Risk model for predicting mortality in patients with necrotizing soft tissue infections in the intensive care unit.

BACKGROUND: The goal of this study is to look into the factors that lead to death in patients with necrotizing soft tissue infections（NSTIs） in the intensive care unit and create a mortality risk model. METHODS: The clinical data of 106 patients with necrotizing soft tissue infections admitted to intensive care unit(ICU) of the First Affiliated Hospital of Wenzhou Medical University between January 2008 and December 2021 were retrospectively analyzed. Univariate analysis and multivariate analysis were performed to evaluate the risk factors impacting patient mortality. The regression coefficient in binary logistic regression analysis was converted into the item score in the model, and then the model score of each patient was calculated. Finally, an ROC curve was constructed to evaluate the efficiency of the model for predicting mortality. Thirteen patients with NSTIs admitted to ICU between January 2022 and November 2022 were used to validate the model. RESULTS: The death group had 44 patients, while the survival group had 62 patients. The overall mortality was 41.5%. Binary logistic regression analysis showed that risk factors for mortality were age≥ 60 years(OR:4.419; 95%CI:1.093-17.862; P = 0.037), creatinine ≥ 132µmol/L(OR:11.166; 95%CI:2.234-55.816; P = 0.003), creatine kinase ≥ 1104 U/L(OR:4.019; 95%CI:1.134-14.250; P = 0.031), prothrombin time ≥ 24.4 s(OR:11.589; 95%CI:2.510-53.506; P = 0.002), and invasive mechanical ventilation (OR:17.404; 95%CI:4.586-66.052; P＜0.000). The AUC of the model for predicting mortality was 0.940 (95% CI:0.894-0.986). When the cut-off value for the model was 4 points, the sensitivity was 95.5% and the specificity was 83.9%. CONCLUSION: The death risk model in this study for NSTIs patients in the intensive care unit shows high sensitivity and specificity. Patients with a score of ≥ 4 points have a higher risk of mortality.

Mutual promotion of mitochondrial fission and oxidative stress contributes to mitochondrial-DNA-mediated inflammation and epithelial-mesenchymal transition in paraquat-induced pulmonary fibrosis.

BACKGROUND: Pulmonary fibrosis (PF) is one of the main causes of death in patients with paraquat (PQ) poisoning. This study aimed to evaluate the relationship between mitochondrial fission and oxidative stress in PQ-induced epithelial-mesenchymal transition (EMT) and PF. METHODS: C57BL/6 mice and MLE-12 cells were exposed to PQ to construct a PF model in vivo and in vitro. Histological changes in the lungs were examined by hematoxylin and eosin (H&E) staining. Mitochondrial morphology was detected by MitoTracker® Deep Red FM or transmission electron microscopy (TEM). Western blotting and immunofluorescence were used to determine the expression of protein. The migration ability of the cells was detected by the cell scratch test. Mitochondrial DNA (mtDNA) levels were assessed by real-time polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay (ELISA) was applied to detect cytokine levels. Superoxide dismutase (SOD) activity and the levels of glutathione (GSH) and malondialdehyde (MDA) were detected by chemichromatometry. RESULTS: PQ exposure caused EMT and PF in vivo and in vitro. PQ destroyed mitochondrial structure and enhanced the expression of dynamin-related protein 1 (Drp1), which were accompanied by oxidative stress. Inhibiting mitochondrial fission using mitochondrial division inhibitor-1 (Mdivi-1), a selective inhibitor of Drp1, attenuated PQ-induced EMT and oxidative damage. Treatment with N-acetyl-L-cysteine (NAC), an antioxidant, reduced Drp1 expression, attenuated mitochondrial structure damage and inhibited PQ-induced EMT and PF. Both Mdivi-1 and NAC treatment markedly suppressed mtDNA release, the expression of Toll-like receptor 9 (TLR9) and phosphorylation (P)-NF-κB p65 as well as cytokines (interleukin 6 [IL-6], interleukin-1ß [IL-1ß], and tumor necrosis factor-α [TNF-α]) production. CONCLUSION: Mutual promotion of mitochondrial fission and oxidative stress contributes to EMT in PQ-induced PF, which is associated with the mtDNA/TLR9/NF-κB pathway.

