4-phenylbutyric acid improves sepsis-induced cardiac dysfunction by modulating amino acid metabolism and lipid metabolism via Comt/Ptgs2/Ppara.

INTRODUCTION: Cardiac dysfunction after sepsis the most common and severe sepsis-related organ failure. The severity of cardiac damage in sepsis patients was positively associated to mortality. It is important to look for drugs targeting sepsis-induced cardiac damage. Our previous studies found that 4-phenylbutyric acid (PBA) was beneficial to septic shock by improving cardiovascular function and survival, while the specific mechanism is unclear. OBJECTIVES: We aimed to explore the specific mechanism and PBA for protecting cardiac function in sepsis. METHODS: The cecal ligation and puncture-induced septic shock models were used to observe the therapeutic effects of PBA on myocardial contractility and the serum levels of cardiac troponin-T. The mechanisms of PBA against sepsis were explored by metabolomics and network pharmacology. RESULTS: The results showed that PBA alleviated the sepsis-induced cardiac damage. The metabolomics results showed that there were 28 metabolites involving in the therapeutic effects of PBA against sepsis. According to network pharmacology, 11 hub genes were found that were involved in lipid metabolism and amino acid transport following PBA treatment. The further integrated analysis focused on 7 key targets, including Comt, Slc6a4, Maoa, Ppara, Pparg, Ptgs2 and Trpv1, as well as their core metabolites and pathways. In an in vitro assay, PBA effectively inhibited sepsis-induced reductions in Comt, Ptgs2 and Ppara after sepsis. CONCLUSIONS: PBA protects sepsis-induced cardiac injury by targeting Comt/Ptgs2/Ppara, which regulates amino acid metabolism and lipid metabolism. The study reveals the complicated mechanisms of PBA against sepsis.

Metabolomics and machine learning approaches for diagnostic and prognostic biomarkers screening in sepsis.

BACKGROUND: Sepsis is a life-threatening disease with a poor prognosis, and metabolic disorders play a crucial role in its development. This study aims to identify key metabolites that may be associated with the accurate diagnosis and prognosis of sepsis. METHODS: Septic patients and healthy individuals were enrolled to investigate metabolic changes using non-targeted liquid chromatography-high-resolution mass spectrometry metabolomics. Machine learning algorithms were subsequently employed to identify key differentially expressed metabolites (DEMs). Prognostic-related DEMs were then identified using univariate and multivariate Cox regression analyses. The septic rat model was established to verify the effect of phenylalanine metabolism-related gene MAOA on survival and mean arterial pressure after sepsis. RESULTS: A total of 532 DEMs were identified between healthy control and septic patients using metabolomics. The main pathways affected by these DEMs were amino acid biosynthesis, phenylalanine metabolism, tyrosine metabolism, glycine, serine and threonine metabolism, and arginine and proline metabolism. To identify sepsis diagnosis-related biomarkers, support vector machine (SVM) and random forest (RF) algorithms were employed, leading to the identification of four biomarkers. Additionally, analysis of transcriptome data from sepsis patients in the GEO database revealed a significant up-regulation of the phenylalanine metabolism-related gene MAOA in sepsis. Further investigation showed that inhibition of MAOA using the inhibitor RS-8359 reduced phenylalanine levels and improved mean arterial pressure and survival rate in septic rats. Finally, using univariate and multivariate cox regression analysis, six DEMs were identified as prognostic markers for sepsis. CONCLUSIONS: This study employed metabolomics and machine learning algorithms to identify differential metabolites that are associated with the diagnosis and prognosis of sepsis patients. Unraveling the relationship between metabolic characteristics and sepsis provides new insights into the underlying biological mechanisms, which could potentially assist in the diagnosis and treatment of sepsis. TRIAL REGISTRATION: This human study was approved by the Ethics Committee of the Research Institute of Surgery (2021-179) and was registered by the Chinese Clinical Trial Registry (Date: 09/12/2021, ChiCTR2200055772).

Activated Drp1 Initiates the Formation of Endoplasmic Reticulum-Mitochondrial Contacts via Shrm4-Mediated Actin Bundling.

