Therapeutic mechanisms of mesenchymal stem cells in acute respiratory distress syndrome reveal potentials for Covid-19 treatment.

The mortality rate of critically ill patients with acute respiratory distress syndrome (ARDS) is 30.9% to 46.1%. The emergence of the coronavirus disease 2019 (Covid-19) has become a global issue with raising dire concerns. Patients with severe Covid-19 may progress toward ARDS. Mesenchymal stem cells (MSCs) can be derived from bone marrow, umbilical cord, adipose tissue and so on. The easy accessibility and low immunogenicity enable MSCs for allogeneic administration, and thus they were widely used in animal and clinical studies. Accumulating evidence suggests that mesenchymal stem cell infusion can ameliorate ARDS. However, the underlying mechanisms of MSCs need to be discussed. Recent studies showed MSCs can modulate immune/inflammatory cells, attenuate endoplasmic reticulum stress, and inhibit pulmonary fibrosis. The paracrine cytokines and exosomes may account for these beneficial effects. In this review, we summarize the therapeutic mechanisms of MSCs in ARDS, analyzed the most recent animal experiments and Covid-19 clinical trial results, discussed the adverse effects and prospects in the recent studies, and highlight the potential roles of MSC therapy for Covid-19 patients with ARDS.

Mesenteric Lymph Drainage Improves Cardiac Papillary Contractility and Calcium Sensitivity in Rats with Hemorrhagic Shock.

BACKGROUND: Myocardial dysfunction is an important adverse factor of hemorrhagic shock that induces refractory hypotension, and post-hemorrhagic shock mesenteric lymph (PHSML) return is involved in this adverse effect. This study investigated whether mesenteric lymph drainage (MLD) improves PHSML return-induced cardiac contractile dysfunction via the restoration of cardiomyocyte calcium sensitivity. MATERIALS AND METHODS: A hemorrhage shock model was established by using a controlled hemorrhage through the femoral artery that maintained a mean arterial pressure of 40 ± 2 mmHg for 3 h. MLD and mesenteric lymph duct ligation (MLDL) were performed from 1 to 3 h during hypotension. The papillary muscles of the heart were collected for measurement of calmodulin expression and for determining contractile responses to either isoprenaline or calcium. RESULTS: The results showed that either MLD or MLDL reversed the hemorrhagic shock-induced downregulation of calmodulin expression, a marker protein of cardiomyocyte calcium sensitization, in papillary muscles. MLD also improved the decreased contractile response and ±df/dt of the papillary muscle strip to gradient isoprenaline or calcium caused by hemorrhagic shock. CONCLUSION: These findings indicate that increased cardiac contractibility may be associated with the restoration of calcium sensitivity produced by PHSML drainage.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro.

BACKGROUND: Immune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. METHODS: The normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. RESULTS: Hemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. CONCLUSIONS: The results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

Stellate Ganglion Blockade repairs Intestinal Mucosal Barrier through suppression of Endoplasmic Reticulum Stress following Hemorrhagic Shock.

Background: Hemorrhagic shock-induced ischemia and hypoxia elicit endoplasmic reticulum stress (ERS) that leads to cell apoptosis, tissue structural damage and organ dysfunction and failure. Stellate ganglion blockade (SGB) has been demonstrated to improve intestinal barrier dysfunction induced by hemorrhagic shock. The present study sought to investigate whether the beneficial effect of SGB on the intestinal mucosal barrier function is via suppression of ERS. Materials and methods: A conscious rat model of hemorrhagic shock (40 ±2 mmHg for 1 hour, followed by resuscitation) was established. The parameters reflecting intestinal morphology and intestinal mucosal barrier function including wet-dry ratio (W/D), intestinal permeability, D-lactic acid (D-LA) and intestinal fatty acid binding protein (I-FABP) in plasma, and expressions of ATF6α, PERK, and IRE1α in intestinal tissues were then observed. Furthermore, the effects of either SGB or ERS inhibitor, 4-phenylbutyric acid (4-PBA), on these parameters in rats with hemorrhagic shock were assessed. The effect of ERS agonist tunicamycin (TM) on the rats subjected with both SGB and hemorrhagic shock was also determined. Results: Either SGB or administration of ERS inhibitor, 4-PBA, alleviated hemorrhagic shock-induced adverse effects such as intestinal mucosal barrier dysfunction and excessive autophagy, which were characterized by damaged intestinal tissue, enhanced intestinal permeability and D-LA and I-FABP levels in plasma, and increased expressions of ATF6α, PERK, IRE1α in intestinal tissue. In contrast, administration of ERS agonist, TM, suppressed the beneficial effects of SGB on intestinal tissue and function during hemorrhagic shock. Conclusion: The SGB repairs intestinal mucosal barrier through suppression of ERS following hemorrhagic shock.

