MKK3 depletion attenuates intestinal injury after traumatic hemorrhagic shock by restoring mitochondrial function.

BACKGROUND: Traumatic hemorrhagic shock (THS) is a complex pathophysiological process resulting in multiple organ failure. Intestinal barrier dysfunction is one of the mechanisms implicated in multiple organ failure. The present study aimed to explore the regulatory role of mitogen-activated protein kinase kinase 3 (MKK3) in THS-induced intestinal injury and to elucidate its potential mechanism. METHODS: Rats were subjected to trauma and hemorrhage to establish a THS animal model. MKK3-targeted lentiviral vectors were injected via the tail vein 72 h before modeling. Twelve hours post-modeling, the mean arterial pressure (MAP) and heart rate (HR) were monitored, and histological injury to the intestine was assessed via H&E staining and transmission electron microscopy. Mitochondrial function and mitochondrial reactive oxygen species (ROS) were evaluated. IEC-6 cells were exposed to hypoxia to mimic intestinal injury following THS in vitro. RESULTS: MKK3 deficiency alleviated intestinal injury and restored mitochondrial function in intestinal tissues from THS-induced rats and hypoxia-treated IEC-6 cells. In addition, MKK3 deficiency promoted Sirt1/PGC-1α-mediated mitochondrial biogenesis and restricted Pink1/Parkin-mediated mitophagy in the injured intestine and IEC-6 cells. Furthermore, the protective effect of MKK3 knockdown against hypoxia-induced mitochondrial damage was strengthened upon simultaneous LC3B/Pink1/Parkin knockdown or weakened upon simultaneous Sirt1 knockdown. CONCLUSION: MKK3 deficiency protected against intestinal injury induced by THS by promoting mitochondrial biogenesis and restricting excessive mitophagy.

Involvement of CD44 and MAPK14-mediated ferroptosis in hemorrhagic shock.

To elucidate the induction of ferroptotic pathways and the transcriptional modulation of pivotal genes in the context of hemorrhagic shock. The R software was used to analyze the GSE64711 dataset, isolating genes relevant to ferroptosis. Enrichment analyses and protein interaction networks were assembled. Using WGCNA hub genes were identified and intersected with ferroptosis-related genes, highlighting hub genes CD44 and MAPK14. In a rat hemorrhagic shock model, cardiac ROS, Fe2+, MDA, and GSH levels were assessed. Key ferroptotic proteins (SLC7A11/GPX4) in myocardial tissues were examined via western blot. Hub genes, CD44 and MAPK14, expressions were confirmed through immunohistochemistry. Analyzing the GSE64711 dataset revealed 337 differentially expressed genes, including 12 linked to ferroptosis. Enrichment analysis highlighted pathways closely related to ferroptosis. Using Genemania, we found these genes mainly affect ROS metabolism and oxidative stress response. WGCNA identified CD44 and MAPK14 as hub genes. Rat myocardial tissue validation showed significant cardiac damage and elevated ROS and MDA levels, and decreased GSH levels in the hemorrhagic shock model. The ferroptotic pathway SLC7A11/GPX4 was activated, and immunohistochemistry showed a significant increase in the expression levels of CD44 and MAPK14 in the hemorrhagic shock rat model. We demonstrated the presence of tissue ferroptosis in hemorrhagic shock by combining bioinformatics analysis with in vivo experimentation. Specifically, we observed the activation of the SLC7A11/GPX4 ferroptotic pathway. Further, CD44 and MAPK14 were identified as hub genes in hemorrhagic shock.

Mesenchymal stem cells overexpressing PBX1 alleviates haemorrhagic shock-induced kidney damage by inhibiting NF-κB activation.