Excessive mitochondrial fission following ischemia and hypoxia relies on the formation of contacts between the endoplasmic reticulum and mitochondria (ER-Mito); however, the specific mechanisms behind this process remain unclear. Confocal microscopy and time course recording are used to investigate how ischemia and hypoxia affect the activation of dynamin-related protein 1 (Drp1), a protein central to mitochondrial dynamics, ER-Mito interactions, and the consequences of modifying the expression of Drp1, shroom (Shrm) 4, and inverted formin (INF) 2 on ER-Mito contact establishment. Both Drp1 activation and ER-Mito contact initiation cause excessive mitochondrial fission and dysfunction under ischemic-hypoxic conditions. The activated form of Drp1 aids in ER-Mito contact initiation by recruiting Shrm4 and promoting actin bundling between the ER and mitochondria. This process relies on the structural interplay between INF2 and scattered F-actin on the ER. This study uncovers new roles of cytoplasmic Drp1, providing valuable insights for devising strategies to manage mitochondrial imbalances in the context of ischemic-hypoxic injury.

Protective Effect of Modified Hemoglobin on Rabbits and Goats in High-Altitude Sickness.

The prevalence and severity of high-altitude sickness increases with increasing altitude. Prevention of hypoxia caused by high-altitude sickness is an urgent problem. As a novel oxygen-carrying fluid, modified hemoglobin can carry oxygen in a full oxygen partial pressure environment and release oxygen in a low oxygen partial pressure environment. It is unclear whether modified hemoglobin can improve hypoxic injury on a plateau. Using hypobaric chamber rabbit (5000 m) and plateau goat (3600 m) models, general behavioral scores and vital signs, hemodynamic, vital organ functions, and blood gas are measured. The results show that the general behavioral scores and vital signs decrease significantly in the hypobaric chamber or plateau, and the modified hemoglobin can effectively improve the general behavioral scores and vital signs in rabbits and goats, and reduce the degree of damage to vital organs. Further studies reveal that arterial partial pressure of oxygen (PaO2 ) and arterial oxygen saturation (SaO2 ) on the plateau decrease rapidly, and the modified hemoglobin could increase PaO2 and SaO2 ; thus, increasing the oxygen-carrying capacity. Moreover, modified hemoglobin has few side effects on hemodynamics and kidney injury. These results indicate that modified hemoglobin has a protective effect against high-altitude sickness.

Identification of featured necroptosis-related genes and imbalanced immune infiltration in sepsis via machine learning.

Background: The precise diagnostic and prognostic biological markers were needed in immunotherapy for sepsis. Considering the role of necroptosis and immune cell infiltration in sepsis, differentially expressed necroptosis-related genes (DE-NRGs) were identified, and the relationship between DE-NRGs and the immune microenvironment in sepsis was analyzed. Methods: Machine learning algorithms were applied for screening hub genes related to necroptosis in the training cohort. CIBERSORT algorithms were employed for immune infiltration landscape analysis. Then, the diagnostic value of these hub genes was verified by the receiver operating characteristic (ROC) curve and nomogram. In addition, consensus clustering was applied to divide the septic patients into different subgroups, and quantitative real-time PCR was used to detect the mRNA levels of the hub genes between septic patients (SP) (n = 30) and healthy controls (HC) (n = 15). Finally, a multivariate prediction model based on heart rate, temperature, white blood count and 4 hub genes was established. Results: A total of 47 DE-NRGs were identified between SP and HC and 4 hub genes (BACH2, GATA3, LEF1, and BCL2) relevant to necroptosis were screened out via multiple machine learning algorithms. The high diagnostic value of these hub genes was validated by the ROC curve and Nomogram model. Besides, the immune scores, correlation analysis and immune cell infiltrations suggested an immunosuppressive microenvironment in sepsis. Septic patients were divided into 2 clusters based on the expressions of hub genes using consensus clustering, and the immune microenvironment landscapes and immune function between the 2 clusters were significantly different. The mRNA levels of the 4 hub genes significantly decreased in SP as compared with HC. The area under the curve (AUC) was better in the multivariate prediction model than in other indicators. Conclusion: This study indicated that these necroptosis hub genes might have great potential in prognosis prediction and personalized immunotherapy for sepsis.

Genistein, a Soybean Isoflavone, Promotes Wound Healing by Enhancing Endothelial Progenitor Cell Mobilization in Rats with Hemorrhagic Shock.