Vesicle transport related protein Synaptotagmin-1 mediates paraquat transport to antagonize paraquat toxicity.

In this study, A549/PQ cells with moderate resistance to paraquat (PQ) were obtained by treating A549 cells with PQ, their growth rate was slowed down, the accumulation concentration of PQ and the levels of growth inhibition, injury and early apoptosis induced by PQ were significantly lower than those of parental A549 cells. Microarray screening and RT-qPCR detection found that Synaptotagmin-1 (SYT1) expression in drug-resistant cells was significantly increased, and PQ further enhanced its expression. After inhibiting SYT1 expression in A549/PQ cells, cell viability, intracellular PQ concentration and the expression of Bcl-2, SNAP25 and RAB26 were significantly reduced, while the mortality, early apoptosis rate and Bax expression were significantly increased. In vivo experiments also further showed that PQ promoted the expression of SYT1, SNAP25 and RAB26 in PQ-poisoned mice; when inhibiting SYT1 expression, PQ concentration in lung tissues was significantly increased, and the levels of lung injury and apoptosis were also significantly enhanced, while the expression of SNAP25 and RAB26 was significantly reduced. This indicates that PQ poisoning leads to compensatory up-regulation of vesicle transport related proteins such as SYT1 in vivo, thereby promoting PQ transmembrane transport, and then reducing the pulmonary accumulation of PQ and PQ-caused lung injury.

Carnosic acid alleviates depression-like behaviors on chronic mild stressed mice via PPAR-γ-dependent regulation of ADPN/FGF9 pathway.

RATIONALE: The pathway of adiponectin (ADPN)/fibroblast growth factor 9 (FGF9) was recently thought as a key role in the development of depression. ADPN is crucially regulated by peroxisome proliferator-activated receptor-gamma (PPAR-γ). Natural material carnosic acid (CA) has been applied for therapeutics of mental disorders. OBJECTIVES: To evaluate the antidepressive effect of CA in stress-treated mice and define whether its effects is involved in the regulation of ADPN/FGF9 pathway. METHODS: In vivo study, the levels of ADPN and FGF9 in both serum and hippocampus tissues, the expressions of ADPN receptor 2 (AdipoR2) in hippocampus and PPAR-γ in abdominal adipose, as well as the pathological changes of hippocampus were determined in 28-day period of chronic unpredictable mild stress (CUMS)-induced depression model of male ICR (Institute of Cancer Research) mice or adipo-/- mice. In vitro study, the level of ADPN and the mRNA expressions of both ADPN and PPAR-γ were determined in mouse 3T3-L1 preadipocytes. RESULTS: In vivo study, treatment with CA (50 or 100 mg/kg per day) for 21 days markedly suppressed depressive-like behaviors, the elevating levels of FGF9 and decreasing levels of ADPN in both serum and hippocampus tissues, the downregulating protein and mRNA expressions of AdipoR2 in hippocampus and PPAR-γ in abdominal adipose, as well as the pathological injury of hippocampus induced by CUMS in male ICR mice. The antidepressive effects of CA were markedly attenuated in male CUMS-treated adipo-/- mice. In vitro study, incubation with CA (3-30 µmol/L) for 24 h could concentration-dependently upregulate the mRNA expressions of both PPAR-γ and ADPN as well as increase the level of ADPN. The experiments using PPAR-γ-specific inhibitor GW9662 and transient transfection with mutated PPAR-γ-binding site promotor constructs showed that the activation of PPAR-γ mediated CA-induced ADPN expression in adipocytes. CONCLUSIONS: CA could significantly improve stress-induced depressive disorder, which may be related to regulating the dysfunction of ADPN-FGF9 pathway via activating PPAR-γ in adipocytes.