Mesenchymal stem cells (MSCs) have favourable outcomes in the treatment of kidney diseases. Pre-B-cell leukaemia transcription factor 1 (PBX1) has been reported to be a regulator of self-renewal of stem cells. Whether PBX1 is beneficial to MSCs in the treatment of haemorrhagic shock (HS)-induced kidney damage is unknown. We overexpressed PBX1 in rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human bone marrow-derived mesenchymal stem cells (hBMSCs) to treat rats with HS and hypoxia-treated human proximal tubule epithelial cells (HK-2), respectively. The results indicated that PBX1 enhanced the homing capacity of rBMSCs to kidney tissues and that treatment with rBMSCs overexpressing PBX1 improved the indicators of kidney function, alleviated structural damage to kidney tissues. Furthermore, administration with rBMSCs overexpressing PBX1 inhibited HS-induced NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and the release of proinflammatory cytokines, and further attenuated apoptosis. We then determined whether NF-κB, an important factor in NLRP3 activation and the regulation of inflammation, participates in HS-induced kidney damage, and we found that rBMSCs overexpressing PBX1 inhibited NF-κB activation by decreasing the p-IκBα/IκBα and p-p65/p65 ratios and inhibiting the nuclear translocation and decreasing the DNA-binding capacity of NF-κB. hBMSCs overexpressing PBX1 also exhibited protective effects on HK-2 cells exposed to hypoxia, as shown by the increase in cell viability, the mitigation of apoptosis, the decrease in inflammation, and the inhibition of NF-κB and NLRP3 inflammasome activation. Our study demonstrates that MSCs overexpressing PBX1 ameliorates HS-induced kidney damage by inhibiting NF-κB pathway-mediated NLRP3 inflammasome activation and the inflammatory response.

LncRNA SNHG7 Knockdown Aggravates Hepatic Ischemia-Reperfusion Injury and Promotes Apoptosis in Hemorrhagic Shock Pregnant Rats by Modulating miR-34a-5p/YWHAG Axis.

Hemorrhagic shock is a frequent threat to pregnant women, and blood transfusions can contribute to organ damage, including hepatic ischemia-reperfusion (HIR) injury. LncRNA SNHG7 (SNHG7) has been reported to exert an essential role in various diseases, while the effect of SNHG7 on HIR injury induced by hemorrhagic shock and reperfusion in pregnant rats is still unclear. In our study, we examined the function and mechanism of SNHG7 in the progression of HIR injury in pregnant rats. The results showed that SNHG7 expression was low in the hepatic tissues of pregnant rats after the hemorrhagic shock and reperfusion modeling. Knockdown of SNHG7 further aggravated hepatic injury, apoptosis, and oxidative stress induced by hemorrhagic shock and reperfusion during pregnancy. Additionally, SNHG7 was bound directly to miR-34a-5p, and miR-34a-5p inhibitors partially reversed the effect of SNHG7 silencing on models of hemorrhagic shock and reperfusion. Furthermore, YWHAG is a direct target of miR-34a-5p and is negatively regulated by miR-34a-5p mimics. Overexpression of YWHAG effectively eliminated the effect of SNHG7 knockdown on pregnant rats. In summary, this investigation proved that SNHG7 knockdown exacerbated HIR injury after hemorrhagic shock in pregnant rats, and reperfusion might by mediating miR-34a-5p/YWHAG axis, indicating that SNHG7 can serve as a target gene for the treatment of HIR injury caused by hemorrhagic shock and reperfusion during pregnancy.

HIF-1α promotes the expression of syndecan-1 and inhibits the NLRP3 inflammasome pathway in vascular endothelial cells under hemorrhagic shock.

Hemorrhagic shock (HS) is a global life-threatening matter that causes massive mortality annually worldwide. Syndecan-1 (SDC1) is an important predictor and evaluation index for HS, but its mechanism involved in the HS development remain unclear. HS mice model and human umbilical vein endothelial cells (HUVECs) under hypoxia were applied to explore the relationship of SDC1 with HIF-1α and NLRP3 inflammasome in vascular ECs under HS. Transcriptome sequencing of isolated vascular ECs were conduct to search for hub genes. Dual luciferase assay was adopted to prove the binding effects of the HIF-1α on SDC1 promoter in HUVECs. Molecular expression was evaluated through routine experiments. Here, HS led to aggravated lung injury and inflammatory response with the shedding of SDC1 on the lung vascular ECs in mice. Circulatory SDC1 and proinflammatory cytokines were significantly increased after HS. HIF-1α and IL-1ß were identified as hub genes in vascular ECs of HS mice. Meanwhile, HIF-1α-mediaed hypoxia and IL-1ß-involved NLRP3 inflammasome pathways were activated following HS. The transcriptional factor HIF-1α promoted the expression of SDC1 through binding to the SDC1 promoter. SDC1 had an inhibitory effect on the NLRP3 inflammasome activity. An exogenous increase of HIF-1α upregulated SDC1 and restrained the activation of the NLRP3 inflammasome under hypoxia, while further interference of SDC1 weakened this effect. Hence, SDC1 is an intermediate connecting HIF-1α and NLRP3 inflammasome in the vascular ECs under hypoxia. HIF-1α promotes the expression of SDC1 and inhibits the NLRP3 inflammasome pathway in vascular ECs under HS.

c-Jun-mediated miR-19b expression induces endothelial barrier dysfunction in an in vitro model of hemorrhagic shock.

