI). Mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1), especially different from other components of the lectin pathway, mediates proinflammatory and procoagulant reactions independent of complement cascades. However, the role of MASP-1 in myocardial IRI remains unknown so far. MethodsMyocardial IRI was established with 45 min ischemia and 24 h reperfusion in mice. C1 inhibitor, as the natural inhibitor of MASP-1, was administrated at 20 IU/Kg via tail vein 5 min before surgical operation. Cardiac function and myocardial infarct size were assessed. Myocardial histology and fibrosis were evaluated by H&E and Masson staining, respectively. Deposition of MASP-1, expression of PAR-1/4 and neutrophil extracellular traps (NET) were investigated on myocardium tissue by IHC staining. Cell apoptosis was detected by TUNEL assay. Levels of myocardial enzymes and proinflammatory cytokines were determined by ELISA. ResultsInhibition of MASP-1 with C1 INH improved cardiac function and alleviated myocardium tissue injury (infarct size, enzymes, histology and fibrosis) after myocardial IRI. Deposition of MASP-1 and expression PAR-1, as well as NET formation in myocardial tissue were suppressed by MASP-1 inhibitor, while PAR-4 was elevated. Levels of apoptosis, HMGB-1 and IL-6 were lower after blocking MASP-1. Yet, IL-8 and TNF-α remained unchanged. ConclusionsMASP-1, as a new contributor, played a critical role in myocardial IRI. Inhibition of MASP-1 protected myocardial tissue from IRI probably via regulation of PARs/NET pathway. This may provide a novel target strategy against myocardial IRI.

Protection of a novel velvet antler polypeptide PNP1 against cerebral ischemia-reperfusion injury

AimWe isolated a novel polypeptide PNP1 from velvet antler and investigated the role of PNP1 in ischemia reperfusion and its associated mechanism. MethodsWe built the ischemia reperfusion mouse model by the middle cerebral artery occlusion (MCAO) approach. Thereafter, PNP-1 was injected via the tail vein, and neurological function was scored. Meanwhile, the tissue injury level was detected through hematoxylin & eosin (HE) and immunohistochemical (IHC) staining, inflammatory factor levels were determined with enzyme-linked immunosorbent assay (ELISA), while protein levels through Western blotting. In addition, vascular endothelial cells were used to construct the oxygen-glucose deprivation (OGD) injury model in vitro, so as to detect the intervention effect of PNP1 on endothelial injury. Additionally, microglial cells were utilized to construct the inflammatory injury model to examine the impact of PNP1 on the polarization of microglial cells. ResultsPNP1 suppressed hypoxic cerebral injury in MCAO mice, decreased the tissue inflammatory factors, promoted tissue angiogenesis, and reduced the ischemic penumbra area. Experimental results in vitro demonstrated that, PNP1 suppressed vascular endothelial cell injury, and inhibited microglial M1 polarization as well as inflammatory response. ConclusionVelvet antler polypeptide PNP1 isolated in this study has the anti-ischemic cerebral injury effect, and its mechanism is associated with suppressing vascular endothelial cell injury and microglial cell inflammatory response.

Treatment With a High-dose of the Omega-3 Fatty Acid, Eicosapentaenoic Acid (EPA), Results in Accelerated Post-ischaemic Reperfusion in Mice

Treatment With a High-dose of the Omega-3 Fatty Acid, Eicosapentaenoic Acid (EPA), Results in Accelerated Post-ischaemic Reperfusion in Mice

Extracellular RNAs/TLR3 signaling contributes to acute intestinal injury induced by intestinal ischemia reperfusion in mice

Toll-like receptor 3 (TLR3), one pattern recognition receptor activated by viral and endogenous RNA, has been recently reported to regulate ischemia/reperfusion (I/R) injury in various organs. However, the role of TLR3 in the development of intestinal I/R injury remains unclear. The aim of this study is to evaluate the effects of extracellular RNAs/TLR3 signaling in intestinal I/R injury. An intestinal I/R injury model was established with superior mesenteric artery occlusion both in wild-type and TLR3 knockout (KO, −/−) mice, and MODE-K cells were subjected to hypoxia/reoxygenation (H/R) to mimic the I/R model in vivo. Extracellular RNAs (exRNAs), especially double-stranded RNAs (dsRNAs) co-localized with TLR3, were significantly increased both in vitro and in vivo. Compared with wild-type mice, TLR3 knockout obviously attenuated intestinal I/R injury. Both TLR3/dsRNA complex inhibitor and TLR3 siRNA administration reduced TLR3 expressions and subsequently inhibited intestinal inflammatory cytokine production and apoptosis. In conclusion, exRNAs/TLR3 signaling is a key mechanism that regulates intestinal I/R injury in adult mice, and the TLR3/dsRNA complex inhibitor can be an effective approach for attenuating intestinal I/R-induced inflammatory response and apoptosis.