The correlation between adiponectin and FGF9 in depression disorder.

Adiponectin (ADPN) and fibroblast growth factor 9 (FGF9) has been reported as anti-depressive and pro-depressive factor, respectively. However, it is unknown whether there is directly interaction between ADPN and FGF9 in depression. The present study aims to investigate the correlation between ADPN and FGF9 in depression disorder. Firstly, the decreased level of ADPN and the increased level of FGF9 in plasma of depressive patients compared with non-depressive subjects were observed. Furthermore, these is a significant negative correlation between the ratio of ADPN to FGF9 and the total score of Hamilton Depression Scale in total investigated subjects. The similar changes of ADPN and FGF9 were also observed in elder adiponectin gene knockout (Adipo-/-) mice with an increasing trend to depressive-like behaviors. Secondly, the decreasing level of ADPN and increasing level of FGF9 in plasma and hippocampus tissues were observed in chronic unpredictable mild stress (CUMS)-induced depression in ICR mice with significant depressive-like behaviors and hippocampus damage, which attenuated by injection of recombinant ADPN or FGF9 antibody into lateral ventricle. In Adipo-/- mice, injection of FGF9 antibody into lateral ventricle also attenuated CUMS-induced depressivelike behaviors. The protein expression of FGF receptor 3 (FGFR3), the main receptor of FGF9, was significantly down-regulated in hippocampus tissues of CUMS-treated mice, which could be attenuated by treatment with either recombinant ADPN or anti-FGF9. In summary, the present results suggest that ADPN maybe a key negative regulator of FGF9/FGFR3 in depressive disorder and the dysfunction of ADPN-FGF9 pathway plays a key role in stress-induced depression.

Nrf2 overexpression protects against paraquat-induced A549 cell injury primarily by upregulating P-glycoprotein and reducing intracellular paraquat accumulation.

Paraquat (PQ) intoxication causes thousands of mortalities every year, worldwide. Its pulmonary-targeted accumulation and the acute lung injury it subsequently causes, remain a challenge for detoxification treatment. A previous study has demonstrated that the upregulation of nuclear factor erythroid-2 related factor 2 (Nrf2) prevents PQ toxicity in cell line and murine models. As Nrf2 target genes include a group of membrane transporters, the current study assessed the protective mechanism exerted by Nrf2 against PQ toxicity and intracellular PQ accumulation via its effects on P-glycoprotein (P-gp), a downstream transporter of Nrf2. Adenovirus vectors containing the Nrf2 gene were transfected into A549 cells. Cell proliferation was assessed by Cell Counting Kit-8. The levels of LDH, MDA, SOD, TNF-α, IL-6 levels were detected using their respective ELISA kits. In addition, the levels of Nrf2 and P-gp protein expression were detected by western blot analysis. The concentration of PQ was measured by HPLC. The results revealed that overexpressed Nrf2 significantly increased P-gp protein levels, decreased the intracellular accumulation of PQ and attenuated PQ-induced toxicity. However, the protective effects of Nrf2 overexpression on PQ-challenged A549 cells were abrogated following cyclosporine A treatment, a competitive inhibitor of P-gp, which also increased intracellular PQ levels. These data indicated that Nrf2 gene overexpression prevented PQ toxicity in A549 cells, potentially via the upregulation of P-gp activity and the inhibition of intracellular PQ accumulation. Thus, Nrf2 and P-gp may serve as potential therapeutic targets for the treatment of PQ-induced injury.

Effect of Ginkgo biloba extract-761 on motor functions in permanent middle cerebral artery occlusion rats.