BACKGROUND: Our previous data demonstrated that miR-19b expression was increased in human lung microvascular endothelial cells in-vitro-, in-vivo and in patients with hemorrhagic shock, leading to a decrease in syndecan-1 mRNA and protein and resulting in loss of endothelial barrier function. However, the mechanism underlying increased miR-19b expression remains unclear. The objective of the current study was to determine if c-Jun mediates the early responsive microRNA, miR-19b, to cause endothelial barrier dysfunction. METHOD: Human lung microvascular endothelial cells (HLMEC) or HEK293T cells were transfected with c-Jun overexpressing vector, c-Jun siRNA, miR-19b promoter vector, miR-19b mutated promoter vector, miR-19b oligo inhibitor, then subjected to hypoxia/reoxygenation as in-vitro model of hemorrhagic shock. Levels of protein, miRNA, and luciferase activity were measured. Transwell permeability of endothelial monolayers were also determined. Plasma levels of c-Jun were measured in injured patients with hemorrhagic shock. RESULT: Hypoxia/reoxygenation induced primary (pri-)miR-19b, mature miR-19b, and c-Jun expression over time in a comparable timeframe. c-Jun silencing by transfection with its specific siRNA reduced both pri-miR-19b and mature miR-19b levels. Conversely, c-Jun overexpression enhanced H/R-induced pri-miR-19b. Studies using a luciferase reporter assay revealed that in cells transfected with vectors containing the wild-type miR-19b promoter and luciferase reporter, c-Jun overexpression or hypoxia/ reoxygenation significantly increased luciferase activity. c-Jun knockdown reduced the luciferase activity in these cells, suggesting that the miR-19b promoter is directly activated by c-Jun. Further, chromatin immunoprecipitation assay confirmed that c-Jun directly bound to the promoter DNA of miR-19b and hypoxia/reoxygenation significantly increased this interaction. Additionally, c-Jun silencing prevented cell surface syndecan-1 loss and endothelial barrier dysfunction in HLMECs after hypoxia/reoxygenation. Lastly, c-Jun was significantly elevated in patients with hemorrhagic shock compared to healthy controls. CONCLUSION: Transcription factor c-Jun is inducible by hypoxia/reoxygenation, binds to and activates the miR-19b promoter. Using an in-vitro model of hemorrhagic shock, our findings identified a novel cellular mechanism whereby hypoxia/ reoxygenation increases miR-19b transcription by inducing c-Jun and leads to syndecan-1 decrease and endothelial cell barrier dysfunction. This finding supports that miR-19b could be a potential therapeutic target for hemorrhage shock.

The Reaction Pathway of miR-30c-5p Activates Lipopolysaccharide Promoting the Course of Traumatic and Hemorrhagic Shock Acute Lung Injury.

Acute lung injury (ALI) is an acute hypoxic respiratory failure caused by diffuse inflammatory injury in alveolar epithelial cells during severe infection, trauma, and shock. Among them, trauma/hemorrhagic shock (T/HS) is the main type of indirect lung injury. Despite a great number of clinical studies, indirect factor trauma/hemorrhagic shock to the function and the mechanism in acute lung injury is not clear yet. Therefore, it is still necessary to carry on relevant analysis in order to thoroughly explore its molecular and cellular mechanisms and the pathway of disease function. In our research, we aimed to identify potential pathogenic genes and do modular analysis by downloading disease-related gene expression profile data. And our dataset is from the NCBI-GEO database. Then, we used the Clusterprofiler R package, GO function, and KEGG pathway enrichment analysis to analyze the core module genes. In addition, we also identified key transcription factors and noncoding RNAs. Based on the high degree of interaction of potential pathogenic genes and their involved functions and pathways, we identified 17 dysfunction modules. Among them, up to 9 modules significantly regulate the response to bacterial-derived molecules, and the response to lipopolysaccharide and other related functional pathways that mediate disease development. In addition, miR-290, miR-30c-5p, miR-195-5p, and miR-1-3p-based ncRNA and Jun, Atf1, and Atf3-based transcription factors have a total of 80 transcription drivers for functional modules. In summary, this study confirmed that miR-30c-5p activates lipopolysaccharide response pathway to promote the pathogenesis of ALI induced by hemorrhagic shock. This result can be an important direction for further research on related deepening diseases such as acute respiratory distress syndrome (ARDS). It further provides a piece of scientific medical evidence for revealing the pathogenic principle and cure difficulty of acute lung injury and also provides important guidance for the design of therapeutic strategies and drug development.

