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.

Melastoma dodecandrum lour. Protects against cerebral ischemia-reperfusion injury by ameliorating oxidative stress and endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Melastoma dodecandrum Lour. (MD), a traditional Chinese medicine used by the She ethnic group, has been used to treat cerebral ischemia-reperfusion (CIR) injury due to its efficacy in promoting blood circulation and removing blood stasiss; however, the therapeutic effects and mechanisms of MD in treating CIR injury remain unclear. AIM: To investigate the protective effects of MD on CIR injury, in addition to its impact on oxidative stress, endoplasmic reticulum (ER) stress, and cell apoptosis. MATERIALS AND METHODS: The research was conducted using both cell experiments and animal experiments. The CCK-8 method, immunofluorescence staining, and flow cytometry were used to analyze the effects of MD-containing serum on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cell viability, reactive oxygen species (ROS) clearance, anti-inflammatory, neuroprotection and inhibition of apoptosis. Furthermore, 2,3,5-Triphenyl tetrazolium chloride staining, hematoxylin and eosin staining, Nissl staining, and immunohistochemistry were used to detect infarct size, pathological changes, Nissl corpuscula and neuronal protein expression in middle cerebral artery occlusion (MCAO) rats. Polymerase chain reaction and Western Blotting were conducted in cell and animal experiments to detect the expression levels of ER stress-related genes and proteins. RESULTS: The MD extract enhanced the viability of PC12 cells under OGD/R modeling, reduced ROS and IL-6 levels, increased MBP levels, and inhibited cell apoptosis. Furthermore, MD improved the infarct area in MCAO rats, increased the number of Nissl bodies, and regulated neuronal protein levels including Microtubule-Associated Protein 2 (MAP-2), Myelin Basic Protein (MBP), Glial Fibrillary Acidic Protein (GFAP), and Neurofilament 200 (NF200). Additionally, MD could regulate the expression levels of oxidative stress proteins malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and catalase (CAT). Both cell and animal experiments demonstrated that MD could inhibit ER stress-related proteins (GRP78, ATF4, ATF6, CHOP) and reduce cell apoptosis. CONCLUSION: This study confirmed that the therapeutic mechanism of the MD extract on CIR injury was via the inhibition of oxidative stress and the ER stress pathway, in addition to the inhibition of apoptosis.

Alpha B-crystallin Ameliorates Imbalance of Redox Homeostasis, Inflammation and Apoptosis in an Acute Lung Injury Model with Rats.

Objective: Ischemia-reperfusion (IR) of the aorta is a significant contributor to the development of postoperative acute lung damage after abdominal aortic surgery. The aim of the present study was to examine the effect of alpha B-crystallin, a small heat shock protein (known as HspB5), on lung injury induced by abdominal aortic IR in rats. Methods: Male Sprague-Dawley rats were divided into three groups: control, ischemia-reperfusion (IR, 90 min ischemia and 180 min reperfusion), and alpha B-crystallin +IR. Alpha B-crystallin (50 µg/100 g) was intraperitoneally administered 1 h before IR. Lung tissue samples were obtained for histological and biochemical analyses of oxidative stress and cytokine and apoptosis parameters in plasma, lung tissues, and bronchoalveolar lavage (BAL) fluid. Results: The levels of malondialdehyde, reactive oxygen species, total oxidant status, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), nuclear factor kappa B (NFKß), caspase-9 (CASP-9), 8-hydroxy-2'-deoxyguanosine, total antioxidant status, superoxide dismutase, and interleukin-10 levels in lung tissues, plasma, and BAL fluid (p<0.05 versus control) increased in Aortic IR. However, alpha B-crystallin significantly reduced the lung tissue levels of oxidative, inflamatuvar, and apoptotic parameters in the plasma, lung tissues, and BAL fluid (p<0.05 versus aortic IR). Histopathological results showed that alpha B-crystallin ameliorated the morphological changes related to lung injury (p<0.001). Conclusion: Alpha B-crystallin substantially restored disrupted the redox balance, inflammation, and apoptotic parameters in rats exposed to IR. The cytoprotective effect of alpha B-crystallin on redox balance might be attributed to improved lung injury.

A gradient model of renal ischemia reperfusion injury to investigate renal interstitial fibrosis.