BACKGROUND: Ginkgo biloba extract (EGb-761) has been in use to treat variety of ailments including memory loss and emotional disorders usually experienced after ischemic stroke. However, data regarding its protective role in stroke associated motor dysfunction is scarce. PURPOSE: The present work was designed to investigate the long-term effects of EGb-761 on the motor dysfunctions associated with permanent middle cerebral artery occlusion (pMCAO) in rats. STUDY DESIGN/METHODS: Focal ischemic stroke was induced in male Sprague-Dawley rats by pMCAO. These rats were orally administered with EGb-761 (25, 50, 100 mg/kg) and positive control butylphthalide (50 mg/kg) for up to 28 consecutive days. The motor function was evaluated by assessing neurological scores, rotarod performance and gait analysis after 7, 14, 21 and 28 days. After 28 days, the histological examination of in frontal cortex and hippocampus was also carried out. RESULTS: EGb-761 treatment significantly improved motor function with better outcome in coordination and gait impairment rats. EGb-761 (25, 50, 100 mg/kg) treatment for 28 days significantly decreased the neurological scores. After 28 days of treatment EGb-761 (50 and 100 mg/kg) significantly increased the latency in rotarod test, walk speed, and the body rotation, whereas, decreased the stride time and the left posterior swing length in gait were observed. EGb-761 (50, 100 mg/kg). EGb-761 (50, 100 mg/kg) significantly improved the pathological changes related to pMCAO. CONCLUSIONS: EGb 761 could improve motor function especially gait impairments among pMCAO rat model related to the decreased neuronal damage. Therefore, it might be the potential to be explored further as an effective therapeutic drug to treat post stroke motor dysfunctions.

Stem cells enhance reperfusion following ischemia: Validation using laser speckle imaging in predicting tissue repair.

OBJECTIVES: The lack of real-time assessment of vascular perfusion changes remains a major weakness in assessing the efficacy of bone marrow stromal cells (BMSC) therapeutic ischemia reperfusion (I/R) injury. This study provides for the first time the real-time in vivo perfusion monitoring in I/R mice with BMSC therapy. STUDY DESIGN: Animal model. METHODS: Surgically created cutaneous flaps perfused by the inferior epigastric vessels were subjected to 3.5 hours of ischemia/reperfusion. Wound healing and vascular perfusion were assessed by Image-J and laser speckle contrast analysis (LSCA) in three groups (sham, I/R, and I/R + BMSC). BMSC tracking was quantified in an additional two groups (with/without I/R) using intravital fluorescent microscopy. The histopathology of skin flaps was examined by hematoxylin and eosin stain. Infiltrated macrophages were analyzed by confocal immunofluorescent microscopy. RESULTS: Postischemic tissues treated with BMSC demonstrated significantly greater survival than I/R control. On days 3 to 7 postreperfusion, both proximal and distal areas in BMSC-treated flaps demonstrated greater levels of perfusion than untreated I/R flaps (P < 0.05). Intravital fluorescent microscopy revealed that numbers of labeled BMSC were significantly increased in the distal area compared to the proximal area in both with and without ischemic mice. Histological examination showed lower necrosis and infiltrated inflammatory cells in I/R + BMSC-treated mice versus I/R controls. CONCLUSION: BMSC accumulated in I/R flaps and exerted beneficial effects including: 1) improving vascular perfusion and 2) attenuating inflammatory cell infiltration. LSCA facilitates monitoring of the real-time restitution of perfusion during flap wound healing in experimental animals and could also similarly applied in clinical investigations. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E198-E205, 2018.

Dynamics of early stem cell recruitment in skin flaps subjected to ischemia reperfusion injury.