Down-regulation of miR-18b-5p protects against splenic hemorrhagic shock by directly targeting HIF-1α/iNOS pathway.

Splenic hemorrhagic shock is a typical emergency in surgery, seriously threatening human beings' life. Emerging evidence shows that microRNAs (miRNAs) are closely related to inflammation and immunity in the body. However, the detailed effects and underlying mechanisms of miRNAs on the immune function of splenic hemorrhagic shock have not been revealed yet. In the present study, we construct the rat hemorrhagic shock model, and the rats are further cured with splenic blood transport clipping recanalization (SBTCR). MiR-18b-5p was highly expressed in the spleen of hemorrhagic shock rats detected by the qRT-PCR assay. Functionally, down-regulation of miR-18b-5p notably inhibited the levels of SOD1, iNOS and IL-6 in macrophages isolated from splenic tissues detected by qRT-PCR and ELISA assays. In addition, inhibition of miR-18b-5p significantly decreased the M1/M2 ratio of macrophages. Besides, knockdown of miR-18b-5p obviously reduced the Th1/Th2 ratio of CD4+ T cells. Moreover, HIF-1α was predicted as a target gene of miR-18b-5p, which was further confirmed by dual-luciferase reporter assay, and HIF-1α was negatively associated with miR-18b-5p. Furthermore, overexpression of HIF-1α partially restored the effects of miR-18b-3p on inflammation and immunity in macrophages. Taken together, miR-18b-5p may be a novel therapeutic candidate target in splenic hemorrhagic shock treatment.

Hemorrhagic shock and encephalopathy syndrome in a patient with a de novo heterozygous variant in KIF1A.

INTRODUCTION: KIF1A, a gene that encodes a neuron-specific motor protein, plays important roles in cargo transport along neurites. Variants in KIF1A have been described in three different disorders, and neurodegeneration and spasticity with or without cerebellar atrophy or cortical visual impairment syndrome (NESCAVS) is the severest phenotype. CASE REPORT: A 3-year-old girl was born at term with a birth weight of 2590 g. At five months of age, she visited our hospital due to developmental delay. An EEG showed multiple epileptic discharge, and a nerve conduction study showed severe axonopathy of both motor and sensory nerves. We performed exome sequencing and identified a de novo heterozygous missense variant in KIF1A (NM_001244008.1: c. 757G > A, p.E253K). At six months of age, she developed acute encephalopathy, multiple organ failure and disseminated intravascular coagulation, necessitating intensive care. Her brain CT showed severe brain edema, followed by profound brain atrophy. We diagnosed hemorrhagic shock and encephalopathy syndrome (HSES) according to the clinico-radiological features. Currently, she is bed-ridden, and requires gastrostomy because of dysphagia. CONCLUSION: The clinical course of our case confirmed that p.E253K is associated with severe neurological features. Severe KIF1A deficiency could cause thermoregulatory dysfunction and may increase the risk of acute encephalopathy including HSES.

Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

BACKGROUND: After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. METHODS: C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. RESULTS: MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. CONCLUSION: The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

Hemorrhage and saline resuscitation are associated with epigenetic and proteomic reprogramming in the rat lung.