Blockade of Stellate Ganglion Remediates Hemorrhagic Shock-Induced Intestinal Barrier Dysfunction.

BACKGROUND: Acute hemorrhage-induced excessive excitation of sympathetic-adrenal-medullary system (SAS) leads to gut hypoperfusion and barrier dysfunction, which is a critical event during hemorrhagic shock-induced multiple organ injury. Stellate ganglion blockade (SGB) has been widely used for suppression of sympathetic-adrenal-medullary system in the clinical practice. However, whether SGB improves intestinal barrier function after hemorrhagic shock remains unclear. Here, we hypothesized that the implementation of SGB restores intestinal barrier function and reduces gut injury. MATERIALS AND METHODS: Male rats received the SGB pretreatment and underwent hemorrhagic shock followed by resuscitation. The 96-h survival rate, intestinal permeability and morphology, D-lactic acid concentration and diamine oxidase activity in plasma, and expressions of F-actin, Claudin-1, and E-cadherin in intestinal tissues were observed. RESULTS: Pretreatment with SGB significantly enhances the 96-h survival rate in rats subjected to hemorrhagic shock (from 8.3% to 66.7%). Hemorrhagic shock reduced the coverage scale of intestinal mucus and intestinal villus width and height, enhanced the intestinal permeability to fluorescein isothiocyanate-dextran 4 and D-lactic acid concentration in plasma, and decreased the expressions of F-actin, Claudin-1, and E-Cadherin in intestinal tissue. These hemorrhagic shock-induced adverse effects were abolished by SGB treatment. CONCLUSIONS: SGB treatment has a beneficial effect during hemorrhagic shock, which is associated with the improvement of intestine barrier function. SGB may be considered as a new therapeutic strategy for treatment of hemorrhagic shock.

Posthemorrhagic shock mesenteric lymph enhances monolayer permeability via F-actin and VE-cadherin.

BACKGROUND: Vascular hyperpermeability plays a critical role in the development of refractory hypotension after severe hemorrhagic shock. Posthemorrhagic shock mesenteric lymph (PHSML) return has been shown to be involved in regulation of vascular hyperpermeability. The present study was conducted to investigate the effect of PHSML on permeability of endothelial cells in vitro. MATERIALS AND METHODS: A hemorrhagic shock model (40 ± 2 mm Hg for 90 min, followed by fluid resuscitation) was used for collection of PHSML. Two separated PHSMLs were collected from period 0-3 h (early) and period 3-6 h (late) after resuscitation and diluted into concentration of 4% or 10%. The human umbilical vein endothelial cells (HUVECs) were then treated with these PHSMLs for 6 h. The monolayer cellular permeability to FITC-albumin was observed by using the costar transwell system. The multiple approaches including scanning electron microscope, fluorescent cytochemistry staining, and Western blotting were also used to assess the changes in cellular morphologic and the expressions of F-actin and VE-cadherin. RESULTS: The treatments with either early or late PHSML resulted in morphologic injuries, increased cellular permeability, and decreased expression of F-actin in HUVECs. In contrast, only early PHSML, but not late PHSML, reduced the VE-cadherin expression. CONCLUSIONS: These results indicate that the PHSML in vitro increases the cellular permeability of HUVECs through suppression of F-actin and VE-cadherin.

Inhibitory effect of post-hemorrhagic shock mesenteric lymph drainage on the HMGB1 and RAGE in mouse kidney.

