Mutual regulation between Polo-like kinase 3 and SIAH2 E3 ubiquitin ligase defines a regulatory network that fine-tunes the cellular response to hypoxia and nickel.

Elevated cellular response to hypoxia, which contributes to cell transformation and tumor progression, is a prominent feature of malignant cells in solid tumors. Polo-like kinase 3 (Plk3) is a serine/threonine protein kinase known to inhibit the cellular response to hypoxia and tumorigenesis. Nickel compounds are well-established human carcinogens that induce tumorigenesis partly through their hypoxia-mimicking effects. Despite previous research efforts, the role of Plk3 in the hypoxic response induced by hypoxia or nickel is not completely understood. Here, we show that NiCl2 (Ni(II)) or hypoxia reduces the protein level and shortens the half-life of cytoplasmic Plk3 in a ubiquitin-proteasome-dependent manner. We identify SIAH2, a RING finger E3 ubiquitin ligase associated with the cellular hypoxic response, to be the ubiquitin E3 ligase that mediates the degradation of Plk3. We show that SIAH2 binds to Plk3 and mediates its ubiquitination primarily through its polo-box domain. We report that USP28, a deubiquitinase known to be inhibitable by Ni(II) or hypoxia, may also contribute to the suppression of the Plk3 protein by Ni(II). We also show that Plk3 in turn suppresses the SIAH2 protein level in a kinase activity-dependent manner. Our study revealed an interesting mutual regulation between Plk3 and SIAH2 and uncovered a regulatory network that functions to fine-tune the cellular hypoxic response. We propose that suppression of Plk3 expression contributes to carcinogenesis and tumor progression induced by nickel compounds.

Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase-dependent mechanism.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)-dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3-binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair.

Melatonin protects mouse spermatogonial stem cells against hexavalent chromium-induced apoptosis and epigenetic histone modification.

Given the potential biological functions of spermatogonial stem cells (SSCs) in spermatogenesis and in delivering parental genetic information to the next generation, how these cells respond to environmental toxins and carcinogens should be investigated. We examined the toxic effect of hexavalent chromium (Cr(VI)) on global histone modifications and apoptotic signaling pathways in SSCs. We determined the effect of melatonin, one of the most powerful endogenous free radical scavengers and wide-spectrum antioxidants, in protecting SSCs from Cr(VI)-induced apoptosis and global histone modification by Western blot analysis. In addition, we examined the in vivo effect of melatonin on Cr(VI)-induced histological changes of seminiferous tubules in mouse testes. We also evaluated the fertility of male mice by monitoring litter size following intraperitoneal injection of these chemicals. Our study demonstrated the Cr(VI)-induced global increases in H3K9me3 and H3K27me3 and activated the apoptotic signaling pathway. Pretreatment of SSCs with melatonin alleviated Cr(VI)-induced apoptosis and the global increase of H3K9me3. Exposure to melatonin also attenuated the Cr(VI)-induced increase of the abundance of histone methyltransferase ESET. Furthermore, exogenous administration of melatonin protected mice against Cr(VI)-induced changes in testicular histology and germ cell apoptosis, which helped maintain normal spermatogenesis and male fertility. Our study revealed a potential new therapeutic approach for male reproductive injury caused by Cr(VI).

[Hardware System for the Test of Artificial Joint Biomechanics].

In the research of artificial joint biomechanics, it is a common method in the world to evaluate the biomechanical properties of the implanted fixtures through experiments in vitro. The domestic research started late, and the corresponding testing methods were lacking. There is still no unified standard. In this paper, a complete hardware test system was designed and built around the existing mechanical testing machine, including:binocular vision catcher, torque bearing clamp, film pressure sensor and so on. The system can accurately measure the relative motion angle value, external torque value, and inter pressure value of each joint. It has some reference significance for the forming and standardization of the artificial joints' evaluation system.

[Development of Software System for Artificial Joint Biomechanics].

