Naoxintong capsule accelerates mitophagy in cerebral ischemia-reperfusion injury via TP53/PINK1/PRKN pathway based on network pharmacology analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence and mortality of cerebrovascular diseases are increasing year by year. Cerebral ischemia-reperfusion injury (CIRI) is common in patients with ischemic stroke. Naoxintong (NXT) is composed of a variety of Chinese medicines and has the ability to treat CIRI. AIM OF THE STUDY: The aim of this study is to investigate whether NXT regulates mitophagy in CIRI based on network pharmacology analysis and experimental validation. MATERIALS AND METHODS: Oxygen and glucose deprivation/re-oxygenation (OGD/R, 2/22 h) model of PC12 cells and transient middle cerebral artery occlusion (tMCAO, 2/22 h) model of rats were established. Pharmacodynamic indicators include neurological deficit score, 2,3,5-triphenyte-trazoliumchloride (TTC) staining, hematoxylin-eosin (HE) staining and cell viability. Network pharmacology was used to predict pharmacological mechanisms. Pharmacological mechanism indexes include transmission electron microscopy (TEM), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), immunohistochemistry (IHC), western blot (WB) and immunofluorescence (IF). Kevetrin (an agonists of p53) and pifithrin-α (an inhibitor of p53) used to detect the key role of p53 in mitophagy of NXT. RESULTS: NXT (1% serum containing NXT and 110 mg/kg) improved the damage of OGD/R PC12 cells and tMCAO rats, and this protective effect was related to the anti-oxidation and ability to promote mitophagy of NXT. NXT and pifithrin-α increased the expression of promoting-mitophagy targets (PINK1, PRKN and LC3B) and inhibited the expression of inhibiting-mitophagy targets (p52) via restraining p53, and finally accelerated mitophagy caused by CIRI. CONCLUSION: This study demonstrates that NXT promotes mitophagy in CIRI through restraining p53 and promoting PINK1/PRKN in vivo and in vitro.

CTGF regulated by ATF6 inhibits vascular endothelial inflammation and reduces hepatic ischemia-reperfusion injury.

Vascular endothelial inflammation is crucial in hepatic ischemia-reperfusion injury (IRI). Our previous research has shown that connective tissue growth factor (CTGF), secreted by endothelial cells, protects against acute liver injury, but its upstream mechanism is unclear. We aimed to clarify the protective role of CTGF in endothelial cell inflammation during IRI and reveal the regulation between endoplasmic reticulum stress-induced activating transcription factor 6 (ATF6) and CTGF. Hypoxia/reoxygenation in endothelial cells, hepatic IRI in mice and clinical specimens were used to examine the relationships between CTGF and inflammatory factors and determine how ATF6 regulates CTGF and reduces damage. We found that activating ATF6 promoted CTGF expression and reduced liver damage in hepatic IRI. In vitro, activated ATF6 upregulated CTGF and downregulated inflammation, while ATF6 inhibition had the opposite effect. Dual-luciferase assays and chromatin immunoprecipitation confirmed that activated ATF6 binds to the CTGF promoter, enhancing its expression. Activated ATF6 increases CTGF and reduces extracellular regulated protein kinase 1/2 (ERK1/2) phosphorylation, decreasing inflammatory factors. Conversely, inhibiting ATF6 decreases CTGF and increases the phosphorylation of ERK1/2, increasing inflammatory factor levels. ERK1/2 inhibition reverses this effect. Clinical samples have shown that CTGF increases after IRI, inversely correlating with inflammatory cytokines. Therefore, ATF6 activation during liver IRI enhances CTGF expression and reduces endothelial inflammation via ERK1/2 inhibition, providing a novel target for diagnosing and treating liver IRI.

JuA alleviates liver ischemia-reperfusion injury by activating AKT/NRF2/HO-1 pathways.