Severe trauma and hemorrhaging are often accompanied by delayed cutaneous wound healing. Soybean isoflavone is a natural phytoestrogen that has attracted great attention due to its protective effects against various injuries. Endothelial progenitor cells (EPCs) are precursor cells with directional differentiation characteristics. This study is to determine whether genistein (GEN), an isoflavone in soybean products, benefits wound healing in hemorrhagic shock (HS) rats by promoting EPC homing and to investigate the underlying mechanisms. In this study, it is found that GEN promotes skin wound healing in HS rats, which is due at least partly to the mobilization of endogenous EPCs to the injury site via angiotensin II (Ang-II), stromal cell-derived factor-1alpha (SDF-1α), and transforming growth factor beta(TGF-ß) signaling.

Low-dose norepinephrine in combination with hypotensive resuscitation may prolong the golden window for uncontrolled hemorrhagic shock in rats.

Hypotension resuscitation is an important principle for the treatment after trauma. Current hypotensive resuscitation strategies cannot obtain an ideal outcome for remote regions. With the uncontrolled hemorrhagic shock (UHS) model in rats, the effects of norepinephrine (NE) on the tolerance time of hypotensive resuscitation, blood loss, vital organ functions, and animal survival were observed. Before bleeding was controlled, only the LR infusion could effectively maintain the MAP to 50-60 mmHg for 1 h, while the MAP gradually decreased with prolonging time, even with increasing infusion volume. Low-dose NE during hypotensive resuscitation prolonged the hypotensive tolerance time to 2-3 h, and the effect of 0.3 µg/kg/min NE was the best. Further studies showed that 0.3 µg/kg/min NE during hypotensive resuscitation significantly lightened the damage of organ function induced by UHS via protecting mitochondrial function, while the LR infusion did not. At the same time, NE administration improved Hb content, DO2, and VO2, and restored liver and kidney blood flow. The survival results showed that low-dose NE administration increased the survival rate and prolonged the survival time. Together, low-dose NE during hypotensive resuscitation was suitable for the early treatment of UHS, which can strive for the golden window of emergency treatment for serious trauma patients by reducing blood loss and protecting vital organ functions.

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive. METHODS: The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1ß, etc., were detected under normal or Drp1 interference conditions. RESULTS: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 - 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1ß increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen-glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05). CONCLUSIONS: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

The Characteristics of Organ Function Damage of Hemorrhagic Shock in Hot Environment and the Effect of Hypothermic Fluid Resuscitation.

BACKGROUND: Hemorrhagic shock is the important factor for causing death of trauma and war injuries. However, pathophysiological characteristics and underlying mechanism in hemorrhagic shock with hot environment remain unclear. METHODS: Hemorrhagic shock in hot environment rat model was used to explore the changes of mitochondrial and vital organ functions, the variation of the internal environment, stress factors, and inflammatory factors; meanwhile, the suitable treatment was further studied. RESULTS: Above 36°C hot environment induced the increase of core temperature of rats, and the core temperature was not increased in 34°C hot environment, but the 34°C hot environment aggravated significantly hemorrhagic shock induced mortality. Further study showed that the mitochondrial functions of heart, liver, and kidney were more damaged in hemorrhagic shock rats with 34°C hot environment as compared with room environment. Moreover, the results showed that in hemorrhagic shock rats with hot environment, the blood concentration of Na+, K+, and plasma osmotic pressure, the expression of inflammatory factors tumor necrosis factor-α and interleukin-6 in the serum, as well as the stress factors Adrenocorticotropic Hormone and Glucocorticoid were all notably enhanced; and acidosis was more serous; oxygen supply and oxygen consumption were remarkably decreased. In addition, the present study demonstrated that mild hypothermia (10°C) fluid resuscitation could significantly improve the survival rate in hemorrhagic shock rats with hot environment as compared with normal temperature fluid resuscitation. CONCLUSIONS: Hot environment accelerated the death of hemorrhagic shock rats, which was related to the disorder of internal environment, the increase of inflammatory and stress factors. Furthermore, moderate hypothermic (10°C) fluid resuscitation was suitable for the treatment of hemorrhagic shock in hot environment.

Mitochondrial Drp1 recognizes and induces excessive mPTP opening after hypoxia through BAX-PiC and LRRK2-HK2.