BACKGROUND: Excessively inflammatory response is one of mechanisms that underlie the acute kidney injury (AKI) induced by severe hemorrhagic shock, which could be ameliorated by post-hemorrhagic shock mesenteric lymph (PHSML) blockage. Recent studies demonstrate that high mobility group box 1 (HMGB1) and the receptor for advanced glycation end products (RAGE) are critical mediators of local inflammations. The present study was sought to investigate whether the PHSML drainage inhibits the HMGB1 and RAGE in mouse kidney to ameliorate the renal inflammatory responses. METHODS: A mouse hemorrhagic shock model (40 ± 2 mmHg for 90 min, fluid resuscitation for 30 min) was employed, and the PHMSL drainage was performed at the end of the resuscitation. After 3 h of resuscitation, the expressions of mRNA and protein for the renal HMGB1 and RAGE and the levels of interleukin (IL)-1ß and IL-18 were assessed by the real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: Hemorrhagic shock elicited significant increases in the mRNA expressions of HMGB1 and RAGE and in the protein expressions of HMGB1, RAGE, IL-1ß and IL-18 in kidney. The PHSML drainage abolished these potentiating effects. CONCLUSION: The present study demonstrates that PHSML blockade reduces the increased HMGB1 and RAGE and pro-inflammatory factors following hemorrhagic shock, suggesting that the PHSML elicits the inflammatory responses via enhancing the HMGB1 and RAGE production in the kidney.

Mesenteric lymph duct ligation after hemorrhagic shock enhances the ATP level and ATPase activity in rat kidneys.

BACKGROUND: Kidney injury commonly occurs following hemorrhagic shock. This study aims to observe the effects of mesenteric lymph duct ligation (MLDL) on the adenosine triphosphate (ATP) levels and the cell membrane adenosine triphosphatase (ATPase) activity in the kidneys of rats subjected to hemorrhagic shock. METHODS: Wistar rats were assigned into sham, shock, and ligation groups. The hemorrhagic shock model was established in the shock and ligation groups, and MLDL was performed in the ligation group after resuscitation. Renal homogenates were prepared to determine the ATP and ATPase levels at 90 min after hemorrhage and at 0, 1, 3, 6, 12, and 24 h after resuscitation. RESULTS: The ATP levels, and the Na(+)-K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and Ca(2+)-Mg(2+)-ATPase activities in the renal tissue of the shock group were lower than those in the sham group at the multiple time points. Furthermore, the corresponding values in the ligation group were significantly higher than those in the shock group at multiple time points. CONCLUSION: MLDL improves energy metabolism and enhances the ATPase activity in the kidneys of hemorrhagic shock rats, along with other mechanisms that alleviate renal injury after hemorrhagic shock.

Effect of mesenteric lymph duct ligation on gene expression profiles of renal tissue in hemorrhagic shock rats with fluid resuscitation.

BACKGROUND: Acute kidney injury (AKI), a common pathological process following hemorrhagic shock, can lead to an internal milieu disorder, which is an important factor of multiple organ failure (MOF). It has been shown that the mesenteric lymph return plays a deleterious effect on MOF induced by hemorrhagic shock. In this study, we investigated the effects of mesenteric lymph duct ligation (MLDL) on gene expression profiles of renal tissue following hemorrhagic shock with fluid resuscitation. METHODS: After establishment of hemorrhagic shock model and fluid resuscitation in rats of shock and shock ligation groups, the MLDL was performed in shock ligation group, and only threading under the mesenteric lymph duct in the shock group. Then, the fixed position renal tissue was taken out for homogenate in two groups at 3 h after resuscitation, the total mRNA was extracted, reversely transcribed into cDNAs and marked with Cy3 and Cy5. The cDNAs were subjected for microarray scanning with 12,028 cDNA probes; the differentially expressed genes between two groups were analyzed. RESULTS: In the 5812 valid dates of rat genomes transcription, there were 34 known differentially expressed genes between the two groups, of which 11 genes were up-regulated whereas 23 genes were down-regulated by MLDL. These different expressed genes encoding protein function were mainly involved in signal transduction, transcription regulation, metabolism, transport, cell growth, cell cycle, cell adhesion, cell movement, cellular component, and biological process. CONCLUSIONS: The mechanism of MLDL alleviating the AKI aftershock might be associated with up- or down-regulation of the above gene expressions.

Normal mesenteric lymph ameliorates acute kidney injury following lipopolysaccharide challenge in mice.