Background: The progression from acute kidney injury to chronic kidney disease poses a significant health challenge. Nonetheless, a constraint in existing animal models of renal ischemia/reperfusion (I/R) injury is the necessity for a severe injury, almost reaching a life-threatening level, to trigger the subsequent onset of renal fibrosis. Hence, we explored an adapted gradient approach to induce I/R injury, aiming to promote the progression of renal fibrosis while preserving the overall normal functioning of the kidney. Methods: In each group, 6-8 male C57BL/6 mice were used for model construction, with all undergoing sodium pentobarbital anesthesia and left kidney removal. Subsequently, a silk thread was passed beneath the lower renal branch, elevating the right kidney under a 20-g weight's tension via a pulley system for durations of 30, 40, or 60 min. Afterwards, we lowered the kidney, sutured the wound, and administered intraperitoneal saline. Mice in different groups were euthanized following reperfusion for 1, 3, 7, or 28 days. Results: We observed a complete cessation of blood flow in the lower pole, while an incomplete cessation in the upper pole in the elevated kidney. Significant renal impairment was evident on day 1 with a 60min ischemic period (187.0 ± 65.3 vs 17.9 ± 4.8 µmol/L serum creatinine in normal; p < .001), but not with 30 or 40min. On day 1, tubular necrosis and hyaline cast formation was evident in both lower and upper poles. On day 3, renal function returned to normal and remained normal through day 28. Histologic damage resolved in the upper pole over days 3 to 7, resulting in normal histology on day 28. By contrast, there was sustained tubular damage tubular in the lower pole on days 3 and 7, which failed to resolve and led to significant renal fibrosis by day 28. Conclusion: We created a model demonstrating clinically "silent" renal fibrosis.

Curcumin pretreatment attenuates myocardial ischemia/reperfusion injury by inhibiting ferroptosis, autophagy and apoptosis via HES1.

The early restoration of hemodynamics/reperfusion in acute myocardial infarction (AMI) is an effective therapeutic strategy to reduce sudden death and improve patient prognosis. However, reperfusion induces additional cardiomyocyte damage and cardiac tissue dysfunction. In this context, turmeric­derived curcumin (Cur) has been shown to exhibit a protective effect against myocardial ischemia/reperfusion injury (I/RI). The molecular mechanism of its activity, however, remains unclear. The current study investigated the protective effect of Cur and its molecular mechanism via in vitro experiments. The Cell Counting Kit­8 and lactate dehydrogenase (LDH) assay kit were used to assess the cell viability and cytotoxicity. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase, glutathione (GSH)/glutathione disulfide (GSSG), total iron, ferrous iron, caspase­3 and reactive oxygen species (ROS) were measured using an appropriate kit. Western blotting was used to detect the expression of relevant proteins. The levels of apoptosis, mitochondrial permeability transition pore (MPTP) opening, and mitochondrial membrane potential (MMP) were detected by flow cytometry. The study findings indicated that anoxia/reoxygenation (A/R) injury significantly decreased cell viability, increased in LDH and caspase­3 activities, induced ferroptosis, increased apoptosis and overactivated autophagy. However, pretreatment with Cur or ferrostatin­1 (Fer­1, a ferroptosis inhibitor) significantly increased A/R­reduced cell viability, SOD, glutathione peroxidase activity, GSH/GSSH ratio and HES1 and glutathione peroxidase 4 protein expression; attenuated A/R­induced LDH, MDA, total iron, ferrous iron, prostaglandin­endoperoxide synthase 2 protein expression and prevented ROS overproduction and MMP loss. In addition, Cur inhibited caspase­3 activity, upregulated the Bcl­2/Bax ratio, reduced apoptotic cell number and inhibited MPTP over­opening. Furthermore, Cur increased P62, LC3II/I, NDUFB8 and UQCRC2 expression and upregulated the p­AMPK/AMPK ratio. However, erastin (a ferroptosis activator), pAD/HES1­short hairpin RNA, rapamycin (an autophagy activator) and Compound C (an AMPK inhibitor) blocked the protective effect of Cur. In conclusion, Cur pretreatment inhibited ferroptosis, autophagy overactivation and oxidative stress; improved mitochondrial dysfunction; maintained energy homeostasis; attenuated apoptosis; and ultimately protected the myocardium from A/R injury via increased HES1 expression.