Mitochondrial mass imbalance is one of the key causes of cardiovascular dysfunction after hypoxia. The activation of dynamin-related protein 1 (Drp1), as well as its mitochondrial translocation, play important roles in the changes of both mitochondrial morphology and mitochondrial functions after hypoxia. However, in addition to mediating mitochondrial fission, whether Drp1 has other regulatory roles in mitochondrial homeostasis after mitochondrial translocation is unknown. In this study, we performed a series of interaction and colocalization assays and found that, after mitochondrial translocation, Drp1 may promote the excessive opening of the mitochondrial permeability transition pore (mPTP) after hypoxia. Firstly, mitochondrial Drp1 maximumly recognizes mPTP channels by binding Bcl-2-associated X protein (BAX) and a phosphate carrier protein (PiC) in the mPTP. Then, leucine-rich repeat serine/threonine-protein kinase 2 (LRRK2) is recruited, whose kinase activity is inhibited by direct binding with mitochondrial Drp1 after hypoxia. Subsequently, the mPTP-related protein hexokinase 2 (HK2) is inactivated at Thr-473 and dissociates from the mitochondrial membrane, ultimately causing structural disruption and overopening of mPTP, which aggravates mitochondrial and cellular dysfunction after hypoxia. Thus, our study interprets the dual direct regulation of mitochondrial Drp1 on mitochondrial morphology and functions after hypoxia and proposes a new mitochondrial fission-independent mechanism for the role of Drp1 after its translocation in hypoxic injury.

The Protective Effect of a Novel Cross-Linked Hemoglobin-Based Oxygen Carrier on Hypoxia Injury of Acute Mountain Sickness in Rabbits and Goats.

Hypoxia is the major cause of acute altitude hypoxia injury in acute mountain sickness (AMS). YQ23 is a kind of novel bovine-derived, cross-linked hemoglobin-based oxygen carrier (HBOC). It has an excellent capacity for carrying and releasing oxygen. Whether YQ23 has a protective effect on the acute altitude hypoxia injury in AMS is unclear. In investigating this mechanism, the hypobaric chamber rabbit model and plain-to-plateau goat model were used. Furthermore, this study measured the effects of YQ23 on the ability of general behavior, general vital signs, Electrocardiograph (ECG), hemodynamics, vital organ injury markers, and blood gases in hypobaric chamber rabbits and plain-to-plateau goats. Our results showed that the ability of general behavior (general behavioral scores, GBS) (GBS: 18 ± 0.0 vs. 14 ± 0.5, p < 0.01) and the general vital signs weakened [Heart rate (HR, beats/min): 253.5 ± 8.7 vs. 301.1 ± 19.8, p < 0.01; Respiratory rate (RR, breaths/min): 86.1 ± 5.2 vs. 101.2 ± 7.2, p < 0.01] after exposure to plateau environment. YQ23 treatment significantly improved the ability of general behavior (GBS: 15.8 ± 0.5 vs. 14.0 ± 0.5, p < 0.01) and general vital signs [HR (beats/min): 237.8 ± 24.6 vs. 301.1 ± 19.8, p < 0.01; RR (breaths/min): 86.9 ± 6.6 vs. 101.2 ± 7.2, p < 0.01]. The level of blood PaO2 (mmHg) (115.3 ± 4.7 vs. 64.2 ± 5.6, p < 0.01) and SaO2(%) (97.7 ± 0.7 vs. 65.8 ± 3.1, p < 0.01) sharply decreased after exposure to plateau, YQ23 treatment significantly improved the blood PaO2 (mmHg) (97.6 ± 3.7 vs. 64.2 ± 5.6, p < 0.01) and SaO2(%) (82.7 ± 5.2 vs. 65.8 ± 3.1, p < 0.01). The cardiac ischemia and injury marker was increased [troponin (TnT, µg/L):0.08 ± 0.01 vs. 0.12 ± 0.02, p < 0.01], as well as the renal [blood urea nitrogen (BUN, mmol/L): 6.0 ± 0.7 vs. 7.3 ± 0.5, p < 0.01] and liver injury marker [alanine aminotransferase (ALT, U/L): 45.8 ± 3.6 vs. 54.6 ± 4.2, p < 0.01] was increased after exposure to a plateau environment. YQ23 treatment markedly alleviated cardiac ischemia [TnT (µg/L):0.10 ± 0.01 vs 0.12 ± 0.02, p < 0.01] and mitigated the vital organ injury. Besides, YQ23 exhibited no adverse effects on hemodynamics, myocardial ischemia, and renal injury. In conclusion, YQ23 effectively alleviates acute altitude hypoxia injury of AMS without aside effects.

Role of AQP3 in the Vascular Leakage of Sepsis and the Protective Effect of Ss-31.