Liver ischemia-reperfusion (I/R) injury is a detrimental complication of organ transplantation, shock, and sepsis. However, the available drugs to mitigate I/R injury remain limited. Jujuboside A (JuA) is renowned for its antioxidant, anti-inflammatory, and anti-apoptotic properties; nevertheless, its potential in liver I/R injury remains unknown. Thus, this study aimed to explore the role and underlying mechanisms of JuA in liver I/R injury. Mouse models of I/R and AML12 cell models of hypoxia/reoxygenation (H/R) were constructed. Haematoxylin and eosin staining, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) detection, and cell viability analysis were used to assess liver injury. To evaluate oxidative stress, inflammation, apoptosis, and mitochondrial damage, immunofluorescence staining, transmission electron microscopy analysis, enzyme-linked immunosorbent assay, and flow cytometry were conducted. Moreover, molecular docking techniques and western blot were employed to identify downstream target molecules and pathways affected by JuA. The results showed that JuA pretreatment effectively attenuated liver necrosis and ALT and AST level elevations induced by I/R while enhancing AML12 cell viability following H/R. Furthermore, JuA pretreatment suppressed oxidative stress triggered by I/R and H/R, thereby inhibiting the level of pro-inflammatory factors and NLRP3 inflammasome activation. Notably, JuA pretreatment alleviated mitochondrial damage and apoptosis. Mechanistically, JuA pretreatment resulted in the activation of the AKT/NRF2/HO-1 signalling pathways, whereas MK2206, the inhibitor of AKT, partially reversed the hepatoprotective effects of JuA during liver I/R. Collectively, our findings illustrated that JuA mitigated oxidative stress, inflammation, apoptosis, and mitochondrial damage by facilitating the AKT/NRF2/HO-1 signalling pathway, thereby alleviating liver I/R injury.

[Retracted] Salidroside mitigates hypoxia/reoxygenation injury by alleviating endoplasmic reticulum stress­induced apoptosis in H9c2 cardiomyocytes.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the cell apoptotic assay data shown in Fig. 1D on p. 3763 were strikingly similar to data that had already been submitted for publication in Fig. 3A in different form in another article written by different authors at different research institutes. Owing to the fact that the contentious data in the above article had already been submitted for publication prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 18: 3760­3768, 2018; DOI: 10.3892/mmr.2018.9403].

DJ-1 regulates astrocyte activation through miR-155/SHP-1 signaling in cerebral ischemia/reperfusion injury.

Reactive astrocyte activation in the context of cerebral ischemia/reperfusion (I/R) injury gives rise to two distinct subtypes: the neurotoxic A1 type and the neuroprotective A2 type. DJ-1 (Parkinson disease protein 7, PARK7), originally identified as a Parkinson's disease-associated protein, is a multifunctional anti-oxidative stress protein with molecular chaperone and signaling functions. SHP-1 (Src homology 2 domain-containing phosphatase-1) is a protein tyrosine phosphatase closely associated with cellular signal transduction. miR-155 is a microRNA that participates in cellular functions by regulating gene expression. Recent studies have uncovered the relationship between DJ-1 and astrocyte-mediated neuroprotection, which may be related to its antioxidant properties and regulation of signaling molecules such as SHP-1. Furthermore, miR-155 may exert its effects by influencing SHP-1, providing a potential perspective for understanding the molecular mechanisms of stroke. A middle cerebral artery occlusion/reperfusion (MCAO/R) model and an oxygen-glucose deprivation/reperfusion (OGD/R) model were established to simulate focal cerebral I/R injury in vivo and in vitro, respectively. The in vivo interaction between DJ-1 and SHP-1 has been experimentally validated through immunoprecipitation. Overexpression of DJ-1 attenuates I/R injury and suppresses miR-155 expression. In addition, inhibition of miR-155 upregulates SHP-1 expression and modulates astrocyte activation phenotype. These findings suggest that DJ-1 mediates astrocyte activation via the miR-155/SHP-1 pathway, playing a pivotal role in the pathogenesis of cerebral ischemia-reperfusion injury. Our results provide a potential way for exploring the pathogenesis of ischemic stroke and present promising targets for pharmacological intervention.

High­fat diet­induced LCN2 exacerbates myocardial ischemia­reperfusion injury by enhancing platelet activation.

Following acute myocardial infarction, the recovery of blood flow leads to myocardial ischemia­reperfusion (MI/R) injury, which is primarily characterized by the activation of inflammatory signals, microvascular obstruction, increased oxidative stress and excessive Ca2+ overload. It has also been demonstrated that platelets can exacerbate MI/R injury by releasing reactive oxygen species, inflammatory factors and chemokines, while also obstructing microvessels through thrombus formation. As a bioactive molecule with proinflammatory and chemotactic properties, lipocalin 2 (LCN2) exhibits a positive correlation with obesity, hyperglycemia, hypertriglyceridemia and insulin resistance index, which are all significant risk factors for ischemic cardiomyopathy. Notably, the potential role of LCN2 in promoting atherosclerosis may be related to its influence on the function of macrophages, smooth muscle cells and endothelial cells, but its effect on platelet function has not yet been reported. In the present study, the effect of a high­fat diet (HFD) on LCN2 expression was determined by detecting LCN2 expression levels in the liver and serum samples of mice through reverse transcription­quantitative PCR and enzyme linked immunosorbent assay, respectively. The effect of LCN2 on platelet function was evaluated by examining whether LCN2 affected platelet activation, aggregation, adhesion, clot retraction and P­selectin expression. To determine whether LCN2 aggravated MI/R injury in HFD­fed mice by affecting platelet and inflammatory cell recruitment, wild­type and LCN2 knockout mice fed a HFD were subjected to MI/R injury, then hearts were collected for hematoxylin and eosin staining and 2,3,5­triphenyltetrazolium chloride staining, and immunohistochemistry was employed to detect the expression of CD42b, Ly6G, CD3 and B220. Based on observing the upregulation of LCN2 expression in mice fed a HFD, the present study further confirmed that LCN2 could accelerate platelet activation, aggregation and adhesion. Moreover, in vivo studies validated that knockout of LCN2 not only mitigated MI/R injury, but also inhibited the recruitment of platelets and inflammatory cells in myocardial tissue following ischemia­reperfusion. In conclusion, the current findings suggested that the effect of HFD­induced LCN2 on aggravating MI/R injury may totally or partially dependent on its promotion of platelet function.