BACKGROUND: The kidney is one of the prior damaged organs subjected to severe infection and sepsis shock. Our previous studies have shown that the normal mesenteric lymph (NML) obtained from healthy dogs could alleviate multiple organ injuries following endotoxic shock. In the current study, we further investigated the beneficial effect of NML from healthy mice on acute kidney injury (AKI) induced by lipopolysaccharide (LPS) in mice. METHODS: The mice in LPS and LPS + NML groups received an intraperitoneal injection of LPS (35 mg/kg). One hour later, the treatment of NML was performed and kept for 6 h. Then, the renal function indices, renal morphology, the levels of phosphorylation mitogen-activated protein kinases (MAPKs), markers of sensitization to LPS, as well as pro-inflammatory mediators in renal tissue were observed. RESULTS: Intraperitoneal injection of LPS induced an increased level of urea in plasma, lipopolysaccharide-binding protein (LBP), cluster of differentiation 14 (CD14), tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), but no obvious changes in the MAPKs in renal tissue. NML treatment decreased the levels of urea, CD14, TNF-α and IL-6 in mice after LPS injection. CONCLUSION: The current results indicate that NML alleviates LPS-induced AKI through its attenuation of sensitization to LPS.

Mesenteric lymph drainage alleviates acute kidney injury induced by hemorrhagic shock without resuscitation.

This study aimed to investigate the effect of mesenteric lymph drainage on the acute kidney injury induced by hemorrhagic shock without resuscitation. Eighteen male Wistar rats were randomly divided into sham, shock, and drainage groups. The hemorrhagic shock model (40 mmHg, 3 h) was established in shock and drainage groups; mesenteric lymph drainage was performed from 1 h to 3 h of hypotension in the drainage group. The results showed that renal tissue damage occurred; the levels of urea, creatinine, and trypsin in the plasma as well as intercellular adhesion molecule-1 (ICAM-1), receptor of advanced glycation end-products (RAGE), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), lactic acid (LA), and 2,3-DPG in the renal tissue were increased in the shock group after 3 h of hypotension. Mesenteric lymph drainage lessened the following: renal tissue damage; urea and trypsin concentrations in the plasma; ICAM-1, RAGE, TNF-α, MDA, and LA levels in the renal tissue. By contrast, mesenteric lymph drainage increased the 2,3-DPG level in the renal tissue. These findings indicated that mesenteric lymph drainage could relieve kidney injury caused by sustained hypotension, and its mechanisms involve the decrease in trypsin activity, suppression of inflammation, alleviation of free radical injury, and improvement of energy metabolism.

Exercise preconditioning improves mesenteric lymphatic contractility through MAM in rats following hemorrhagic shock.

ABSTRACT: Restoration of mesenteric lymphatic microcirculation is crucial for alleviating severe hemorrhagic shock-induced death. Exercise preconditioning (EP) enhances adaptability and resistance to injury and disease. The mitochondria-associated endoplasmic reticulum membrane (MAM) plays a crucial role in the energy and information exchange between the two organelles. Therefore, we hypothesized that EP ameliorates mesenteric lymphatic contractility through MAM in rats following hemorrhagic shock, aiming to confirm that EP enhances resistance to hemorrhagic shock and further popularizes the idea that exercise is beneficial for health. To test this hypothesis, we observed the effects of EP for four weeks on survival time and mesenteric lymphatic contractility in conscious rats following hemorrhagic shock, and further explored the effects of MAM agonists and inhibitors. The results showed that EP prolonged the survival time and improved the mesenteric lymphatic contractility and reactivity in vivo and in vitro in rats underwent hemorrhagic shock, ameliorated the MAM ultrastructure in lymphatic smooth muscle cells (LSMCs) and reduced the voltage-dependent anion channel 1 (VDAC1, a vital protein of MAM) and IP3R1 expressions in mesenteric lymphatic tissue. Importantly, treatment with 2-APB (IP3R1 inhibitor) or VBIT-12 (VDAC1 inhibitor) prolonged the survival time, improved mesenteric lymphatic contractility in vivo, ameliorated the MAM ultrastructure injury, and decreased the IP3R1 or VDAC1 expressions in LSMCs in rats following hemorrhagic shock. In contrast, the administration of drinking water containing CdCl2 (IP3R1 activator) abolished the beneficial effect of EP on hemorrhagic shock. Taken together, the protective effect of EP on lymphatic contractility following hemorrhagic shock was achieved by improving MAM in LSMCs.

STELLATE GANGLION BLOCK REVERSES PHSML-INDUCED VASCULAR HYPOREACTIVITY THROUGH INHIBITING AUTOPHAGY-MEDIATED PHENOTYPIC TRANSFORMATION OF VSMCs.