BACKGROUND: Epigenetic changes have been described in trauma patients in the form of histone acetylation events, but whether DNA-methylation occurs remains unknown. We hypothesized that the combination of hemorrhage and saline resuscitation would alter DNA-methylation and associated proteomic profiles in the rat lung. METHODS: Ten rats were subjected to a pressure-controlled hemorrhage and resuscitation model consisting of hemorrhage to a mean arterial pressure (MAP) of 35mmHg for 90 minutes, followed by saline resuscitation to a MAP >70mmHg for 90 minutes (n=5) or sham (only anesthesia and cannulation). Lungs were harvested and subjected to reduced genome wide DNA-methylation analysis through bisulphite sequencing as well as proteomics analysis. Data was analyzed for differentially methylated regions and associated alterations in proteomic networks through a weighted correlation network analysis (WCNA). Pathway analysis was used to establish biological relevance of findings. RESULTS: Hemorrhage and saline resuscitation were associated with differential methylation of 353 sites across the genome compared to the sham group. Of these, 30 were localized to gene promoter regions, 31 to exon regions and 87 to intron regions. Network analysis identified an association between hemorrhage/resuscitation and DNA-methylation events located to genes involved in areas of endothelial and immune response signaling. The associated proteomic response was characterized by activations of mRNA processing as well as endothelial Nitric Oxide Synthase (eNOS) metabolism. CONCLUSION: We demonstrated an association between DNA-methylation and hemorrhage/saline resuscitation. These results suggest a potential role of DNA-methylation in the host response to injury.

A road map from single-cell transcriptome to patient classification for the immune response to trauma.

Immune dysfunction is an important factor driving mortality and adverse outcomes after trauma but remains poorly understood, especially at the cellular level. To deconvolute the trauma-induced immune response, we applied single-cell RNA sequencing to circulating and bone marrow mononuclear cells in injured mice and circulating mononuclear cells in trauma patients. In mice, the greatest changes in gene expression were seen in monocytes across both compartments. After systemic injury, the gene expression pattern of monocytes markedly deviated from steady state with corresponding changes in critical transcription factors, which can be traced back to myeloid progenitors. These changes were largely recapitulated in the human single-cell analysis. We generalized the major changes in human CD14+ monocytes into 6 signatures, which further defined 2 trauma patient subtypes (SG1 vs. SG2) identified in the whole-blood leukocyte transcriptome in the initial 12 hours after injury. Compared with SG2, SG1 patients exhibited delayed recovery, more severe organ dysfunction, and a higher incidence of infection and noninfectious complications. The 2 patient subtypes were also recapitulated in burn and sepsis patients, revealing a shared pattern of immune response across critical illness. Our data will be broadly useful to further explore the immune response to inflammatory diseases and critical illness.

Effect of Beta-Blockade on the Expression of Regulatory MicroRNA after Severe Trauma and Chronic Stress.

BACKGROUND: Beta-blockade administration after lung contusion, hemorrhagic shock, and chronic stress has been shown to improve bone marrow function, decrease hypercatecholaminemia, and reduce inflammation. MicroRNAs (miR) are critical biologic regulators that can downregulate gene expression by causing messenger RNA degradation or inhibition of translation. This study sought to expand our understanding of the molecular mechanisms underlying the reduced inflammatory response after the administration of beta-blockade (BB) in our rodent trauma model. STUDY DESIGN: Male Sprague-Dawley rats aged 8 to 9 weeks were randomized to lung contusion, hemorrhagic shock with daily restraint stress (LCHS/CS) or LCHS/CS plus propranolol (LCHS/CS+BB). Restraint stress occurred 2 hours daily after LCHS. Propranolol (10 mg/kg) was given daily until day 7. Total RNA and miR were isolated from bone marrow and genome-wide miR expression patterns were assayed. Bone marrow cytokine expression was determined with quantitative polymerase chain reaction. RESULTS: LCHS/CS led to significantly increased bone marrow expression of interleukin (IL) 1ß, tumor necrosis factor-α, IL-6, nitric oxide, and plasma C-reactive protein. There were marked differences in expression of 45 miRs in the LCHS/CS+BB group compared with the LCHS/CS group when using a p value <0.001. Rno-miR-27a and miR-25 were upregulated 7- to 8-fold in the rodents who underwent LCHS/CS+BB compared with LCHS/CS alone, and this correlated with reduced bone marrow expression of IL-1ß, tumor necrosis factor-α, IL-6, nitric oxide, and reduced plasma C-reactive protein in the LCHS/CS+BB group. CONCLUSIONS: The genomic and miR expression patterns in bone marrow after LCHS/CS differed significantly compared with rodents that received propranolol after LCHS/CS. The use of BB after severe trauma can help mitigate persistent inflammation by upregulating Rno-miR-27a and miR-25 and reducing inflammatory cytokines in those who remain critically ill.