Enforced HCELL Expression Enhances Bone Marrow Stromal Cells Homing and Amelioration of Cerebral Ischemia-Reperfusion Injury via induction of PGE2.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion (MCAO) model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by up-regulating BCL-2 and down-regulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1ß (IL-1ß), IL-2, IL-6 and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs towards cerebral ischemic lesions and their subsequent transendothelial migration through the up-regulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

LncRNA ZFAS1/miR-186-5p axis is involved in oxidative stress inhibition of myocardial ischemia-reperfusion injury by targeting BTG2.

OBJECTIVE: To probe the involvement of long noncoding RNA zinc finger antisense 1 (ZFAS1)/microRNA (miR)-186-5p axis in inhibiting oxidative stress in myocardial ischemia-reperfusion injury (MIRI) by targeting B-cell translocation gene 2 (BTG2). METHODS: The MIRI mice model was established by ligating the left anterior descending branch of the left coronary artery in C57BL/6 mice. The in vitro MIRI model was constructed by hypoxia and reoxygenation of HL-1 cardiomyocytes. Cardiomyocyte apoptosis and the extent of myocardial injury in mice were detected. The apoptosis rates, malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in HL-1 cells were assessed. The relationship among ZFAS1, miR-186-5p, and BTG2 was verified. RESULTS: High ZFAS1 and BTG2 levels and low miR-186-5p levels were demonstrated in I/R-injured myocardial tissues and in H/R-treated cardiomyocytes. Interference with ZFAS1 or elevation of miR-186-5p inhibited apoptosis and oxidative stress in H/R model cardiomyocytes and I/R-injured myocardial tissues. Overexpressing BTG2 impaired the ameliorative effects of miR-186-5p on MIRI. ZFAS1 negatively regulated miR-186-5p expression by acting as a molecular sponge. miR-186-5p targeted to regulate BTG2 negatively. CONCLUSION: Interfering with ZFAS1 can upregulate miR-186-5p and thus inhibit BTG2 expression, thereby ameliorating MIRI.

Exercise preconditioning mitigates brain injury after cerebral ischemia-reperfusion injury in rats by restraining TIMP1.

BACKGROUND: Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia-reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. METHODS: The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE-TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE-TIMP1) groups. RESULTS: The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE-TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE-TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B-cell lymphoma-2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2-associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE-TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia-inducible factor-alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE-TIMP1. CONCLUSION: Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI.

Fibroblast growth factor 21 attenuates pulmonary ischemia/reperfusion injury via inhibiting endoplasmic reticulum stress-induced ferroptosis though FGFR1/PPARδ signaling pathway.

BACKGROUND: Acute lung injury is a critical life-threatening complication of pulmonary and cardiac surgery with a high rate of morbidity and mortality. Fibroblast growth factor 21 (FGF21) has been reported to play an important role in protecting vital organs from damage. This study aims to investigate the potential protective role and mechanism of FGF21 in pulmonary ischemia/reperfusion (I/R)-induced acute lung injury. METHODS: A pulmonary epithelial cell line was treated with hypoxia/regeneration (H/R) in vitro and a mouse model of acute lung injury was induced with pulmonary I/R in vivo. Lung injury after pulmonary I/R was compared between FGF21-konckout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect. RESULTS: Circulating levels of FGF21 in mice with pulmonary I/R injury were significantly higher than in those without pulmonary I/R injury. Lung injury was aggravated in FGF21-KO mice compared with WT mice and the administration of FGF21 alleviated lung injury in mouse treated with I/R and pulmonary epithelial cell injury treated with H/R. FGF21 treatment decreased endoplasmic reticulum (ER) stress, Fe2+ and lipid reactive oxygen species (ROS) contents and GPX4 expression and increased PTGS2 levels. Mechanistically, FGF21 upregulated the expression of FGFR1 and PPARδ, ameliorated ER stress and ER stress induced-ferroptosis. Furthermore, FGF21 increased the expression level of PPARδ in pulmonary epithelial cell exposed to H/R, which was inhibited by FGFR1 inhibitor (PD173074). The protective effects of FGF21 were abolished by co-treatment with PPARδ inhibitor (GSK0660), indicating FGF21 attenuated ER stress-induced ferroptosis by dependent on FGFR1/PPARδ signaling pathway. CONCLUSION: Our study reveals that FGF21 protects against pulmonary I/R injury via inhibiting ER stress-induced ferroptosis though FGFR1/PPARδ signaling pathway. Boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for pulmonary IRI.