Borneol promotes berberine-induced cardioprotection in a rat model of myocardial ischemia/reperfusion injury via inhibiting P-glycoprotein expression.

Berberine is reported to protect the heart against ischemia/reperfusion (I/R) injury, although efficacy is limited by low bioavailability. This study aims to determine whether borneol, a classic guiding drug, can enhance the cardioprotection induced by berberine and to clarify the underlying mechanisms involving P-glycoprotein (P-gp) in the heart. Adult male Sprague Dawley rats were gavaged with berberine (200 mg/kg) with or without borneol (100 mg/kg) for 7 consecutive days. A rat model of myocardial I/R injury was established by 30 min left coronary artery occlusion followed with 120 min reperfusion. The arrhythmia score, cardiac enzyme content, and myocardial infarct size were determined following reperfusion. Heart tissues were collected for Western blot and immunofluorescence analyses to measure the protein expression levels of Bcl-2, Bax, and P-gp. The results showed that administration of berberine protected the heart against I/R injury, as demonstrated by lower arrhythmia scores, serum cTnI contents, myocardial infarct size, and cardiomyocytes apoptosis. Moreover, borneol substantially enhanced the cardioprotective effects of berberine. Western blot and immunofluorescence analyses showed that both berberine and I/R injury did not alter P-gp expression in heart. In contrast, borneol combined with berberine significantly reduced P-gp levels by 43.4% (P = 0.0240). Interestingly, treatment with borneol alone decreased P-gp levels, but did not protect against myocardial I/R injury. These findings suggest that borneol, as an adjuvant drug, improved the cardioprotective effects of berberine by inhibiting P-gp expression in heart. Borneol combined with berberine administration provides a new strategy to protect the heart against I/R injury.

Ubiquitination of ATAD3A by TRIM25 exacerbates cerebral ischemia-reperfusion injury via regulating PINK1/Parkin signaling pathway-mediated mitophagy.

BACKGROUND: Cerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. HYPOTHESIS: We hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitochondrial autophagy via the PINK1/Parkin pathway, and aggravating CI/RI. STUDY DESIGN: Rat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively. METHODS: To evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM). RESULTS: Our findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathway-mediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression. CONCLUSION: TRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage.

Strategies for the development of in vitro models of spinal cord ischemia-reperfusion injury: Oxygen-glucose deprivation and reoxygenation.

BACKGROUND: In vitro models tailored for spinal cord ischemia-reperfusion injury are pivotal for investigation of the mechanisms underlying spinal cord injuries. We conducted a two-phased study to identify the optimal conditions for establishing an in vitro model of spinal cord ischemia-reperfusion injury using primary rat spinal motor neurons. NEW METHOD: In the first phase, cell cultures were subjected to oxygen deprivation (OD) only, glucose deprivation (GD) only, or simultaneous deprivation of oxygen and glucose [oxygen-glucose deprivation (OGD)] for different durations (1, 2, and 6 h). In the second phase, different durations of re-oxygenation (1, 12, and 24 h) were applied after 1 h of OGD to determine the optimal duration simulating reperfusion injury. RESULTS AND COMPARISON WITH EXISTING METHOD(S): GD for 6 h significantly reduced cell viability (91 % of control, P<0.001) and increase cytotoxicity (111 % of control, P<0.001). OGD for 1 h and 2 h, resulted in a significant decrease in cell viability (80 % of control P<0.001, respectively), and increase in cytotoxicity (130 % of control, P<0.001, respectively). Re-oxygenation for 1, 12, and 24 h worsened ischemic injury following 1 h of OGD (all P<0.05). CONCLUSIONS: Our results may provide a valuable guide to devise in vitro models of spinal cord ischemia-reperfusion injury using primary spinal motor neurons.