Alda-1 Prevents Pulmonary Epithelial Barrier Dysfunction following Severe Hemorrhagic Shock through Clearance of Reactive Aldehydes.

Severe hemorrhagic shock and resuscitation (HS/R) can lead to lung injury, resulting in respiratory insufficiency. We investigated whether treatment with Alda-1, an ALDH2 activator, decreased lung injury induced by severe HS/R in a rat model. Male Sprague-Dawley rats were randomized into three groups, hemorrhagic shock + placebo, hemorrhagic shock + Alda-1, and sham. All animals were heparinized, and then 50% of the total calculated blood volume was collected over 60 minutes. After 40 minutes of hemorrhagic shock, animals were reinfused with the shed blood over 40 minutes and then observed for an additional 2 hours. Concentrations of 4-HNE, TNF-α, IL-6, and ALDH2 activity were detected; lung injury and lung wet-to-dry weight ratios were assessed. Expression of occludin and ZO-1 proteins in lung tissues was also determined. At 2 hours after resuscitation, lung injury was significantly reduced and the wet-to-dry weight ratio was notably decreased in the Alda-1 group compared with placebo (P<0.05). Alda-1 treatment also significantly increased the activity of ALDH2 and decreased the levels of toxic 4-HNE (P<0.05). In the Alda-1 group, IL-6 and TNF-α were dramatically decreased compared with placebo-treated animals (P<0.05). Expression of occludin and ZO-1 proteins was significantly decreased in the placebo group compared with the Alda-1 group (P<0.05). Thus, in a rat model of severe HS/R, treatment with Alda-1 increased the activity of ALDH2, significantly accelerated the clearance of reactive aldehydes, and concomitantly alleviated lung injury through improvement of pulmonary epithelial barrier integrity resulting in decreased alveolar epithelial tissue permeability, lung edema, and diffuse infiltration of inflammatory cells.

Activation of Pregnane X Receptor Sensitizes Mice to Hemorrhagic Shock-Induced Liver Injury.

Hemorrhagic shock (HS) is a life-threatening condition associated with tissue hypoperfusion and often leads to injury of multiple organs including the liver. Pregnane X receptor (PXR) is a species-specific xenobiotic receptor that regulates the expression of drug-metabolizing enzymes (DMEs) such as the cytochrome P450 (CYP) 3A. Many clinical drugs, including those often prescribed to trauma patients, are known to activate PXR and induce CYP3A. The goal of this study is to determine whether PXR plays a role in the regulation of DMEs in the setting of HS and whether activation of PXR is beneficial or detrimental to HS-induced hepatic injury. PXR transgenic, knockout, and humanized mice were subject to HS, and the liver injury was assessed histologically and biochemically. The expression and/or activity of PXR and CYP3A were manipulated genetically or pharmacologically in order to determine their effects on HS-induced liver injury. Our results showed that genetic or pharmacological activation of PXR sensitized wild-type and hPXR/CYP3A4 humanized mice to HS-induced hepatic injury, whereas knockout of PXR protected mice from HS-induced liver injury. Mechanistically, the sensitizing effect of PXR activation was accounted for by PXR-responsive induction of CYP3A and increased oxidative stress in the liver. The sensitizing effect of PXR was attenuated by ablation or pharmacological inhibition of CYP3A, treatment with the antioxidant N-acetylcysteine amide, or treatment with a PXR antagonist. Conclusion: We have uncovered a function of PXR in HS-induced hepatic injury. Our results suggest that the unavoidable use of PXR-activating drugs in trauma patients has the potential to exacerbate HS-induced hepatic injury, which can be mitigated by the coadministration of antioxidative agents, CYP3A inhibitors, or PXR antagonists.

Pathophysiological significance of clock genes BMAL1 and PER2 as erythropoietin-controlling factors in acute blood hemorrhage.