The combination of post-mortem sevoflurane ventilation and in situ topical cooling provides improved 6h lung preservation in an uncontrolled DCD porcine model.

BACKGROUND: Recent clinical series on donation after uncontrolled cardiovascular death (uDCD) reported successful transplantation of lungs preserved by pulmonary inflation up to 3h post-mortem. This study aims to investigate the additive effects of in situ lowering of intrathoracic temperature and sevoflurane preconditioning on lung grafts in a porcine uDCD model. METHODS: After uDCD induction, donor pigs were allocated to one of the following groups: Control - static lung inflation only (SLI); TC - SLI + continuous intrapleural topical cooling (TC); or TC+Sevo - SLI + TC + sevoflurane. Lungs were retrieved 6h post-asystole and evaluated via ex vivo lung perfusion (EVLP) for 6h. A left single lung transplant was performed using lungs from the best performing group, followed by 4h of graft evaluation. RESULTS: Animals that received topical cooling achieved intrathoracic temperature < 15°C within 1 hour after chest filling of coolant. Only lungs from donors that received TC and TC+Sevo completed the planned post-preservation 6h EVLP assessment. Despite similar early performance of the two groups on EVLP, the TC+Sevo group was superior - associated with overall lower airway pressures, higher pulmonary compliances, less edema development, and less release of inflammatory cytokines. Transplantation was performed using lungs from the TC+Sevo group, and excellent graft function was observed post-reperfusion. CONCLUSION: Preservation of uDCD lungs with a combination of static lung inflation, topical cooling and sevoflurane treatment maintains good pulmonary function up to 6h post-mortem with excellent early post-lung transplant function. These interventions may significantly expand the clinical utilization of uDCD donor lungs.

Bringing PERT to Pediatrics: Initial Experience and Outcomes of a Pediatric Multidisciplinary Pulmonary Embolism Response Team (PERT).

INTRODUCTION: Multidisciplinary Pulmonary Embolism Response Teams (PERTs) streamline care of adults with life-threatening pulmonary embolism (PE). Given rarity of pediatric PE, developing a clinical, educational, and research PERT paradigm is a novel and underutilized concept in pediatrics. RESEARCH QUESTION: Is PERT feasible in pediatrics, and does it improve PE care? STUDY DESIGN AND METHODS: A strategy-to-execution proposal to launch a pediatric PERT was developed for institutional buy-in. Key stakeholders collectively implemented PERT. Data were collected for the two-year pre- and post-PERT eras, and outcomes were compared. RESULTS: PERT implementation took 12 months. Our PERT, led by hematology, comprises of pediatric experts in emergency medicine, critical care, interventional cardiology, anesthesiology, and interventional radiology. Data on 30 patients pre-PERT and 31 post-PERT were analyzed. Pre-PERT, 10%(3/30), 13%(4/30), 20%(6/30), and 57%(17/30), and post-PERT, 3%(1/31), 10%(3/31), 16%(5/31), and 71%(22/31) were categorized as high-risk, intermediate-LOW risk, intermediate-HIGH risk, and low-risk PE, respectively. Post-PERT, there were 13 unique PERT activations. PERT was activated on all eligible PE patients and, additionally, on four low-risk PEs. Time-to-echocardiogram was shorter post-PERT (4.7 hrs vs 2 hrs, P=0.0147). Anticoagulation was ordered (90 min vs 54 min, P=0.003) and given sooner (154 min vs 113 min, P=0.049) post-PERT. There were no differences in time-to-reperfusion therapies (12 hrs pre-PERT vs 8.7 hrs post-PERT, P=0.1). Five (83.3%) of six eligible (intermediate-HIGH and high-risk) patients received reperfusion therapies in the post-PERT era compared to three (37.5%) of eight eligible patients in the pre-PERT era (P=0.0001). There were no differences in major bleeding, mortality, or length of stay in either era. INTERPRETATION: The pediatric PERT paradigm was successfully created and adopted locally. Our PERT enhanced access to experts, facilitated timely advanced therapies, and held value for low-risk PE. The University of Texas Southwestern Medical Center (UTSW) and Children's Health System of Texas pediatric PERT may serve as a best-practice model for streamlining care for pediatric PE.