ABSTRACT: Aquaporins (AQPs) are a group of membrane proteins related to water permeability. Studies have shown that AQPs play a vital role in various diseases. Whether AQPs participate in regulating vascular permeability after sepsis and whether the subtype of AQPs is related are unknown. Ss-31, as a new antioxidant, had protective effects on a variety of diseases. However, whether Ss-31 has a protective effect on pulmonary vascular permeability in sepsis and whether its effect is related to AQPs are unclear. Using the cecum ligation perforation-induced septic rat and LPS-treated pulmonary vein endothelial cells, the role of AQPs in the regulation of the permeability of pulmonary vascular and its relationship to Ss-31 were studied. The results showed that the pulmonary vascular permeability significantly increased after sepsis, meanwhile the expressions of AQP3, 4, and 12 increased. Among those, the AQP3 was closely correlated with pulmonary vascular permeability. The inhibition of AQP3 antagonized the increase of the permeability of monolayer pulmonary vein endothelial cells. Further study showed that the expression of caveolin-1 (Cav-1) increased and occludin decreased after sepsis. The inhibition of AQP3 antagonized the decrease of Cav-1 and the increase of occludin in sepsis. Antioxidant Ss-31 decreased the expression of AQP3 and ROS levels. At the same time, Ss-31 improved pulmonary vascular permeability and prolonged survival of sepsis rats. In conclusion, AQP3 participates in the regulation of pulmonary vascular permeability after sepsis, and the antioxidant Ss-31 has a protective effect on pulmonary vascular permeability by downregulating the expression of AQP3 and inhibiting ROS production.

A Novel Cross-Linked Hemoglobin-Based Oxygen Carrier, YQ23, Extended the Golden Hour for Uncontrolled Hemorrhagic Shock in Rats and Miniature Pigs.

Background: Hypotensive resuscitation is widely applied for trauma and war injury to reduce bleeding during damage-control resuscitation, but the treatment time window is limited in order to avoid hypoxia-associated organ injury. Whether a novel hemoglobin-based oxygen carrier (HBOC), YQ23 in this study, could protect organ function, and extend the Golden Hour for treatment is unclear. Method: Uncontrolled hemorrhagic shock rats and miniature pigs were infused with 0.5, 2, and 5% YQ23 before bleeding was controlled, while Lactate Ringer's solution (LR) and fresh whole blood plus LR (WB + LR) were set as controls. During hypotensive resuscitation the mean blood pressure was maintained at 50-60 mmHg for 60 min. Hemodynamics, oxygen delivery and utilization, blood loss, fluid demand, organ function, animal survival as well as side effects were observed. Besides, in order to observe whether YQ23 could extend the Golden Hour, the hypotensive resuscitation duration was extended to 180 min and animal survival was observed. Results: Compared with LR, infusion of YQ23 in the 60 min pre-hospital hypotensive resuscitation significantly reduced blood loss and the fluid demand in both rats and pigs. Besides, YQ23 could effectively stabilize hemodynamics, and increase tissue oxygen consumption, increase the cardiac output, reduce liver and kidney injury, which helped to reduce the early death and improve animal survival. In addition, the hypotensive resuscitation duration could be extended to 180 min using YQ23. Side effects such as vasoconstriction and renal injury were not observed. The beneficial effects of 5% YQ23 are equivalent to similar volume of WB + LR. Conclusion: HBOC, such as YQ23, played vital roles in damage-control resuscitation for emergency care and benefited the uncontrolled hemorrhagic shock in the pre-hospital treatment by increasing oxygen delivery, reducing organ injury. Besides, HBOC could benefit the injured and trauma patients by extending the Golden Hour.

Synergistic effects of EMPs and PMPs on pulmonary vascular leakage and lung injury after ischemia/reperfusion.