OBJECTIVE: Bone marrow-derived stromal cell (BMSCs) therapy improves survival of skin flaps subject to ischemia/reperfusion (I/R) injury. However, very little is known about the trafficking and distribution of BMSCs in post-ischemic skin tissue following intravenous administration. The aim of this study was to assess the behavior of BMSCs in post-ischemic skin flaps and to compare the magnitude and kinetics of accumulation of BMSCs and leukocytes following I/R. METHODS: Cutaneous flaps perfused by the inferior epigastric vessels were created in C57Bl6 mice. The flaps were subjected to 3.5h of ischemia followed by reperfusion. Wound healing and vascular perfusion were assessed in 3 groups of mice (sham, I/R, and I/R+BMSCs treatment) on days 3, 5, 7 and 14 post-reperfusion. The kinetics and magnitude of BMSCs and leukocyte recruitment were quantified in additional 2 groups (Sham and I/R) after I/R using intravital fluorescence microscopy at 2 and 4h after the intravenous injection of fluorescently labeled BMSCs. RESULTS: Wound healing after I/R was significantly enhanced in skin flaps of mice treated with BMSCs, compared to controls. The rolling velocity of BMSCs was higher compared to leukocytes both in control mice (32.4±3.7µm/s vs 24.0±2.2µm/s, p<0.05) and in I/R mice (34.6±3.8µm/s vs 20.2±2.3µm/s, p<0.005). However, the rolling velocity of both cell populations was not altered by I/R. The firm adhesion and transendothelial migration of BMSCs did not differ from the values detected for leukocytes for both control and I/R mice. CONCLUSIONS: The magnitude and kinetics of BMSCs recruitment in skin flaps subjected to I/R are not significantly different from the responses noted for leukocytes, suggesting that similar mechanisms may be involved in the recruitment of both cell populations following I/R.

Interleukin-6 mediates enhanced thrombus development in cerebral arterioles following a brief period of focal brain ischemia.

OBJECTIVE: The cerebral microvasculature is rendered more vulnerable to thrombus formation following a brief (5.0 min) period of focal ischemia. This study examined the contribution of interleukin-6 (IL-6), a neuroprotective and prothrombotic cytokine produced by the brain, to transient ischemia-induced thrombosis in cerebral arterioles. APPROACH & RESULTS: The middle cerebral artery of C57BL/6J mice was occluded for 5 min, followed by 24h of reperfusion (MCAo/R). Intravital fluorescence microscopy was used to monitor thrombus development in cerebral arterioles induced by light/dye photoactivation. Thrombosis was quantified as the time of onset of platelet aggregation on the vessel wall and the time for complete blood flow cessation. MCAo/R in wild type (WT) mice yielded an acceleration of thrombus formation that was accompanied by increased IL-6 levels in plasma and in post-ischemic brain tissue. The exaggerated thrombosis response to MCAo/R was blunted in WT mice receiving an IL-6 receptor-blocking antibody and in IL-6 deficient (IL-6(-/-)) mice. Bone marrow chimeras, produced by transplanting IL-6(-/-) marrow into WT recipients, did not exhibit protection against MCAo/R-induced thrombosis. CONCLUSIONS: The increased vulnerability of the cerebral vasculature to thrombus development after MCAo/R is mediated by IL-6, which is likely derived from brain cells rather than circulating blood cells. These findings suggest that anti-IL-6 therapy may reduce the likelihood of cerebral thrombus development after a transient ischemic attack.

Transient ischemia elicits a sustained enhancement of thrombus development in the cerebral microvasculature: effects of anti-thrombotic therapy.

OBJECTIVE: While transient ischemic attack (TIA) is a well-known harbinger of ischemic stroke, the mechanisms that link TIA to subsequent strokes remain poorly understood. The overall aim of this study was to determine whether: 1) brief periods of transient cerebral ischemia render this tissue more vulnerable to thrombus development and 2) antiplatelet agents used in TIA patients alter ischemia-induced thrombogenesis. APPROACH & RESULTS: The middle cerebral artery of C57BL/6 mice was occluded for 2.5-10min, followed by reperfusion periods of 1-28days. Intravital microscopy was used to monitor thrombus development in cerebral microvessels induced by light/dye photoactivation. Thrombosis was quantified as the time to platelet aggregation on the vessel wall and the time for complete blood flow cessation. While brief periods of cerebral ischemia were not associated with neurological deficits or brain infarction (evaluated after 1day), it yielded a pronounced and prolonged (up to 28days) acceleration of thrombus formation, compared to control (sham) mice. This prothrombotic phenotype was not altered by pre- and/or post-treatment of mice with either aspirin (A), clopidogrel (C), dipyridamole (D), or atorvastatin (S), or with A+D+S. CONCLUSIONS: The increased vulnerability of the cerebral vasculature to thrombus development after a brief period of transient ischemia can be recapitulated in a murine model. Antiplatelet or antithrombotic agents used in patients with TIA show no benefit in this mouse model of brief transient ischemia.