Evaluation of the effect of carvedilol orodispersible tablets on ischemia-reperfusion injury and flap viability in rats: An in vivo study.

Flap surgery is an integral part of plastic surgery, and ischemia-reperfusion (I/R) injury significantly affects the viability of the flap. Carvedilol (CRV), a nonselective beta-blocker with alpha-1 blocking and antioxidant properties, and known for its potential in reducing I/R damage, was chosen as the active substance for our study. The aim of this study was to investigate the vasodilator and antioxidant effects of CRV on rat inferior epigastric artery skin flap using orally disintegrating tablets (ODTs). The optimized ODT formulation was subjected to in vivo experiments using Sprague-Dawley female rats (n = 24) divided into three groups: Group I (control, I/R), Group II (treatment, I/R + CRV), and Group III (treatment, I/R), I/R + CRV ODT). Reperfusion was then observed following the release of the microclamp from the pedicle, and the flap was then re-adapted to its original position. Control rats were given oral isotonic solution via gavage and were subjected to 8 h of ischemia and 12 h of reperfusion. Group II was given 2 mg/kg CRV oral tablets for 7 days before and after surgery. Group III was given 2 mg/kg/day CRV ODT for the same period. Biopsies were taken from the flap and histopathological and biochemical analyses including superoxide dismutase, glutathionenitric oxide, malondialdehyde, paraoxonase 1, total oxidant, and total antioxidant capacities were performed. This study demonstrates that CRV ODTs significantly increased flap viability by approximately 25% compared to the control group, highlighting their promising therapeutic potential.

Regulation of STAT3 and NF-κB signaling pathways by trans-Anethole in testicular ischemia-reperfusion injury and its gonadoprotective effect.

Testicular ischemia reperfusion (I/R) injury is a significant urological problem where clinical interventions may be inadequate, and the antioxidants might be potential co-treatment modalities. This study examined the gonadoprotective effect of trans-Anethole in testicular I/R injury. Twenty-eight male rats were divided into four groups. Rats in the I/R, I/R + t100, I/R + t200 groups underwent bilateral testicular I/R injury. The I/R + t100 and I/R + t200 groups received 100 or 200 mg/kg trans-Anethole at the 2nd hour of ischemia. Microscopic evaluations demonstrated that testicular I/R injury leads to severe testicular degeneration. Tissue oxidative stress, pro-apoptotic Bcl-2 associated X (Bax) and Caspase 3, pro-inflammatory Tumor necrosis factor-alpha (TNF-α), Interleukin-1 beta (IL-1ß) and Interleukin 6 (IL-6) cytokines levels were significantly (p < 0.05) upregulated when compared to the Control group. Additionally, transcription factors Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) levels increased significantly (p < 0.05) compared to the Control group. Tissue disrupted parameters in the I/R + t200 group were significantly different (p < 0.05) from the I/R group, contrasting with the slight improvement in the I/R + t100 group. The STAT3 and NF-κB expression levels in the I/R + t200 group were significantly suppressed (p < 0.05) compared to the I/R group. In conclusion, our study indicates that trans-Anethole could enhance gonadoprotective activity in testicular I/R injury, potentially involving transcription factors STAT3 and NF-κB. However, before the consumption of trans-Anethole-containing natural or manufactured goods, the potential benefits and side effects should be carefully evaluated.

Antiarrhythmic potentials of irisin in ischemia/reperfusion injury of diabetic rats through modulating mitochondria-endoplasmic reticulum interaction and inhibiting pyroptosis.