ABSTRACT: Posthemorrhagic shock mesenteric lymph (PHSML) return-contributed excessive autophagy of vascular smooth muscle cells (VSMCs) is involved in vascular hyporeactivity, which is inhibited by stellate ganglion block (SGB) treatment. The contractile phenotype of VSMCs transforms into a synthetic phenotype after stimulation with excessive autophagy. Therefore, we hypothesized that SGB ameliorates PHSML-induced vascular hyporeactivity by inhibiting autophagy-mediated phenotypic transformation of VSMCs. To substantiate this hypothesis, a hemorrhagic shock model in conscious rats was used to observe the effects of SGB intervention or intravenous infusion of the autophagy inhibitor 3-methyladenine (3-MA) on intestinal blood flow and the expression of autophagy- and phenotype-defining proteins in mesenteric secondary artery tissues. We also investigated the effects of intraperitoneal administration of PHSML intravenous infusion and the autophagy agonist rapamycin (RAPA) on the beneficial effect of SGB. The results showed that hemorrhagic shock decreased intestinal blood flow and enhanced the expression of LC3 II/I, Beclin 1, and matrix metalloproteinase 2, which were reversed by SGB or 3-MA treatment. In contrast, RAPA and PHSML administration abolished the beneficial effects of SGB. Furthermore, the effects of PHSML or PHSML obtained from rats treated with SGB (PHSML-SGB) on cellular contractility, autophagy, and VSMC phenotype were explored. Meanwhile, the effects of 3-MA on PHSML and RAPA on PHSML-SGB were observed. The results showed that PHSML, but not PHSML-SGB, incubation decreased VSMC contractility and induced autophagy activation and phenotype transformation. Importantly, 3-MA administration reversed the adverse effects of PHSML, and RAPA treatment attenuated the effects of PHSML-SGB incubation on VSMCs. Taken together, the protective effect of SGB on vascular reactivity is achieved by inhibiting excessive autophagy-mediated phenotypic transformation of VSMCs to maintain their contractile phenotype.

Inhibitory effect of melanoma differentiation associated gene-7/interleukin-24 on invasion in vitro of human melanoma cancer cells.

The acquisition of metastasis potential is a critical point for malignant tumors. Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a potential tumor suppress gene and frequently down-regulated in malignant tumors. It has been implicated that overexpression of MDA-7 led to proliferation inhibition in many types of human tumor. Invasion is an important process which is potential to promote tumor metastasis. However, the role and potential molecular mechanism of mda-7/IL-24 to inhibit the invasion of human melanoma cancer is not fully clear. In this report, we identified a solid role for mda-7/IL-24 in invasion inhibition of human melanoma cancer LiBr cells, including decreasing of adhesion and invasion in vitro, blocking cell cycle, down-regulating the expression of ICAM-1, MMP-2/9, CDK1, the phosphorylation of ERK and Akt, NF-κB and AP-1 transcription activity. Meanwhile, there was an increased expression of PTEN in mda-7/IL-24 over-expression LiBr cells. Our results demonstrated that mda-7/IL-24 is a potential invasion suppress gene, which inhibits the invasion of LiBr cells by the down-regulation of ICAM-1, MMP-2/9, PTEN, and CDK1 expression. The molecular pathways involved were the MAPK/ERK, PI3K-Akt, NF-κB, and AP-1. These findings suggest that mda-7/IL-24 may be used as a possible therapeutic strategy for human melanoma cancer.

Exogenous normal lymph alleviates lipopolysaccharide-induced acute kidney injury in rats.

BACKGROUND: Acute kidney injury (AKI) is a common pathological process which occurs in hemorrhage, intoxication, etc. It has been shown that the lymphatic circulation plays an important regulatory role in the pathogenesis of hemorrhage shock, and that exogenous normal lymph (ENL) has a beneficial effect on multiple organ injuries. In the present study, we investigated the effect of ENL on lipopolysaccharide (LPS)-induced AKI in rats. METHODS: The AKI was induced by the jugular vein injection of LPS (iv, 15 mg/kg). After 15 min of LPS injection, saline or ENL without cell components (5 mL/kg) was iv infused at the speed of 0.5 mL per minute. Then, the renal function indices in plasma and renal histomorphology, and the levels of P-selectin, intercellular adhesion molecule-1 (ICAM-1), myeloperoxidase (MPO) and Na(+)-K(+)-ATPase in renal tissue were assessed at 3 or 6 h after LPS injection. RESULTS: LPS induced a severe kidney injury including increased levels of urea, creatinine in plasma, aggrandized activities of ICAM-1 and MPO in renal tissue, and decreased the Na(+)-K(+)-ATPase activity in renal cells. These deleterious effects of LPS were significantly ameliorated by ENL treatment. CONCLUSION: The present results indicate that ENL protect against LPS-induced AKI, suggesting an alternative therapeutic strategy for treatment of kidney injury accompanied with severe infection or sepsis.