BACKGROUND: Vascular leakage is an important pathophysiological process of critical conditions such as shock and ischemia-reperfusion (I/R)-induced lung injury. Microparticles (MPs), including endothelial cell-derived microparticles (EMPs), platelet-derived microparticles (PMPs) and leukocyte-derived microparticles (LMPs), have been shown to participate in many diseases. Whether and which of these MPs take part in pulmonary vascular leakage and lung injury after I/R and whether these MPs have synergistic effect and the underlying mechanism are not known. METHODS: Using hemorrhage/transfusion (Hemo/Trans) and aorta abdominalis occlusion-induced I/R rat models, the role of EMPs, PMPs and LMPs and the mechanisms in pulmonary vascular leakage and lung injury were observed. RESULTS: The concentrations of EMPs, PMPs and LMPs were significantly increased after I/R. Intravenous administration of EMPs and PMPs but not LMPs induced pulmonary vascular leakage and lung injury. Furthermore, EMPs induced pulmonary sequestration of platelets and promoted more PMPs production, and played a synergistic effect on pulmonary vascular leakage. MiR-1, miR-155 and miR-542 in EMPs, and miR-126 and miR-29 in PMPs, were significantly increased after hypoxia/reoxygenation (H/R). Of which, inhibition of miR-155 in EMPs and miR-126 in PMPs alleviated the detrimental effects of EMPs and PMPs on vascular barrier function and lung injury. Overexpression of miR-155 in EMPs down-regulated the expression of tight junction related proteins such as ZO-1 and claudin-5, while overexpression of miR-126 up-regulated the expression of caveolin-1 (Cav-1), the trans-cellular transportation related protein such as caveolin-1 (Cav-1). Inhibiting EMPs and PMPs production with blebbistatin (BLE) and amitriptyline (AMI) alleviated I/R induced pulmonary vascular leakage and lung injury. CONCLUSIONS: EMPs and PMPs contribute to the pulmonary vascular leakage and lung injury after I/R. EMPs mediate pulmonary sequestration of platelets, producing more PMPs to play synergistic effect. Mechanically, EMPs carrying miR-155 that down-regulates ZO-1 and claudin-5 and PMPs carrying miR-126 that up-regulates Cav-1, synergistically mediate pulmonary vascular leakage and lung injury after I/R. Video Abstract.

Mdivi-1 attenuates oxidative stress and exerts vascular protection in ischemic/hypoxic injury by a mechanism independent of Drp1 GTPase activity.

Vascular dysfunctions such as vascular hyporeactivity following ischemic/hypoxic injury are a major cause of death in injured patients. In this study, we showed that treatment with mitochondrial division inhibitor 1 (Mdivi-1), a selective inhibitor of dynamin-related protein 1 (Drp1), significantly improved vascular reactivity in ischemic rats by attenuating oxidative stress. The antioxidative effects of Mdivi-1 were relatively Drp1-independent, and possibly due to an increase in the levels of the antioxidant enzymes, SOD1 and catalase, as well as to enhanced Nrf2 expression. In addition, we found that while Mdivi-1 had little effect on Drp1 GTPase activity in vascular smooth muscle cells, it inhibited hypoxia-induced Drp1 phosphorylation at Ser-616, reducing excessive mitochondrial fission and slightly enhancing mitochondrial fusion. These effects possibly contributed to vascular protection at an early stage of ischemic/hypoxic injury. Finally, Mdivi-1 stabilized hemodynamics, increased vital organ perfusion, and improved rat survival after ischemic/hypoxic injury, proving a promising therapeutic agent for ischemic/hypoxic injury.

Correction: Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-, BAX-, and GSH- pathways.

The original version of this Article omitted the following from the Acknowledgements: "This work was supported by the National Natural Science foundation of China (No. 81700429) and the Key Program of the National Natural Science Foundation of China (No. 81730059)." This has now been corrected in both the PDF and HTML versions of the Article.

The Beneficial Effect of HES on Vascular Permeability and Its Relationship With Endothelial Glycocalyx and Intercellular Junction After Hemorrhagic Shock.

BACKGROUND: Vascular leakage is a common complication of hemorrhagic shock. Endothelial glycocalyx plays a crucial role in the protection of vascular endothelial barrier function. Hydroxyethyl starch (HES) is a commonly used resuscitation fluid for hemorrhagic shock. However, whether the protective effect of HES on vascular permeability after hemorrhagic shock is associated with the endothelial glycocalyx is unclear. METHODS: Using hemorrhagic shock rat model and hypoxia treated vascular endothelial cells (VECs), effects of HES (130/0.4) on pulmonary vascular permeability and the relationship to endothelial glycocalyx were observed. RESULTS: Pulmonary vascular permeability was significantly increased after hemorrhagic shock, as evidenced by the increased permeability of pulmonary vessels to albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and Evans blue, the decreased transendothelial electrical resistance of VECs and the increased transmittance of FITC-BSA. The structure of the endothelial glycocalyx was destroyed, showing a decrease in thickness. The expression of heparan sulfate, hyaluronic acid, and chondroitin sulfate, the components of the endothelial glycocalyx, was significantly decreased. HES (130/0.4) significantly improved the vascular barrier function, recovered the thickness and the expression of components of the endothelial glycocalyx by down-regulating the expression of heparinase, hyaluronidase, and neuraminidase, and meanwhile increased the expression of intercellular junction proteins ZO-1, occludin, and VE-cadherin. Degradation of endothelial glycocalyx with degrading enzyme (heparinase, hyaluronidase, and neuraminidase) abolished the beneficial effect of HES on vascular permeability, but had no significant effect on the recovery of the expression of endothelial intercellular junction proteins induced by HES (130/0.4). HES (130/0.4) decreased the expression of cleaved-caspase-3 induced by hemorrhagic shock. CONCLUSIONS: HES (130/0.4) has protective effect on vascular barrier function after hemorrgic shock.The mechanism is mainly related to the protective effect of HES on endothelial glycocalyx and intercellular junction proteins. The protective effect of HES on endothelial glycocalyx was associated with the down-regulated expression of heparinase, hyaluronidase, and neuraminidase. HES (130/0.4) had an anti-apoptotic effect in hemorrhagic shock.