This study aimed to characterize the pathophysiology, including possible correlations, of clock gene expression and erythropoietin (EPO) production in the acute stage of blood hemorrhage. Specimens of human cortical tissues (right and left kidneys) and cardiac blood were collected at autopsy from 52 cases following mortality due to acute-stage blood hemorrhage following sharp instrument injury. BMAL1 and PER2 mRNA levels were determined by reverse transcription-polymerase chain reaction; BMAL1 and PER2 protein levels were assessed using immunohistochemistry; BMAL1 protein levels were quantitatively measured by western blotting; and serum EPO levels were measured by chemiluminescent enzyme immunoassay. Separately, a rat model of hemorrhagic conditions was generated and used to confirm the results obtained with autopsy-derived specimens. A positive correlation was observed between BMAL1 protein and serum EPO levels, but not between BMAL1 mRNA levels and serum EPO levels. We also noted that Per2 mRNA expression became elevated in humans who survived for > 3 h after acute hemorrhagic events, with subsequent decreases in serum EPO levels. The rat model showed that even short (30-min) intervals of blood loss yielded increases in both Bmal1 mRNA and serum EPO levels; longer (60-min) intervals resulted in increases in Per2 mRNA expression along with decreases in serum EPO. Thus, the acute-stage human hemorrhage cases and the rat hemorrhage model yielded similar tendencies for clock gene expression and EPO secretion. In conclusion, our results indicated that clock genes are involved in the regulation of EPO production during the early stages of hypoxia/ischemia resulting from the acute hemorrhagic events.

Deficiency of metabolite sensing receptor HCA2 impairs the salutary effect of niacin in hemorrhagic shock.

Inflammation and cellular energetics play critical roles in organ dysfunction following hemorrhagic shock. Recent studies suggest a putative role for sirtuin 1 (SIRT1) in potentiating mitochondrial function and improving organ function following hemorrhagic shock in animal models. SIRT1 is an NAD+ dependent protein deacetylase and increased availability of NAD+ has been shown to augment SIRT1 activity. As niacin is a precursor of NAD+, in this study, we tested whether niacin can improve survival following hemorrhagic shock. However niacin also mediates its biological action by binding to its receptor, hydroxyl-carboxylic acid receptor 2 (HCA2 or Gpr109a); so we examined whether the effect of niacin is mediated by binding to Gpr109a or by increasing NAD+ availability. We found that niacin administered intravenously to rats subjected to hemorrhagic injury (HI) in the absence of fluid resuscitation resulted in a significantly prolonged duration of survival. However, treatment of rats with similar doses of nicotinamide mononucleotide (NMN), a precursor to NAD+ that does not bind Gpr109a, did not extend survival following HI. The duration of survival due to niacin treatment was significantly reduced in Gpr109a-/- mice subjected to HI. These experiments demonstrated that the Gpr109a receptor-mediated pathway contributed significantly to niacin mediated salutary effect. Further studies showed improvement in markers of cellular energetics and attenuation of inflammatory response with niacin treatment. In conclusion, we report that Gpr109a-dependent signalling is important in restoring cellular energetics and immunometabolism following hemorrhagic shock.

The Effects of Genetic 3-Mercaptopyruvate Sulfurtransferase Deficiency in Murine Traumatic-Hemorrhagic Shock.

INTRODUCTION: Hemorrhagic shock is a major cause of death after trauma. An additional blunt chest trauma independently contributes to mortality upon the development of an acute lung injury (ALI) by aggravating pathophysiological consequences of hemorrhagic shock. The maintenance of hydrogen sulfide availability is known to play an important role during hemorrhage and ALI. We therefore tested the impact of a genetic 3-mercaptopyruvate sulfurtransferase mutation (Δ3-MST) in a resuscitated murine model of traumatic-hemorrhagic shock. METHODS: Anesthetized wild-type (WT) and Δ3-MST mice underwent hemorrhagic shock with/without blunt chest trauma. Hemorrhagic shock was implemented for 1 h followed by retransfusion of shed blood and intensive care therapy for 4 h, including lung-protective mechanical ventilation, fluid resuscitation, and noradrenaline titrated to maintain a mean arterial pressure at least 50 mmHg. Systemic hemodynamics, metabolism, and acid-base status were assessed together with lung mechanics and gas exchange. Postmortem tissue samples were analyzed for immunohistological protein expression and mitochondrial oxygen consumption. RESULTS: 3-MST-deficient mice showed similar results in parameters of hemodynamics, gas exchange, metabolism, acid base status, and survival compared with the respective WT controls. Renal albumin extravasation was increased in Δ3-MST mice during hemorrhagic shock, together with a decrease of LEAK respiration in heart tissue. In contrast, mitochondrial oxygen consumption in the uncoupled state was increased in kidney and liver tissue of Δ3-MST mice subjected to the combined trauma. CONCLUSIONS: In summary, in a resuscitated murine model of traumatic-hemorrhagic shock, 3-MST deficiency had no physiologically relevant impact on hemodynamics and metabolism, which ultimately lead to unchanged mortality regardless of an additional blunt chest trauma.