In the research of artificial joint biomechanics, it is a common method in the world to evaluate the biomechanical properties of the implanted fixtures through experiments in vitro. The domestic research started late, and the corresponding testing software were lacking. There is still no special software. In this paper, an integrated software test system was designed and built based on the existing hardware system, including:hardware control, data collection, data save, data processing and display. It can directly show the 3D motion trajectory and the angle curve of joints. The system can accurately measure the relative motion angle value, external torque value, and inter pressure value of each joint. It has some reference significance for the development of the artificial joints' evaluation system.

[Multifunctional Cervical Health Care Pillow].

Cervical spondylosis is due to degenerative cervical disc and its stimulation or oppression of the adjacent nerves, spinal cord, spinal artery and other tissue caused by clinical symptoms. The cervical spine is an anatomical structure with activity, while the pillow has a certain plastic fixation effect on the cervical spine anatomy. Therefore, the pillow not only plays a health role in the cervical spine, but also plays an important role in restoring the normal physiological curvature of the cervical spine. Based on this, a multi-functional cervical vertebra treatment pillow is designed, which not only has the functions of traction, maintaining different positions of the cervical spine, correcting the cervical curvature and equipment exercises, but also has the functions of voice broadcast and network data terminal.

Single-cell RNA sequencing reveals an altered gene expression pattern as a result of CRISPR/cas9-mediated deletion of Gene 33/Mig6 and chronic exposure to hexavalent chromium in human lung epithelial cells.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently reported that depletion of this protein promotes lung epithelial cell transformation induced by hexavalent chromium [Cr(VI)]. However, the early molecular events that mediate this process are not clear. In the present study, we used single-cell RNA sequencing to compare gene expression profiles between BEAS-2B lung epithelial cells chronically exposed to a sublethal dose of Cr(VI) with or without CRISPR/cas9-mediated deletion of Gene 33. Our data reveal 83 differentially expressed genes. The most notable changes are genes associated with cell adhesion, oxidative stresses, protein ubiquitination, epithelial-mesenchymal transition/metastasis, and WNT signaling. Up-regulation of some neuro-specific genes is also evident, particularly ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a deubiquitinase and potential biomarker for lung cancer. Gene 33 deletion and/or Cr(VI) exposure did not cause discernable changes in cell morphology. However, Gene 33 deletion led to a modest but significant reduction of cells in the G2/M phase of the cell cycle regardless of Cr(VI) exposure. Gene 33 deletion also significantly reduced cell proliferation. Interestingly, Cr(VI) exposure eliminated the difference in cell proliferation between the two genotypes. Gene 33 deletion also significantly elevated cell migration. Our data indicate that combined Gene 33 deletion and chronic Cr(VI) exposure produces a gene expression pattern and a phenotype resemble those of the transformed lung epithelial cells. Given the known association of UCHL1 with lung cancer, we propose that UCHL1 is an important player in the early stage of lung epithelial cell transformation and tumorigenesis.

Arsenic induces polyadenylation of canonical histone mRNA by down-regulating stem-loop-binding protein gene expression.

The replication-dependent histone genes are the only metazoan genes whose messenger RNA (mRNA) does not terminate with a poly(A) tail at the 3'-end. Instead, the histone mRNAs display a stem-loop structure at their 3'-end. Stem-loop-binding protein (SLBP) binds the stem-loop and regulates canonical histone mRNA metabolism. Here we report that exposure to arsenic, a carcinogenic metal, decreased cellular levels of SLBP by inducing its proteasomal degradation and inhibiting SLBP transcription via epigenetic mechanisms. Notably, arsenic exposure dramatically increased polyadenylation of canonical histone H3.1 mRNA possibly through down-regulation of SLBP expression. The polyadenylated H3.1 mRNA induced by arsenic was not susceptible to normal degradation that occurs at the end of S phase, resulting in continued presence into mitosis, increased total H3.1 mRNA, and increased H3 protein levels. Excess expression of canonical histones have been shown to increase sensitivity to DNA damage as well as increase the frequency of missing chromosomes and induce genomic instability. Thus, polyadenylation of canonical histone mRNA following arsenic exposure may contribute to arsenic-induced carcinogenesis.

Intraluminal nonbacterial intestinal components control gut and lung injury after trauma hemorrhagic shock.