Drp1 regulates mitochondrial dysfunction and dysregulated metabolism in ischemic injury via Clec16a-, BAX-, and GSH- pathways.

The adaptation of mitochondrial homeostasis to ischemic injury is not fully understood. Here, we studied the role of dynamin-related protein 1 (Drp1) in this process. We found that mitochondrial morphology was altered in the early stage of ischemic injury while mitochondrial dysfunction occurred in the late stage of ischemia. Drp1 appeared to inhibit mitophagy by upregulating mito-Clec16a, which suppressed mito-Parkin recruitment and subsequently impaired the formation of autophagosomes in vascular tissues after ischemic injury. Moreover, ischemia-induced Drp1 activation enhanced apoptosis through inducing mitochondrial translocation of BAX and thereby increasing release of Cytochrome C to activate caspase-3/-9 signalling. Furthermore, Drp1 mediated metabolic disorders and inhibited the levels of mitochondrial glutathione to impair free radical scavenging, leading to further increases in ROS and the exacerbation of mitochondrial dysfunction after ischemic injury. Together, our data suggest a critical role for Drp1 in ischemic injury.

Activated Drp1-mediated mitochondrial ROS influence the gut microbiome and intestinal barrier after hemorrhagic shock.

A role of the mitochondrial dynamin-related protein (Drp1) on gut microbiome composition and intestinal barrier function after hemorrhagic shock has not been identified previously and thus addressed in this study. Here, we used a combination of 16S rRNA gene sequencing and mass spectrometry-based metabolomics profiling in WT and Drp1 KO mouse models to examine the functional impact of activated Drp1 on the gut microbiome as well as mitochondrial metabolic regulation after hemorrhagic shock. Our data showed that changes in mitochondrial Drp1 activity participated in the regulation of intestinal barrier function after hemorrhagic shock. Activated Drp1 significantly perturbed gut microbiome composition in the Bacteroidetes phylum. The abundance of short-chain fatty acid (SCFA) producing microbes, such as Bacteroides, Butyricimonas and Odoribacter, was markedly decreased in mice after shock, and was inversely correlated with both the distribution of the tight junction protein ZO1 and intestinal permeability. Together, these data suggest that Drp1 activation perturbs the gut microbiome community and SCFA production in a ROS-specific manner and thereby substantially disturbs tight junctions and intestinal barrier function after hemorrhagic shock. Our findings provide novel insights for targeting Drp1-mediated mitochondrial function as well as the microbiome in the treatment of intestinal barrier dysfunction after shock.

[Beneficial effects of hemoglobin-based oxygen carriers on early resuscitation in rats with uncontrolled hemorrhagic shock].