Single-Cell WGCNA Combined with Transcriptome Sequencing to Study the Molecular Mechanisms of Inflammation-Related Ferroptosis in Myocardial Ischemia-Reperfusion Injury.

Purpose: Myocardial ischemia-reperfusion injury (MIRI) is characterized by inflammation and ferroptosis, but the precise mechanisms remain unknown. This study used single-cell transcriptomics technology to investigate the changes in various cell subtypes during MIRI and the regulatory network of ferroptosis-related genes and immune infiltration. Methods: Datasets GSE146285, GSE83472, GSE61592, and GSE160516 were obtained from Gene Expression Omnibus. Each cell subtype in the tissue samples was documented. The Seurat package was used for data preprocessing, standardization, and clustering. Cellphonedb was used to investigate the ligand-receptor interactions between cells. The hdWGCNA analysis was used to create a gene co-expression network. GSVA and GSEA were combined to perform functional enrichment and pathway analysis on the gene set. Furthermore, characteristic genes of the disease were identified using Lasso regression and SVM algorithms. Immune cell infiltration analysis was also performed. MIRI rat models were created, and samples were taken for RT-qPCR and Western blot validation. Results: The proportion of MIRI samples in the C2, C6, and C11 subtypes was significantly higher than that of control samples. Three genes associated with ferroptosis (CD44, Cfl1, and Zfp36) were identified as MIRI core genes. The expression of these core genes was significantly correlated with mast cells and monocyte immune infiltrating cells. The experimental validation confirmed the upregulation of Cd44 and Zfp36 expression levels in MIRI, consistent with current study trends. Conclusion: This study used single-cell transcriptomics technology to investigate the molecular mechanisms underpinning MIRI. Numerous important cell subtypes, gene regulatory networks, and disease-associated immune infiltration were also discovered. These findings provide new information and potential therapeutic targets for MIRI diagnosis and treatment.

Reactive oxygen species (ROS)-responsive biomaterials for treating myocardial ischemia-reperfusion injury.

Myocardial ischemia-reperfusion injury (MIRI) is a critical issue that arises when restoring blood flow after an ischemic event in the heart. Excessive reactive oxygen species (ROS) production during this process exacerbates cellular damage and impairs cardiac function. Recent therapeutic strategies have focused on leveraging the ROS microenvironment to design targeted drug delivery systems. ROS-responsive biomaterials have emerged as promising candidates, offering enhanced therapeutic efficacy with reduced systemic adverse effects. This review examines the mechanisms of ROS overproduction during myocardial ischemia-reperfusion and summarizes significant advancements in ROS-responsive biomaterials for MIRI treatment. We discuss various chemical strategies to impart ROS sensitivity to these materials, emphasizing ROS-induced solubility switches and degradation mechanisms. Additionally, we highlight various ROS-responsive therapeutic platforms, such as nanoparticles and hydrogels, and their unique advantages in drug delivery for MIRI. Preclinical studies demonstrating the efficacy of these materials in mitigating MIRI in animal models are reviewed, alongside their mechanisms of action and potential clinical implications. We also address the challenges and future prospects of translating these state of the art biomaterial-based therapeutics into clinical practice to improve MIRI management and cardiac outcomes. This review will provide valuable insights for researchers and clinicians working on novel therapeutic strategies for MIRI intervention.

Malate dehydrogenase-2 inhibition shields renal tubular epithelial cells from anoxia-reoxygenation injury by reducing reactive oxygen species.

Ischemia-reperfusion (I-R) injury is the most common cause of acute kidney injury. In experiments involving primary human renal proximal tubular epithelial cells (RPTECs) exposed to anoxia-reoxygenation, we explored the hypothesis that mitochondrial malate dehydrogenase-2 (MDH-2) inhibition redirects malate metabolism from the mitochondria to the cytoplasm, towards the malate-pyruvate cycle and reversed malate-aspartate shuttle. Colorimetry, fluorometry, and western blotting showed that MDH2 inhibition accelerates the malate-pyruvate cycle enhancing cytoplasmic NADPH, thereby regenerating the potent antioxidant reduced glutathione. It also reversed the malate-aspartate shuttle and potentially diminished mitochondrial reactive oxygen species (ROS) production by transferring electrons, in the form of NADH, from the mitochondria to the cytoplasm. The excessive ROS production induced by anoxia-reoxygenation led to DNA damage and protein modification, triggering DNA damage and unfolded protein response, ultimately resulting in apoptosis and senescence. Additionally, ROS induced lipid peroxidation, which may contribute to the process of ferroptosis. Inhibiting MDH-2 proved effective in mitigating ROS overproduction during anoxia-reoxygenation, thereby rescuing RPTECs from death or senescence. Thus, targeting MDH-2 holds promise as a pharmaceutical strategy against I-R injury.