[Comparison of therapeutic effects of low-dose versus high-dose interferon alpha-2b treatment on chronic myelocytic leukemia: a prospective randomized study].

OBJECTIVE: To compare the therapeutic effects of low-dose and high-dose interferon alpha-2b (IFN) treatment on chronic myelocytic leukemia (CML). METHODS: A real-time quantitative reverse transcriptase PCR (RQ-PCR) method was established to detect the fusion gene bcr-abl expression, thereby studying the reduction of leukemic cells. Thirty newly diagnosed CML patients, 21 males and 9 females, aged 14 - 69, were treated with hydroxyurea to keep the white blood cell count less than 20 x 10(9)/L, and then randomized into 2 groups: high-dose IFN group receiving IFN alpha-2b 5MIU 6 times per week for 3 - 6 months and low-dose IFN group receiving IFN alpha-2b 3MIU every other day for 3 - 6 months. Bone marrow was collected every month to Real-time PCR was used to detect the expression of bcr-abl mRNA. Mononuclear cells were isolated and RNA was extracted to detect the expression of fusion gene bcr-abl and a control gene GAPDH. The results were reported as the number of bcr-abl copies/GAPDH copy. RESULTS: The established real-time quantitative PCR method could detect the bcr-abl molecules as low as 50 copies. The intra-assay coefficient of variation (CV) was less than 5% and the inter-assay CV was 5.13%. The median bcr-abl fusion gene expression level of 30 CML patients before IFN therapy was 0.098 (range: 0.010 - 5.799). The bcr-abl expression level decreased by 19.37% and 24.86% in the low-dose and high-dose IFN groups respectively after 3 months' therapy. No significant difference was observed between the two groups (P = 0.398). Relatively more side effects were observed in the high-dose IFN group than in low-dose group. CONCLUSION: RQ-PCR is a reliable method to monitor CML therapy by analyzing fusion gene bcr-abl expression. There is a difference in bcr-abl fusion gene expression levels among the newly diagnosed patients, and low-dose IFN is as effective as high-dose IFN in reducing bcr-abl expression but with less side effects.

[Inhibitive effect of previously activated psoralens on K562 cell proliferation].

The objective was to observe the influence of previously activated psoralens on the proliferation of K562 cells, and to provide laboratory data for its clinical usage. K562 cells were treated separately with previously and late activated psoralens, then their trypan blue exclusion inhibited rates (TBIR), cell proliferation inhibited rates (CPIR) and colony forming inhibited rates (CFIR) after culture were compared. The results showed that previously activated psoralens displayed an inhibiting effect on the proliferation of K562 cells with a dose-effect relationship. There was no obvious difference between previously and late activated psoralens on TBIR, CPIR and CFIR. In order to exert the inhibitive effect of previously activated psoralens, the time of ultraviolet ray exposure should be 10 minutes at least, and longer than 12 hours for inhibiting K562. The inhibitive effect of previously activated psoratens decreased as the time interval from activation to its use was prolonged. The inhibiting effect of previously activated psoralens was strongest within 6 hours after activation. In conclusion, both previously and late activated psoralens show inhibiting effects on the proliferation of K562, which may be able to use an antineoplastic drug in clinic.