OBJECTIVE: To investigate the early resuscitation effect of hemoglobin-based oxygen carriers (HBOC) in rats with uncontrolled hemorrhagic shock. METHODS: 170 Sprague-Dawley (SD) rats were randomly divided into five groups: lactate Ringer solution (LR) control group, whole blood control group, and 0.5%, 2.0%, 5.0% HBOC groups, with 34 rats in each group. The uncontrolled hemorrhagic shock model in SD rats was reproduced by cutting off the splenic artery branch, and induced mean arterial pressure (MAP) reducing to 40 mmHg (1 mmHg = 0.133 kPa). The corresponding solution was infused after model reproduction in each group, maintaining MAP at 50 mmHg for 1 hour, then completely ligating and hemostasis, and maintaining MAP at 70 mmHg for 1 hour and 80 mmHg for 1 hour respectively, after maintaining MAP 80 mmHg, all were supplemented with LR to 2 times blood loss volume. The survival rate and blood loss rate were observed in 16 rats in each group, hemodynamics parameters including MAP, left ventricular systolic pressure (LVSP) and the maximum rate of left ventricular pressure rise (+dp/dt max) were determined in another 10 rats, and cardiac output (CO) and tissue oxygen supply (DO2) were observed in the rest 8 rats. RESULTS: (1) When resuscitation by LR alone, the blood loss rate of animals was as high as 60% to 70%. Compared with the LR control group, whole blood recovery could significantly reduce the blood loss rate before hemostasis in uncontrolled hemorrhagic shock rats [(46.6±4.5)% vs. (62.3±4.0)%, P < 0.01]; 0.5%, 2.0%, 5.0% HBOC could significantly decrease the blood loss rate, especially in 5.0% HBOC group with significant difference as compared with that in the LR control group [(45.6±4.1)% vs. (62.3±4.0)%, P < 0.01]. (2) When LR was used alone for resuscitation, the rats died quickly and survived for a short time. Only one rat survived for 12 hours, and no rat survived for more than 24 hours. Compared with the LR control group, whole blood resuscitation could improve the survival rate of uncontrolled hemorrhagic shock rats, and the survival time was significantly prolonged (hours: 20.4±4.6 vs. 3.5±1.1, P < 0.01); 0.5%, 2.0% and 5.0% HBOC also significantly prolonged the survival time of rats. The 5.0% HBOC group had the best effect, 4 rats survived in 24 hours, and the survival time was significantly longer than that of the LR control group (hours: 18.4±4.0 vs. 3.5±1.1, P < 0.01), and it was the same as the whole blood control group. (3) Compared with pre-shock, CO, DO2 and hemodynamic parameters of uncontrolled hemorrhagic shock rats were significantly decreased, and the above parameters were gradually increased with the prolongation of rehydration time. Compared with the LR control group, whole blood resuscitation could significantly increase CO and DO2, and improve hemodynamics in rats with uncontrolled hemorrhagic shock at different time points. Three concentrations of HBOC could also increase CO, DO2 and other hemodynamic parameters of rats at 1 hour of maintaining MAP of 80 mmHg after hemostasis and 1 hour and 2 hours after resuscitation. The effect of 5.0% HBOC group was more significant than that of the LR control group with statistically significant difference [CO (×10-3, L/min): 72.84±2.84 vs. 63.11±2.38 at 1 hour of maintaining MAP of 80 mmHg, 70.25±4.55 vs. 59.88±9.31 at 1 hour after resuscitation, 71.51±2.90 vs. 53.24±6.32 at 2 hours after resuscitation; DO2 (L×min-1×m-2): 271.9±13.5 vs. 159.1±25.4 at 1 hour of maintaining MAP of 80 mmHg, 261.0±15.0 vs. 145.7±20.1 at 1 hour after resuscitation, 249.6±12.0 vs. 107.4±18.2 at 2 hours after resuscitation; MAP (mmHg): 82.1±2.1 vs. 74.0±2.8 at 1 hour of maintaining MAP of 80 mmHg, 107.5±9.3 vs. 64.0±5.7 at 1 hour after resuscitation, 104.0±9.7 vs. 73.0±4.2 at 2 hours after resuscitation; LVSP (mmHg): 128.6±7.9 vs. 103.8±0.8 at 1 hour of maintaining MAP of 80 mmHg, 129.3±15.0 vs. 99.4±0.0 at 1 hour after resuscitation, 127.5±11.3 vs. 97.4±0.0 at 2 hours after resuscitation; +dp/dt max (mmHg/s): 6 534.2±787.6 vs. 5 074.0±71.7 at 1 hour of maintaining MAP of 80 mmHg, 5 961.5±545.4 vs. 4 934.5±510.2 at 1 hour after resuscitation, 5 897.4±350.5 vs. 4 534.7±489.2 at 2 hours after resuscitation, all P < 0.05]. CONCLUSIONS: HBOC infusion prolonged the survival time, increased survival rate, and improved hemodynamics, cardiac function and tissue oxygen supply in a dose-dependent manner in the early stage of uncontrolled hemorrhagic shock. The recovery effect of 5.0% HBOC was similar to that of the whole blood.