Combined effect of intermittent hemostasis and a modified external hemorrhage control device in a lethal swine model.

Background: Non-compressible torso hemorrhage (NCTH) presents the ultimate challenge in pre-hospital care. While external hemorrhage control devices (EHCDs) such as the Abdominal Aortic and Junctional Tourniquet (AAJT) and SAM Junctional Tourniquet (SJT) have been invented, the current design and application strategy requires further improvement. Therefore, researchers devised a novel apparatus named Modified EHCD (M-EHCD) and implemented intermittent hemostasis (IH) as a preventive measure against ischemia-reperfusion injury. The objective of this study was to ascertain the combined effect of M-EHCD and IH on the hemostatic effect of NCTH. Methods: Eighteen swine were randomized to M-EHCD, AAJT or SJT. The NCTH model was established by inducing Class Ⅲ hemorrhagic shock and performing a hemi-transection of common femoral artery (CFA). EHCDs were rapidly fastened since the onset of free bleeding (T0min). The IH strategy was implemented by fully releasing M-EHCD at T40min, T70min and T100min, respectively, whereas AAJT and SJT maintained continuous hemostasis (CH) until T120min. All groups underwent CFA bridging at T110min, and EHCDs were removed at T120min. Reperfusion lasted for 60 min, after which euthanasia was performed. Hemodynamics, intra-vesical pressure (IVP), and blood samples were collected periodically. Histological examinations were also conducted. Results: M-EHCD demonstrated the fastest application time (M-EHCD: 26.38 ± 6.32s vs. SJT: 30.84 ± 5.62s vs. AAJT: 54.28 ± 5.45s, P < 0.001) and reduced free blood loss (M-EHCD: 17.77 ± 9.85g vs. SJT: 51.80 ± 33.70g vs. AAJT: 115.20 ± 61.36g, P = 0.011) compared to SJT and AAJT. M-EHCD exhibited inhibitory effects on heart rate (M-EHCD: 91.83 ± 31.61bpm vs. AAJT: 129.00 ± 32.32bpm vs. SJT: 135.17 ± 21.24bpm, P = 0.041) and shock index. The device's external pressure was lowest in M-EHCD and highest in SJT (P = 0.001). The resultant increase in IVP were still the lowest in M-EHCD (M-EHCD: -0.07 ± 0.45 mmHg vs. AAJT: 27.04 ± 5.03 mmHg vs. SJT: 5.58 ± 2.55 mmHg, P < 0.001). Furthermore, M-EHCD caused the least colonic injury (M-EHCD: 1.17 ± 0.41 vs. AAJT: 2.17 ± 0.41 vs. SJT: 2.17 ± 0.41, P = 0.001). The removal of M-EHCD showed the slightest impact on pH (P < 0.001), while AAJT group was more susceptible to the lethal triad based on the arterial lactate and thrombelastogram results. Conclusions: M-EHCD + IH protected the organs and reduced the risk of the lethal triad by decreasing disruptions to IVP, hemodynamics, acid-base equilibrium and coagulation. M-EHCD + IH was superior to the hemostatic safety and efficacy of AAJT/SJT + CH.

Transcriptomic signatures during normothermic liver machine perfusion correspond with graft quality and predict the early graft function.

BACKGROUND: A better understanding of the molecular events during liver normothermic machine perfusion (NMP) is warranted to develop a data-based approach for the identification of biomarkers representative of graft quality and posttransplant outcome. We analysed the dynamic transcriptional changes during NMP and linked them to clinical and biochemical parameters. METHODS: 50 livers subjected to NMP for up to 24 h were enrolled. Bulk RNA sequencing was performed in serial biopsies collected pre and during NMP, and after reperfusion. Perfusate was sampled to monitor liver function. qPCR and immunohistochemistry were performed to validate findings. Molecular profiles were compared between transplanted and non-transplanted livers, and livers with and without early allograft dysfunction. FINDINGS: Pathways related to immune and cell stress responses, cell trafficking and cell regulation were activated during NMP, while cellular metabolism was downregulated over time. Anti-inflammatory responses and genes involved in tissue remodelling were induced at later time-points, suggesting a counter-response to the immediate damage. NMP strongly induced a gene signature associated with ischemia-reperfusion injury. A 7-gene signature corresponds with the benchmarking criteria for transplantation or discard at 6 h NMP (area under curve 0.99). CD274 gene expression (encoding programmed cell-death ligand-1) showed the highest predictive value. LEAP2 gene expression at 6 h NMP correlated with impaired graft function. INTERPRETATION: Assessment of gene expression markers could serve as a reliable tool to evaluate liver quality during NMP and predicts early graft function after transplantation. FUNDING: The research was supported by "In Memoriam Dr. Gabriel Salzner Stiftung", Tiroler Wissenschaftsfond, Jubiläumsfonds-Österreichische Nationalbank and MUI Start grant.