OBJECTIVE: To test whether the mucus layer, luminal digestive enzymes, and intestinal mast cells are critical components in the pathogenesis of trauma shock-induced gut and lung injury. BACKGROUND: Gut origin sepsis studies have highlighted the importance of the systemic component (ischemia-reperfusion) of gut injury, whereas the intraluminal component is less well studied. METHODS: In rats subjected to trauma hemorrhagic shock (T/HS) or sham shock, the role of pancreatic enzymes in gut injury was tested by diversion of pancreatic enzymes via pancreatic duct exteriorization whereas the role of the mucus layer was tested via the enteral administration of a mucus surrogate. In addition, the role of mast cells was assessed by measuring mast cell activation and the ability of pharmacologic inhibition of mast cells to abrogate gut and lung injury. Gut and mucus injury was characterized functionally, morphologically, and chemically. RESULTS: Pancreatic duct exteriorization abrogated T/HS-induced gut barrier loss and limited chemical mucus changes. The mucus surrogate prevented T/HS-induced gut and lung injury. Finally, pancreatic enzyme-induced gut and lung injury seems to involve mast cell activation because T/HS activates mast cells and pharmacologic inhibition of intestinal mast cells prevented T/HS-induced gut and lung injury. CONCLUSIONS: These results indicate that gut and gut-induced lung injury after T/HS involves a complex process consisting of intraluminal digestive enzymes, the unstirred mucus layer, and a systemic ischemic-reperfusion injury. This suggests the possibility of intraluminal therapeutic strategies.

Effects of hexavalent chromium on mitochondria and their implications in carcinogenesis.

Hexavalent chromium (Cr(VI)) is a well-known occupational and environmental human carcinogen. The cellular effect of Cr(VI) is complex and often nonspecific due to its ability to modulate multiple cellular targets. The toxicity of Cr(VI) is strongly linked to the generation of reactive oxygen species (ROS) during its reduction process. ROS can cause oxidation of cellular macromolecules, such as proteins, lipids, and DNA, thereby altering their functions. A major genotoxic effect of Cr(VI) that contributes to carcinogenesis is the formation of DNA adducts, which can lead to DNA damage. Modulations of cellular signaling pathways and epigenetics may also contribute to the carcinogenic effects of Cr(VI). Cr(VI) has a major impact on many aspects of mitochondrial biology, including oxidative phosphorylation, mitophagy, and mitochondrial biogenesis. These effects have the potential to alter the trajectory of Cr(VI)-induced carcinogenic process. This perspective article summarizes current understandings of the effect of Cr(VI) on mitochondria and discusses the future directions of research in this area, particularly with regard to carcinogenesis.

Systemic inflammatory response does not correlate with acute lung injury associated with mechanical ventilation strategies in normal lungs.

BACKGROUND: Mechanical ventilation (MV) can lead to ventilator-induced lung injury secondary to trauma and associated increases in pulmonary inflammatory cytokines. There is controversy regarding the associated systemic inflammatory response. In this report, we demonstrate the effects of MV on systemic inflammation. METHODS: This report is part of a previously published study (Hong et al. Anesth Analg 2010;110:1652-60). Female pigs were randomized into 3 groups. Group H-Vt/3 was ventilated with a tidal volume (Vt) of 15 mL/kg predicted body weight (PBW)/positive end-expiratory pressure (PEEP) of 3 cm H(2)O; group L-Vt/3 with a Vt of 6 mL/kg PBW/PEEP of 3 cm H2O; and group L-Vt/10 with a Vt of 6 mL/kg PBW/PEEP of 10 cm H(2)O, for 8 hours. Each group had 6 subjects (n = 6). Prelung and postlung sera were analyzed for inflammatory markers. Hemodynamics, airway mechanics, and arterial blood gases were monitored. RESULTS: There were no significant differences in systemic cytokines among groups. There were similar trends of serum inflammatory markers in all subjects. This is in contrast to findings previously published demonstrating increases in inflammatory mediators in bronchoalveolar lavage. CONCLUSION: Systemic inflammatory markers did not correlate with lung injury associated with MV.