Analyses of changes in myocardial long non-coding RNA and mRNA profiles after severe hemorrhagic shock and resuscitation via RNA sequencing in a rat model.

BACKGROUND: Ischemia-reperfusion injury has been proven to induce organ dysfunction and death, although the mechanism is not fully understood. Long non-coding RNAs (lncRNAs) have drawn wide attention with their important roles in the gene expression of some biological processes and diseases, including myocardial ischemia-reperfusion (I/R) injury. In this paper, a total of 26 Sprague-Dawley (SD) rats were randomized into two groups: sham and ischemia-reperfusion (I/R) injury. Hemorrhagic shock was induced by removing 45% of the estimated total blood volume followed by reinfusion of shed blood. High-throughput RNA sequencing was used to analyze differentially expressed (DE) lncRNAs and messenger RNAs (mRNAs) in the heart tissue 4 h after reperfusion. Myocardial function was also evaluated. RESULTS: After resuscitation, the decline of myocardial function of shocked animals, expressed by cardiac output, ejection fraction, and myocardial performance index (MPI), was significant (p < 0.05). DE lncRNAs and mRNAs were identified by absolute value of fold change ≥ 2 and the false discovery rate ≤ 0.001. In rats from the I/R injury group, 851 lncRNAs and 1015 mRNAs were significantly up-regulated while 1533 lncRNAs and 1702 m RNAs were significantly down-regulated when compared to the sham group. Among the DE lncRNAs, we found 12 location-associated with some known apoptosis-related protein-coding genes which were up-regulated or down-regulated accordingly, including STAT3 and Il1r1. Real time PCR assays confirmed that the expression levels of five location-associated lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2, NONRATT006035.2 and NONRATT029969.2) and their location-associated mRNAs (STAT3 and Il1r1) in the rats from the I/R injury group were all significantly up-regulated versus the sham group. CONCLUSIONS: The DE lncRNAs (NONRATT006032.2, NONRATT006033.2, NONRATT006034.2 and NONRATT006035.2) could be compatible with their role in myocardial protection by stimulating their co-located gene (STAT3) after hemorrhagic shock and resuscitation. The final prognosis of I/R injury might be regulated by different genes, which is regarded as a complex network.

Metformin Exerts Beneficial Effects in Hemorrhagic Shock in An AMPKα1-Independent Manner.

Despite therapeutic advances in hemorrhagic shock, mortality from multiple organ failure remains high. AMP-activated protein kinase (AMPK) is involved in cellular energy homeostasis. Two catalytic subunits, α1 and α2, have been identified, with α1 subunit largely expressed in major organs. Here, we hypothesized that genetic deficiency of AMPKα1 worsens hemorrhage-induced multiple organ failure. We also investigated whether treatment with metformin, a clinically used drug for metabolic homeostasis, affords beneficial effects. AMPKα1 wild-type (WT) and knock-out mice (KO) were subjected to hemorrhagic shock by blood withdrawing followed by resuscitation with shed blood and Lactated Ringer's solution and treatment with vehicle or metformin. Mice were sacrificed at 3 h after resuscitation. Compared with vehicle-treated WT animals, KO animals exhibited a more severe hypotension, higher lung and liver injury and neutrophil infiltration, and higher levels of plasma inflammatory cytokines. Metformin treatment ameliorated organ injury and mean arterial blood pressure in both WT and KO mice, without affecting systemic cytokine levels. Furthermore, metformin treatment reduced liver lipid peroxidation and increased levels of complex II cosubstrate FAD and levels of ATP in WT and KO mice. Beneficial effects of metformin were associated with organ-specific nuclear-cytoplasmic shuttling and activation of liver kinase B1 and AMPKα2. Thus, our data suggest that AMPKα1 is an important regulator of hemodynamic stability and organ metabolic recovery during hemorrhagic shock. Our data also suggest that metformin affords beneficial effects, at least in part, independently of AMPKα1 and secondary to AMPKα2 activation, increase of Complex II function and reduction of oxidative stress.