Objectives: Myocardial arrhythmia is a major complication of ischemia-reperfusion (I/R) injury in patients with diabetes. Irisin has significant cardioprotective effects, while its role in the pathophysiology of I/R injury-induced myocardial arrhythmia in the presence of diabetes is not well identified. Here, we aimed to investigate the potential antiarrhythmic impacts and mechanisms (mitochondrial biogenesis, endoplasmic reticulum (ER) stress, and pyroptosis) by which irisin reduces I/R injury-induced myocardial arrhythmia in diabetic rats. Materials and Methods: Thirty high-fat diet-induced diabetic rats were subjected to I/R injury and myocardial arrhythmia. Irisin (0.5 µg/kg/day) was injected intraperitoneally before induction of I/R injury. Electrocardiography was used to measure the incidence and severity of ventricular arrhythmias. ELISA and western blotting analyses were employed to quantify the expression of mitochondrial biogenesis, ER stress, and pyroptosis-related proteins in ischemic myocardium. Results: Irisin treatment in diabetic rats significantly decreased the lactate dehydrogenase level and the number and severity of arrhythmia induced by I/R injury. Irisin up-regulated the expression of mitochondrial biogenesis-related proteins while down-regulating the expression of ER stress and pyroptosis-related proteins. Furthermore, the inhibition of mitochondrial quality control by mdivi-1 significantly abolished the cardioprotective effect of irisin. Conclusion: Our findings suggest that irisin reduced myocardial arrhythmia induced by I/R injury in diabetic rats by modulating the interaction of mitochondrial biogenesis and ER stress proteins and inhibiting the pyroptosis pathway. These findings provide a promising strategy for managing myocardial arrhythmia in diabetic patients, but supplementary studies are needed to confirm the clinical efficacy of irisin in these patients.

Bone marrow mesenchymal stem cells-derived exosomal miR-381 alleviates lung ischemia-reperfusion injury by activating Treg differentiation through inhibiting YTHDF1 expression.

AIM: Our study aimed to investigate whether BMSCs-derived exosomal miR-381 promotes Treg cell differentiation in lung ischemia-reperfusion injury (LIRI), and the underlying mechanism. METHODS: The in vitro and in vivo models of LIRI were established by hypoxia/reoxygenation (H/R) treatment and lung ischemia/reperfusion (I/R) surgery, respectively. BMSCs-derived exosomes were isolated and identified by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Cell viability, proliferation, and apoptosis were assessed by CCK-8, EdU, and flow cytometry assay, respectively. IL-18 secretion level in lung microvascular endothelial cells (LMECs) and lung tissue homogenate was examined by ELISA. Treg cell differentiation was determined using flow cytometry. The relationships between miR-381, YTHDF1, and IL-18 were investigated using dual-luciferase reporter gene, RIP, and/or RNA pull-down assays. MeRIP assay was employed to determine m6A modification of IL-18 mRNA in LMECs. The ubiquitination level of Foxp3 protein in CD4+ T cells was analyzed by Co-IP assay. RESULTS: BMSCs-derived exosomes reduced LMECs injury and increased Treg cell differentiation in LIRI, whereas miR-381 inhibition in BMSCs weakened these impacts. Mechanistically, miR-381 inhibited IL-18 translation in LMECs by inhibiting YTHDF1 expression via binding to its 3'-UTR. As expected, YTHDF1 overexpression in LMECs abolished the effects of miR-381-overexpressed exosomes on LMECs injury and Treg cell differentiation. Moreover, LMECs-secreted IL-18 inhibited Treg cell differentiation by promoting the ubiquitination degradation of Foxp3 protein. CONCLUSION: BMSCs-derived exosomal miR-381 suppressed IL-18 translation in LMECs through binding to YTHDF1 3'-UTR, thus suppressing the ubiquitination degradation of Foxp3 in CD4+ T cells, which promoted Treg cell differentiation and mitigated LIRI development.

S100A9 regulates M1 macrophage polarization and exacerbates steatotic liver ischemia-reperfusion injury.

OBJECTIVE: Steatotic livers exhibit higher susceptibility to ischemia reperfusion (IR) injury, which increase the risk of primary graft non-function following liver transplantation. S100A9 is identified as a pivotal innate immune sensor that regulates the progression of liver diseases. However, its significance in steatotic liver IR injury remains under-investigated. METHODS: In mice model, we generated S100A9 knockout (S100A9 KO) mice to investigate the role of S100A9 in IR-stimulated steatotic livers. In vitro, primary bone marrow-derived macrophages were utilized to explore the effect of S100A9 in regulating macrophage polarization and inflammation. RESULTS: S100A9 expression was markedly increased in steatotic livers of mice subjected to IR insult. S100A9 deletion significantly attenuated liver inflammatory injury, as evidenced by the diminished infiltration of both monocytes/macrophages and neutrophils (p < 0.05). The expression of proinflammatory factors was reduced (p < 0.05) at the same time. Additionally, S100A9-deficient livers demonstrated M1 polarization decrease and Toll-like receptor 4 (TLR4) suppression (p < 0.05). In vitro, genetic TLR4 inhibition led to nuclear factor kappa B (NF-κB) inactivation and subsequent M1 polarization decrease (p < 0.05) in macrophages treated with recombinant S100A9. Conclusion In this study, we highlight the pivotal role of TLR4/NF-κB as a critical mediator of S100A9 in inducing M1 macrophage polorization- dependent inflammation in steatotic livers IR injury.