Avanafil Mitigates Testicular Ischemia/Reperfusion Injury via NLRP3 Pathway Modulation in Rats.

OBJECTIVE: In our study, the effectiveness of avanafil, a second-generation phosphodiesterase-5 (PDE5) inhibitor, on testicular torsion (TT) related ischemia/reperfusion injury via NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), which triggers inflammatory response, are studied molecularly, biochemically and histopathologically. MATERIAL AND METHOD: This study was performed on 24 male Wistar albino rats randomized into four groups. Testicular ischemia/reperfusion (I/R) model was created for groups 2, 3 and 4. Groups 3 and 4, respectively, were administered a dose of 5 and 10 mg/kg avanafil before reperfusion by gavage. The testicles which were left in ischemia for two hours, were detorsioned for four hours. All animals were sacrificed after reperfusion. Testicular tissues were examined molecularly, biochemically and histopathologically. RESULTS: The NLRP3, Interleukin-1ß (IL-1ß) and Tumor Necrosis alpha (TNF-α) mRNA expression levels were observed to be significantly increased in the I/R group compared to the healthy group (p < 0.001). After both doses of avanafil, NLRP3, IL-1ß and TNF-α mRNA expression levels, which increased as a result of I/R, decreased in both avanafil groups. (p < 0.001). The greatest decrease was seen at the dose of 10 mg/kg (p < 0.001). Increased Malondialdehyde (MDA) levels due to I/R were statistically significantly decreased in both doses of avanafil (p < 0.001). Decreased Superoxide Dismutase (SOD) levels due to I/R damage increased statistically significantly in both doses of avanafil (p < 0.001). CONCLUSION: It was found that avanafil can reduce the damage caused by testicular I/R and that it will find new applications in the future with the support of advanced experimental and clinical studies.

[Effects of electroacupuncture on learning-memory function and synaptic plasticity in rats with cerebral ischemia-reperfusion injury based on the BDNF/TrkB pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Baihui" (GV 20) and "Sishencong" (EX-HN 1) on the expression of brain-derived neurotrophic factor (BDNF)/tyrosine kinase receptor B (TrkB) pathway, synaptophysin (SYN), and the levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in the hippocampus of the ischemic side in rats with cerebral ischemia-reperfusion injury (CIRI), and to explore the effects and action mechanism of EA on post-CIRI learning-memory function. METHODS: Forty-eight SPF-grade male SD rats were randomly divided into a sham operation group, a model group, an EA group, and a non-acupoint group, with 12 rats in each group. The CIRI model was established in the model group, the EA group, and the non-acupoint group using the modified ZeaLonga suture method. The rats in the EA group were treated with EA at "Sishencong" (EX-HN 1) and "Baihui" (GV 20), with disperse-dense wave at frequency of 2 Hz/10 Hz and intensity of 1 mA. The rats in the non-acupoint group were treated with EA at non-meridian and non-acupoint points under the ribs bilaterally with the same parameters as the EA group. EA were conducted for 30 min each session, once daily, for 7 days. During the intervention, body weight was measured daily at a fixed time, and neurological deficits were assessed on the 1st, 3rd, and 7th days into intervention. Brain infarct volume was measured using small animal magnetic resonance imaging before and after the intervention. After the intervention, learning-memory function were evaluated using the Morris water maze. Hippocampal morphology was observed with HE staining. The positive expression of SYN in the hippocampus of the ischemic side was detected by immunohistochemistry. BDNF, TrkB, and SYN protein expressions in the hippocampus of the ischemic side were detected by Western blot. IL-1ß and IL-18 levels in the hippocampus of the ischemic side were measured by ELISA. RESULTS: From the 2nd to the 7th day into intervention, compared with the sham operation group, the body weight of rats in the model group was decreased (P<0.01); compared with the model group and the non-acupoint group, the body weight of rats in the EA group was increased (P<0.01). On the 1st day into intervention, compared with the sham operation group, neurological function scores of rats in the model group, the EA group, and the non-acupoint group were increased (P<0.01); on the 3rd and 7th days into intervention, neurological function scores of rats in the model group were higher than those in the sham operation group (P<0.01); on the 7th day, neurological function scores of rats in the EA group were lower than those in the model group and the non-acupoint group (P<0.05). Compared with the sham operation group, escape latency was prolonged (P<0.05), and the number of platform crossings was decreased (P<0.01) in the model group; compared with the model group and the non-acupoint group, escape latency was shortened (P<0.05), and the number of platform crossings was increased (P<0.01) in the EA group. Before intervention, the high signal infarcts were observed in the left ventricles of rats in the model group, the EA group, and the non-acupoint group; after intervention compared with the model group and the non-acupoint group, infarct volume in the EA group was decreased (P<0.01). Neuronal cells in the model group and the non-acupoint group were sparsely and disorderedly arranged, with deep-stained cytoplasm and shrunken nuclei; the number and arrangement of neuronal cells in the EA group were similar to the sham operation group, with less deep-stained cytoplasm and shrunken nuclei compared to the model group. Compared with the sham operation group, the positive expression of SYN, and BDNF TrkB, and SYN protein expressions in the hippocampus of the ischemic side were decreased (P<0.01, P<0.05), while levels of IL-1ß and IL-18 were increased (P<0.01) in the model group; compared with the model group and the non-acupoint group, the positive expression of SYN, and BDNF, TrkB and SYN protein expressions in the hippocampus of the ischemic side were increased (P<0.01, P<0.05), while levels of IL-1ß and IL-18 were decreased (P<0.01) in the EA group. CONCLUSION: EA at "Baihui" (GV 20) and "Sishencong" (EX-HN 1) may improve learning-memory function in rats with CIRI by activating the BDNF/TrkB signaling pathway, reducing neuroinflammatory response, and promoting the recovery of synaptic plasticity.