Immune regulatory functions of biologically active proteins from edible fungi.

Proteins from edible mushrooms have a variety of biological activities. Here, thirteen precious edible mushrooms such as Ophiocordyceps sinensis, Ganoderma lucidum, and Morchella esculenta and nine common edible mushrooms such as Flammulina velutipes, Pleurotus ostreatus, and Pleurotus eryngii, etc., from which their proteins were extracted, their composition analyzed and their immunomodulatory activity assessed. Rare mushrooms are a species of edible mushrooms with higher edible value and medicinal value than common edible mushrooms. The results showed that all the different edible mushroom crude proteins increased the proliferation and phagocytosis of mouse macrophages, and we found that these edible mushroom proteins affected the secretion of reactive oxygen species and nitric oxide by mouse macrophages. Further studies on cytokines secreted by mouse macrophages showed a significant increase in pro-inflammatory cytokines, suggesting that edible mushroom proteins promote the polarisation of macrophages into classical M1-type macrophages, further demonstrating that edible mushroom proteins enhance immunity. It was also found that the immunomodulatory activity of the precious edible mushroom proteins was significantly higher than that of the common edible mushroom proteins. These results have important implications for the processing and product development of edible mushroom proteins.

Effect of Gene 33/Mig6/ERRFI1 on hexavalent chromium-induced transformation of human bronchial epithelial cells depends on the length of exposure.

Hexavalent chromium (Cr(VI)) compounds are environmental and occupational lung carcinogens. The present study followed the chronic effect of Cr(VI) on the neoplastic transformation of BEAS-2B lung bronchial epithelial cells with or without deletion of Gene 33 (Mig6, EFFRI1), a multifunctional adaptor protein. We find that Gene 33-deleted cells exhibit increased anchorage-independent growth compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure. Gene 33-deleted cells show a higher level of cell proliferation and are more resistant to acute Cr(VI) toxicity compared to control cells after transformed by 8-week but not 24-week Cr(VI) exposure, despite that 24-week-transformed cells have increased resistance to acute Cr(VI) toxicity. However, Gene 33-deleted cells show increased migration after transformed by both 8-week and 24-week Cr(VI) exposures. Furthermore, only cells transformed by 24 weeks of Cr(VI) exposure can form subcutaneous tumors in nude mice. Although no significant difference in the size of tumors formed by the two cell types, there is a marked difference in the histological manifestation and more MMP3 expression in tumors from Gene 33-deleted cells. Our results demonstrate progressive neoplastic transformation of BEAS-2B cells and the adaptation of these cells to Gene 33 deletion during chronic exposure to Cr(VI).

Regulation of PTEN stability and activity by Plk3.

By studying primary isogenic murine embryonic fibroblasts (MEFs), we have shown that PLK3 null MEFs contain a reduced level of phosphatase and tensin homolog (PTEN) and increased Akt1 activation coupled with decreased GSK3ß activation under normoxia and hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, efficiently phosphorylates PTEN in vitro. Mass spectrometry identifies threonine 366 and serine 370 as two putative residues that are phosphorylated by Plk3. Immunoblotting using a phosphospecific antibody confirms these sites as Plk3 phosphorylation sites. Moreover, treatment of MEFs with LiCl, an inhibitor of GSK3ß and CK2, only partially suppresses the phosphorylation, suggesting Plk3 as an additional kinase that phosphorylates these sites in vivo. Plk3-targeting mutants of PTEN are expressed at a reduced level in comparison with the wild-type counterpart, which is associated with an enhanced activity of PDK1, an upstream activator of Akt1. Furthermore, the reduced level of PTEN in PLK3 null MEFs is stabilized by treatment with MG132, a proteosome inhibitor. Combined, our study identifies Plk3 as a new player in the regulation of the PI3K/PDK1/Akt signaling axis by phosphorylation and stabilization of PTEN.

Plk3 functions as an essential component of the hypoxia regulatory pathway by direct phosphorylation of HIF-1alpha.