The nitration of SIRT6 aggravates neuronal damage during cerebral ischemia-reperfusion in rat.

Ischemic stroke is a major cause of death and disability. The activation of neuronal nitric oxide synthase (nNOS) and the resulting production of nitric oxide (NO) via NMDA receptor-mediated calcium influx play an exacerbating role in cerebral ischemia reperfusion injury. The NO rapidly reacts with superoxide (O2-) to form peroxynitrite (ONOO-), a toxic molecule may modify proteins through tyrosine residue nitration, ultimately worsening neuronal damage. SIRT6 has been proven to be crucial in regulating cell proliferation, death, and aging in various pathological settings. We have previous reported that human SIRT6 tyrosine nitration decreased its intrinsic catalytic activity in vitro. However, the exact role of SIRT6 function in the process of cerebral ischemia reperfusion injury is not yet fully elucidated. Herein, we demonstrated that an increase in the nitration of SIRT6 led to reduce its enzymatic activity and aggravated hippocampal neuronal damage in a rat model of four-artery cerebral ischemia reperfusion. In addition, reducing SIRT6 nitration resulted in increase the activity of SIRT6, alleviating hippocampal neuronal damage. Moreover, SIRT6 nitration affected its downstream molecule activity such as PARP1 and GCN5, promoting the process of neuronal ischemic injury in rat hippocampus. Additionally, treatment with NMDA receptor antagonist MK801, or nNOS inhibitor 7-NI, and resveratrol (an antioxidant) diminished SIRT6 nitration and the catalytic activity of downstream molecules like PARP1 and GCN5, thereby reducing neuronal damage. Finally, in the biochemical regulation of SIRT6 activity, tyrosine 257 was essential for its activity and susceptibility to nitration. Replacing tyrosine 257 with phenylalanine in rat SIRT6 attenuated the death of SH-SY5Y neurocytes under oxygen-glucose deprivation (OGD) conditions. These results may offer further understanding of SIRT6 function in the pathogenesis of cerebral ischemic diseases.

Formononetin alleviates no reflow after myocardial ischemia-reperfusion via modulation of gut microbiota to inhibit inflammation.

Gut microflora plays an important role in relieving myocardial no-reflow (NR), formononetin (FMN) has potential effects on NR, however, the relationship between this effect and gut microflora remains unclear. This study aimed to evaluate the role of FMN in alleviating NR by regulating gut microflora. We used a myocardial NR rat model to confirm the effect and mechanism of action of FMN in alleviating NR. The rats were randomly divided into sham operation group (Sham), NR group, FMN group and sodium nitroprusside (SNP) group. Thioflavin S staining, Hematoxylin Eosin (HE), myocardial enzyme activity, ultrasonic cardiogram and RT-PCR detection showed that FMN could effectively reduce inflammatory cell infiltration, NR and ischemic area, improve cardiac structure and function and reduce TNF-α and NF-κB gene expression in NR rats. The results of 16S rRNA high-throughput sequencing showed that FMN could increase the abundance of anti-inflammatory bacteria such as Ligilactobacillus, Coprococcus, Blautia and Muribaculaceae and decrease the abundance of pro-inflammatory bacteria such as Treponema in Spirochaetota and Campylobacterota. The correlation between the differential bacteria in the gut microflora(anti-inflammatory bacteria and pro-inflammatory bacteria) and TNF-α and NF-κB, showed that they had a strong correlation. Therefore, the anti-NR mechanism of FMN may be related to increasing the abundance of anti-inflammatory bacteria and reducing the abundance of pro-inflammatory bacteria to inhibit inflammation. This study provides innovative mechanistic insights into the relationship between gut microbiota and myocardial protection, suggesting potential strategy for future treatment of NR.