A novel framework for uncovering the coordinative spectrum-effect correlation of the effective components of Yangyin Tongnao Granules on cerebral ischemia-reperfusion injury in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Ischemic stroke is currently a major public health hazard.Yangyin Tongnao Granules (YYTN), a traditional Chinese medicinal prescription, exerts potential therapeutic effects on subsequent cerebral ischemia-reperfusion injury (CIRI) after ischemic stroke. However, further studies are required to comprehend the underlying mechanism of YYTN for treating CIRI and the associated spectrum-effect mechanisms. AIM OF THE STUDY: To investigate the coordinated correlation between the fingerprint and the pharmacodynamic indexes of the effective components (total flavonoids, total saponins, total alkaloids, and total phenolic acids) in YYTN for treating CIRI in rats. METHODS: The fingerprints of five specific components (ligustrazine, puerarin, ferulic acid, calycosin, and formononetin) of YYTN in rats with middle cerebral artery occlusion (MCAO) were established using high-performance liquid chromatography (HPLC), and their peak areas were quantified in plasma samples. The pharmacodynamic indexes of tumor necrosis factor-alpha (TNF-α), cytochrome c (Cyt-C), and total superoxide dismutase (T-SOD) were integrated using the Criteria Importance Through Intercriteria Correlation (CRITIC) method to create a comprehensive evaluation index. Spectrum-effect correlation was analyzed by performing gray relation analysis (GRA), correlation analysis (CA), and partial least squares regression (PLSR). The Borda method was then applied to integrate the obtained results. RESULTS: In MCAO rats, the effective components of YYTN reduced TNF-α and Cyt-C and increased T-SOD, which indicates their anti-inflammatory, antiapoptotic, and antioxidant effects. Spectrum-effect CA revealed certain associations between the chromatographic peaks of the five main components and the comprehensive pharmacodynamic evaluation index. Of these components, formononetin displayed the highest correlation, whereas ferulic acid exhibited the lowest correlation. All components showed a positive correlation. Using the Borda method, the components were ranked as follows based on correlation: formononetin > calycosin > ligustrazine > puerarin > ferulic acid. CONCLUSIONS: The effective components of YYTN exhibited synergistic effects in the treatment of MCAO rats, which could potentially be attributed to their multitarget and multipathway mechanisms. The Borda method-based spectrum-effect correlation analysis provides a coordinated approach to investigate the relationship between fingerprint and pharmacodynamics of traditional Chinese medicine (TCM).

DJ-1: Potential target for treatment of myocardial ischemia-reperfusion injury.