Polo-like kinase 3 (Plk3) plays an important role in the regulation of cell cycle progression and stress responses. Plk3 also has a tumor-suppressing activity as aging PLK3-null mice develop tumors in multiple organs. The growth of highly vascularized tumors in PLK3-null mice suggests a role for Plk3 in angiogenesis and cellular responses to hypoxia. By studying primary isogenic murine embryonic fibroblasts, we tested the hypothesis that Plk3 functions as a component in the hypoxia signaling pathway. PLK3(-/-) murine embryonic fibroblasts contained an enhanced level of HIF-1α under hypoxic conditions. Immunoprecipitation and pulldown analyses revealed that Plk3 physically interacted with HIF-1α under hypoxia. Purified recombinant Plk3, but not a kinase-defective mutant, phosphorylated HIF-1α in vitro, resulting in a major mobility shift. Mass spectrometry identified two unique serine residues that were phosphorylated by Plk3. Moreover, ectopic expression followed by cycloheximide or pulse-chase treatment demonstrated that phospho-mutants exhibited a much longer half-life than the wild-type counterpart, strongly suggesting that Plk3 directly regulates HIF-1α stability in vivo. Combined, our study identifies Plk3 as a new and essential player in the regulation of the hypoxia signaling pathway.

Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury.

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1α-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1α deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1ß mRNA levels. Furthermore, partial HIF-1α deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1α(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1α protein levels nor caused significant gut and lung injury in wild-type or HIF-1α(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.

MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation.

Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. Recently, we have found that microRNA (miRNA) miR-145 is the most abundant miRNA in normal vascular walls and in freshly isolated VSMCs; however, the role of miR-145 in VSMC phenotypic modulation and vascular diseases is currently unknown. Here we find that miR-145 is selectively expressed in VSMCs of the vascular wall and its expression is significantly downregulated in the vascular walls with neointimal lesion formation and in cultured dedifferentiated VSMCs. More importantly, both in cultured rat VSMCs in vitro and in balloon-injured rat carotid arteries in vivo, we demonstrate that the noncoding RNA miR-145 is a novel phenotypic marker and a novel phenotypic modulator of VSMCs. VSMC differentiation marker genes such as SM alpha-actin, calponin, and SM-MHC are upregulated by premiR-145 or adenovirus expressing miR-145 (Ad-miR-145) but are downregulated by the miR-145 inhibitor 2'OMe-miR-145. We have further identified that miR-145-mediated phenotypic modulation of VSMCs is through its target gene KLF5 and its downstream signaling molecule, myocardin. Finally, restoration of miR-145 in balloon-injured arteries via Ad-miR-145 inhibits neointimal growth. We conclude that miR-145 is a novel VSMC phenotypic marker and modulator that is able of controlling vascular neointimal lesion formation. These novel findings may have extensive implications for the diagnosis and therapy of a variety of proliferative vascular diseases.

Recombinant factor XIII mitigates hemorrhagic shock-induced organ dysfunction.

BACKGROUND: Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Since FXIII has also been shown to modulate inflammation, endothelial permeability, as well as diminish multiple organ dysfunction (MOD) after gut ischemia-reperfusion injury, we hypothesized that FXIII would reduce MOD caused by trauma-hemorrhagic shock (THS). MATERIALS AND METHODS: Rats were subjected to a 90 min THS or trauma sham shock (TSS) and treated with either recombinant human FXIII A(2) subunit (rFXIII) or placebo immediately after resuscitation with shed blood or at the end of the TSS period. Lung permeability, lung and gut myeloperoxidase (MPO) activity, gut histology, neutrophil respiratory burst, microvascular blood flow in the liver and muscles, and cytokine levels were measured 3 h after the THS or TSS. FXIII levels were measured before THS or TSS and after the 3-h post-shock period. RESULTS: THS-induced lung permeability as well as lung and gut MPO activity was significantly lower in rFXIII-treated than in placebo-treated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and less ileal mucosal injury. rFXIII-treated rats also had a higher liver microvascular blood flow compared with the placebo group. Cytokine response was more favorable in rFXIII-treated animals. Trauma-hemorrhagic shock did not cause a drop in FXIII activity during the study period. CONCLUSIONS: Administration of rFXIII diminishes THS-induced MOD in rats, presumably by preservation of the gut barrier function, limitation of polymorphonuclear leukocyte (PMN) activation, and modulation of the cytokine response.