Omega-3 polyunsaturated fatty acids alleviates lung injury mediated by post-hemorrhagic shock mesenteric lymph.

Severe hemorrhage-induced acute lung injury (ALI) remains the major contributor to critical patient mortality and is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) play overall protection on acute hemorrhage, but a reliable mechanism needs to be identified. The aims of this study were to investigate the role of ω-3 PUFAs in alleviating ALI and whether is related to the endotoxin contained in PHSML. Mesenteric lymph was harvested from rats subjected to hemorrhagic shock (hemorrhage-induced hypotension of 40 ± 2 mmHg for 90 min plus by resuscitation) or sham shock. The effect of ω-3 PUFAs on pulmonary function, water content, morphology, and LBP, CD14, TNF-α, and IL-6 levels were observed in rats subjected to hemorrhagic shock, while the effect of PHSML intravenous infusion on the beneficial effect of ω-3 PUFAs also was investigated. In addition, the effect of ω-3 PUFAs on the endotoxin contents in mesenteric lymph were detected. Hemorrhagic shock-induced ALI was characterized by increased functional residual capacity (FRC), lung resistance (RI), inspiratory capacity (IC), respiratory frequency, water contents and structural damage, along with increases in LBP, IL-6, and TNF-α. ω-3 PUFAs treatment reduced FRC, RI, IC, frequency, water contents, LBP, IL-6, TNF-α, and alleviated morphological damage. In contrast, PHSML infusion abolished the advantageous effects of ω-3 PUFAs on the above indices and CD14. Furthermore, the endotoxin level of PHSML was significantly enhanced, but declined following ω-3 PUFAs administration. These findings together suggested that treatment with ω-3 PUFAs ameliorates hemorrhagic shock-induced ALI, which is associated with reduced endotoxin contained in PHSML.

Analysis and Identification of Ferroptosis-Related Gene Signature for Acute Lung Injury.

BACKGROUND: Recent studies have shown that ferroptosis is involved in the evolution of acute lung injury (ALI), a serious respiratory pathological process leading to death. However, the regulatory mechanisms underlying ferroptosis in ALI remain largely unknown. The current study analyzed and identified a ferroptosis-related gene signature for ALI. METHODS: Key genes associated with ferroptosis in ALI were identified by bioinformatics analysis. GSE104214, GSE18341, and GSE17355 datasets were downloaded from the Gene Expression Omnibus database. The signature genes were screened by least absolute shrinkage and selection operator (LASSO) regression, and the key genes of ALI were screened by weighted correlation network analysis (WGCNA), followed by immune infiltration analysis and functional enrichment analysis. In addition, mRNA expression of key genes in the lungs of mice with hemorrhagic shock and sepsis was verified. RESULTS: A total of 2132 differential genes were identified by various analyses, and nine characteristic genes were detected using Lasso regression. We intersected nine signature genes with WGCNA module genes and finally determined four key genes (PROK2, IL6, TNF, SLC7A11). All four key genes were closely correlated with immune cells and regulatory genes of ALI, and the expression of the four genes was significantly different in the lung tissues of hemorrhagic shock and sepsis models. Besides, the ferroptosis related molecules GPX4 and ACSL4 showed remarkable difference in these models. CONCLUSION: These results indicate that PROK2, IL6, TNF, SLC7A11 may be key regulatory targets of ferroptosis during ALI. This study proved that ferroptosis is a common pathophysiological process in three ALI models.

POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH IMPAIRS SPLENIC DENDRITIC CELL FUNCTION IN MICE.