Ischemic heart disease (IHD) is a significant global health concern, resulting in high rates of mortality and disability among patients. Although coronary blood flow reperfusion is a key treatment for IHD, it often leads to acute myocardial ischemia-reperfusion injury (IRI). Current intervention strategies have limitations in providing adequate protection for the ischemic myocardium. DJ-1, originally known as a Parkinson's disease related protein, is a highly conserved cytoprotective protein. It is involved in enhancing mitochondrial function, scavenging reactive oxygen species (ROS), regulating autophagy, inhibiting apoptosis, modulating anaerobic metabolism, and exerting anti-inflammatory effects. DJ-1 is also required for protective strategies, such as ischemic preconditioning, ischemic postconditioning, remote ischemic preconditioning and pharmacological conditioning. Therefore, DJ-1 emerges as a potential target for the treatment of myocardial IRI. Our comprehensive review delves into its protective mechanisms in myocardial IRI and the structural foundations underlying its functions.

Unveiling the Renoprotective Mechanisms of Schisandrin B in Ischemia-Reperfusion Injury Through Transcriptomic and Pharmacological Analysis.

Objective: This study investigates the targets, pathways, and mechanisms of Schisandrin B (Sch B) in alleviating renal ischemia-reperfusion injury (RIRI) using RNA sequencing and network pharmacology. Methods: The effects of Sch B on RIRI were assessed using hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining, along with measurements of blood creatinine and urea nitrogen (BUN). Differential gene expression in mouse models treated with RIRI and Sch B+RIRI was analyzed through RNA-Seq. Key processes, targets, and pathways were examined using network pharmacology techniques. The antioxidant capacity of Sch B was evaluated using assays for reactive oxygen species (ROS), mitochondrial superoxide, and JC-1 membrane potential. Molecular docking was employed to verify the interactions between key targets and Sch B, and the expression of these targets and pathway was confirmed using qRT-PCR, Western blot, and immunofluorescence. Results: Sch B pre-treatment significantly reduced renal pathological damage, inflammatory response, and apoptosis in a mouse RIRI model. Pathological damage scores dropped from 4.33 ± 0.33 in the I/R group to 2.17 ± 0.17 and 1.5 ± 0.22 in Sch B-treated groups (p < 0.01). Creatinine and BUN levels were also reduced (from 144.6 ± 21.05 µmol/L and 53.51 ± 2.34 mg/dL to 50.44 ± 5.61 µmol/L and 17.18 ± 0.96 mg/dL, p < 0.05). Transcriptomic analysis identified four key targets (AKT1, ALB, ACE, CCL5) and the PI3K/AKT pathway. Experimental validation confirmed Sch B modulated these targets, reducing apoptosis and oxidative stress, and enhancing renal recovery. Conclusion: Sch B reduces oxidative stress, inflammation, and apoptosis by modulating key targets such as AKT1, ALB, ACE, and CCL5, while activating the PI3K/AKT pathway, leading to improved renal recovery in RIRI.

Rehmanniae Radix Praeparata aqueous extract improves hepatic ischemia/reperfusion injury by restoring intracellular iron homeostasis.

Hepatic ischemia/reperfusion injury (HIRI) is a common pathophysiological condition occurring during or after liver resection and transplantation, leading to hepatic viability impairment and functional deterioration. Recently, ferroptosis, a newly recognized form of programmed cell death, has been implicated in IRI. Rehmanniae Radix Praeparata (RRP), extensively used in Chinese herbal medicine for its hepatoprotective, anti-inflammatory, and antioxidant properties, presents a potential therapeutic approach. However, the mechanisms by which RRP mitigates HIRI, particularly through the regulation of ferroptosis, remain unclear. In this study, we developed a HIRI mouse model and monocrotaline (MCT)- and erastin-induced in vitro hepatocyte injury models. We conducted whole-genome transcriptome analysis to elucidate the protective effects and mechanisms of RRP on HIRI. The RRP aqueous extract was characterized by the presence of acteoside, rehmannioside D, and 5-hydroxymethylfurfural. Our results demonstrate that the RRP aqueous extract ameliorated oxidative stress, reduced intracellular iron accumulation, and attenuated HIRI-induced liver damage. Additionally, RRP significantly inhibited hepatocyte death by restoring intracellular iron homeostasis both in vivo and in vitro. Mechanistically, the RRP aqueous extract reduced intrahepatocellular iron accumulation by inhibiting ZIP14-mediated iron uptake, promoting hepcidin- and ferroportin-mediated iron efflux, and ameliorating mitochondrial iron aggregation through upregulation of Cisd1 expression. Moreover, siRNA-mediated inhibition of hamp synergistically enhanced the RRP aqueous extract's inhibitory effect on ferroptosis. In conclusion, our study elucidates the mechanisms by which RRP aqueous extracts alleviate HIRI, highlighting the restoration of iron metabolic balance. These findings position RRP as a promising candidate for clinical intervention in HIRI treatment.