Intravenous injection of mesenteric lymph produced during hemorrhagic shock decreases RBC deformability in the rat.

OBJECTIVE: To test the hypothesis that gut-derived factors carried in trauma-hemorrhagic shock (T/HS) lymph are sufficient to induce red blood cells (RBC) injury, to investigate their potential mechanisms of action, and to define the time post-T/HS that these factors appear in the lymph. METHODS: Mesenteric lymph collected from T/HS or trauma-sham shock (T/SS) rats over different time periods was injected intravenously into male rats at a rate of 1 mL/h for 3 hours. RBC deformability was measured using laser-assisted ektacytometer to calculate the elongation index. From the shear-stress elongation curve, the stress required for the erythrocytes to reach 50% of their maximal elongation was also determined. RBC deformability was measured before lymph infusion and at 1 hour and 3 hours after the initiation of lymph infusion. The effect of the lymph samples (5% v/v) was also determined in vitro by incubating naïve whole blood with the lymph samples. The potential role of T/HS lymph-induced RBC oxidant injury mediated by inducible nitric oxide synthase (iNOS)-generated oxidants and/or white blood cells (WBC) was investigated using iNOS inhibitors and WBC depletion, respectively. In all the in vivo studies, five to seven rats were studied per group. RESULTS: The intravenous injection of T/HS lymph but not T/SS lymph caused in vivo RBC injury. The biological activity of T/HS lymph varied over time with the RBC-injurious factors being produced only during the first 3 hours postshock. The in vivo inhibition of iNOS did not prevent lymph-induced RBC injury. T/HS lymph incubated in vitro with naïve whole blood resulted in RBC injury, but this injury was not observed in blood depleted of WBC. CONCLUSIONS: These results indicate that T/HS lymph produced during the initial 3-hour postshock period is sufficient to induce RBC injury in otherwise normal rats and that the lymph-induced RBC injury is not dependent on activation of the iNOS pathway but seems to require WBC.

The anatomic sites of disruption of the mucus layer directly correlate with areas of trauma/hemorrhagic shock-induced gut injury.

BACKGROUND: The intestinal mucus layer is an important but understudied component of the intestinal barrier. Consequently, we tested the hypothesis that the anatomic sites of loss of the mucus layer would directly correlate with sites of intestinal villous injury after trauma-hemorrhagic shock (T/HS) and may, therefore, serve as loci of gut barrier failure. Consequently, to investigate this hypothesis, we used Carnoy's fixative solution to prepare fixed tissue blocks where both the gut morphology and the mucus layer could be assessed on the same tissues slides. METHODS: Male Sprague-Dawley rats were subjected to a laparotomy (trauma) and 90 minutes of sham shock (T/SS) or 35 mm Hg × 90 minutes of actual shock (T/HS). Three hours after resuscitation, the rats were killed, and samples of the terminal ileum were processed by fixation in Carnoy's solution. Gut injury was evaluated by determining the percentage of villi injured. The status of the intestinal mucus layer was quantified by determining the percentage of the villi covered by the mucus and the mucus thickness. RESULTS: Histologic analysis of gut injury showed that the incidence of gut injury was ∼10-fold higher in the T/HS than the T/SS rats (T/SS=2.5% ± 0.5% vs. T/HS=22.4% ± 0.5% of injured villi; p<0.01). The T/SS rats had 98% of their ileal mucosa covered with a mucus layer, and this was decreased after T/HS to 63% ± 3% (T/HS vs. T/SS; p<0.001). Furthermore, loss of the mucus layer was found to directly correlate with villous injury with a regression coefficient of r=0.94 (p<0.001). CONCLUSION: This study shows that T/HS significantly reduces the intestinal mucus layer and causes villous injury and that a correlation exists between specific anatomic sites of T/HS-induced loss of the mucus layer and gut injury.