ABSTRACT: Dendritic cell (DC)-mediated immune dysfunction is involved in the process of severe hemorrhagic shock that leads to sepsis. Although post-hemorrhagic shock mesenteric lymph (PHSML) induces immune organs injuries and apoptosis, whether PHSML exerts adverse effects on splenic DCs remains unknown. In this study, we established a hemorrhagic shock model (40 ± 2 mm Hg for 60 min) followed by fluid resuscitation with the shed blood and equal Ringer's solution and drained the PHSML after resuscitation. At 3 h after resuscitation, we harvested the splenic tissue to isolate DCs using anti-CD11c immunomagnetic beads and then detected the necrotic and apoptotic rates in splenocytes and splenic DCs. We also detected the levels of TNF-α, IL-10, and IL-12 in the culture supernatants and surface marker expressions of MHC-II, CD80, and CD86 of splenic DCs following LPS stimulation for 24 h. Second, we purified the DCs from splenocytes of normal mice to investigate the effects of PHSML treatment on cytokine production and surface marker expression following LPS stimulation. The results showed that PHSML drainage attenuated LPS-induced cell death of splenocytes and DCs. Meanwhile, PHSML drainage enhanced the DC percentage in splenocytes and increased the TNF-α and IL-12 production by DCs and the expressions of CD80, CD86, and MHCII of DCs treated by LPS. Furthermore, PHSML treatment reduced the productions of TNF-α, IL-10, and IL-12 and the expressions of CD80 and CD86 in normal DCs after treatment with LPS. In summary, the current investigation demonstrated that PHSML inhibited the cytokine production and surface marker expressions of DCs stimulated by LPS, suggesting that PHSML plays an important role in hemorrhagic shock-induced immunosuppression through the impairment of DC function and maturation.

ESTROGEN ALLEVIATES POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH-MEDIATED LUNG INJURY THROUGH AUTOPHAGY INHIBITION.

ABSTRACT: Background: Hemorrhagic shock-induced acute lung injury (ALI) is commonly associated with the posthemorrhagic shock mesenteric lymph (PHSML) return. Whether excessive autophagy is involved in PHSML-mediated ALI remains unclear. The relationship between estrogen treatment and PHSML or autophagy needs to verify. The current study will clarify the role of estrogen in reducing PHSML-mediated ALI through inhibition of autophagy. Methods: First, a hemorrhagic shock model in conscious rats was used to observe the effects of 17ß-estradiol (E2) on intestinal blood flow, pulmonary function, intestinal and pulmonary morphology, and expression of autophagy marker proteins. Meanwhile, the effect of PHSML and autophagy agonist during E2 treatment was also investigated. Secondly, rat primary pulmonary microvascular endothelial cells were used to observe the effect of PHSML, PHSML plus E2, and E2-PHSML (PHSML obtained from rats treated by E2) on the cell viability. Results: Hemorrhagic shock induced intestinal and pulmonary tissue damage and increased wet/dry ratio, reduced intestinal blood flow, along with pulmonary dysfunction characterized by increased functional residual capacity and lung resistance and decreased inspiratory capacity and peak expiratory flow. Hemorrhagic shock also enhanced the autophagy levels in intestinal and pulmonary tissue, which was characterized by increased expressions of LC3 II/I and Beclin-1 and decreased expression of p62. E2 treatment significantly attenuated these adverse changes after hemorrhagic shock, which was reversed by PHSML or rapamycin administration. Importantly, PHSML incubation decreased the viability of pulmonary microvascular endothelial cells, while E2 coincubation or E2-treated lymph counteracted the adverse roles of PHSML. Conclusions: The role of estrogen reducing PHSML-mediated ALI is associated with the inhibition of autophagy.

Exogenous normal lymph alleviating kidney injury by improving coagulation function in disseminated intravascular coagulation rats.

AIM: To investigate the effect of exogenous normal lymph on kidney injury in disseminated intravascular coagulation (DIC) rats and to probe its mechanism. METHODS: The DIC model was established by intravenous injection of Dextran 500. After 6 min, normal lymph without cell components was infused in the lymph group. After 40 min, the renal and coagulation function indices and renal histomorphology were observed. RESULTS: Serum urea and creatinine in the model group were significantly higher than in the control and lymph groups. Renal morphological study showed red blood cell silting and casts forming in the model group. The prothrombin time (PT), prothrombin time ratio (PTR), activated partial thromboplastin time (APTT), and thrombin time of lymph and model groups were higher than those in the control group, whereas fibrinogen was lower. The PT, PTR, and APTT were prolonged in the lymph group than in the model group. The platelet functions of the lymph and model groups were higher than in the control group, but platelet aggregation rate and thrombosis-forming indices were lower than in the control group; the platelet adhesive and aggregation rates and thrombosis dry weight of the lymph group were lower than those of the model group. CONCLUSION: Exogenous normal lymph could alleviate kidney injury in DIC rats, which may be related to the improving coagulation function.