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In this study, the expression of Cripto-1 and the role of macrophage polarization in immune response after allogeneic transfusion were analyzed by constructing a mouse model of allogeneic transfusion. In order to analyze the effects of miR-449a on the PI3K/AKT/NF-κB signaling pathway and the expression of downstream related regulatory factors under normal and abnormal conditions, we adopt in vitro and in vivo experiments separately. The molecular mechanism of PI3K/AKT/NF-κB signaling pathway was analyzed by blocking or activating gene expression and western blotting. Experiment in vitro has confirmed that inhibition of miR-449a increased the protein expression of Cripto-1. In vivo experiments confirmed that allogeneic transfusion reduced the expression of Cripto-1, which further inhibited NF-κB signaling pathway through AKT/PI3K phosphorylation, regulated macrophage polarization, inhibited M1 polarization of macrophages, promoted M2 polarization, and thus affected immune response of the body.

IntroductionSystemic inflammatory and immune suppressive incidents constitute a significant hallmark of red blood cell transfusion's adverse effects. These adverse side effects are seemingly associated with old blood transfusion; therefore, we aim to investigate the underlying mechanism of fresh autologous blood transfusion (fABT) and its subsequent effect in diabetic mice.Material and methodsIn the present study, we utilized 60 Swiss male mice aged 6-8 weeks, categorized into normal, diabetic, and freshly transfused with autologous blood. After treating the mice accordingly, further experimentations took place as we assessed the M1/M2 macrophage polarization concerning CD16/CD32/CD206 cells by flow cytometry, determined the mitochondrial metabolism using Lowry's analysis, measured hepatic oxidative stress using MDA and SOD assays, and examined the erythrocytic oxygen-carrying capacity (Q value) and oxygen consumption rate (OCR) and osmotic fragility.ResultsResults showed that fresh autologous blood transfusion markedly reduced M2 macrophage polarization, enhanced hepatic mitochondrial metabolism, reduced hepatic oxidative stress, and promoted red blood cells' oxygen-carrying capacity while reducing its osmotic fragility and oxygen consumption. Moreover, we found that fABT promoted the IGF2/PI3K signaling pathway, These results indicate the vital necessity of providing fresh autologous blood transfusion during therapy or surgeries.ConclusionsIn conclusion, our results are the first to highlight the underlying mechanism in which how fABT modulates physiological patterns in diabetic animals model, proposing a sound basis for utilizing fABT in clinical applications.

IntroductionLiver cancer is a common tumor of digestive system. Hepatectomy sometimes results in massive hemorrhage, and intraoperative autotransfusion is often required.Material and methodsMethods: In this basic research, the liver cancer rat model was established, and the visible liver cancer tissue was surgically removed, and the blood of the mice was recovered.ResultsThe results showed that the model group had the highest recurrence and metastasis of liver cancer, and the lowest was autotransfusion group. Flow cytometry showed that the number of HLA-DQB1 positive cells, the content of CD4+CD25+T cells, and the proliferation of CD4+T cells were the most in the autotransfusion group, followed by the model group, and the least in the auto+HLA-DQB1 group, which showed no difference from the control group. Immunohistochemistry and western blot analysis showed that the lowest expression of PDCD1 protein was in the autotransfusion group, which showed no significant difference from the control group, and the highest expression was in auto-HLA-DQB1, followed by the model group.ConclusionsIt elucidates the molecular mechanism that autotransfusion will activate the up-regulated expression of signal molecule HLA-DQB1, enhance the immune surveillance of CD4+T cells, reduce the expression of PDCD1 on the surface molecule of liver cancer cells, and inhibit liver cancer metastasis.Autotransfusion activated the expression of signaling molecule HLA-DQB1, enhanced the immune surveillance of CD4+T cells, reduced the expression of PDCD1 on the surface of liver cancer cells, and reduced the metastasis and recurrence of liver cancer.

Background After storing blood for a period of time, the structure and properties of the red blood cells (RBC) will change, which results in a decrease in the oxygen-carrying capacity, and further has a certain impact on their exosomes. Objectives Effective oxygen uptake (Q), P50, 2,3-DPG, and Na+-K+-ATP of RBC after different storage times were detected. Electron microscopy was used to observe the morphology of RBC and the characteristics of secreting exosomes. Western blot was used to detect the expression of phenotypes CD63 and CD81 of exosomes, and the expression of mitochondrial riboprotein MRPS35 of exosomes was also detected to explore the mechanism of decreased function of RBC with the extension of preservation time. Material and methods After the RBC suspension was prepared, the effective oxygen-carrying capacity (Q) and P50, as well as 2,3-DPG and Na+-K+-ATP were prepared. This was followed by morphology observation of erythrocyte exosomes using transmission electron microscope (TEM), and by western blot analysis of exosome phenotypes CD63 and CD81. Results Erythrocytes secrete exosomes, which results in abnormal expression of related proteins in mitochondria. This leads to increased ROS production, mitochondrial apoptosis and, finally, changes in or damage to erythrocytes. Conclusions Changes in the rheological properties and oxygen-carrying functions of erythrocytes during preservation are all observable manifestations, and underlying these manifestations are mechanisms of damage to erythrocytes at a molecular level. Erythrocytes secrete exosomes, which results in abnormal expression of related proteins in mitochondria, increasing ROS production, mitochondrial apoptosis and, finally, changes or damage to erythrocytes.

Background Red blood cell (RBC) transfusion is associated with systemic inflammation and immune suppression as adverse outcomes. Objectives To investigate the immunomodulatory function of the transfused autologous RBC in altering pro-inflammatory and immunosuppressive effects. Material and methods A total of 24 Sprague Dawley male rats were randomly divided into 3 groups (n = 8 in each group). Group 1 did not receive blood transfusions, while the other 2 groups of rats separately received transfusion of RBC stored for 14 days (group 2) and 35 days (group 3). The rats were treated with HO-1 inhibitor, HO-1 inducer and nuclear factor erythroid 2-related factor 2 (Nrf2) activator after they separately received autologous transfusion of RBC that were cryopreserved for 14 days or 35 days. The blood samples of the rats were collected 12 h after the transfusion, and the macrophage phenotype of M1 and M2 were analyzed with flow cytometry (FCM). Also, the surface protein expression of CD68 and CD200R in macrophages were analyzed and the inflammatory signals in the serum were measured with enzyme-linked immunosorbent assay (ELISA). Moreover, the location and expression of proteins heme oxygenase 1 (HO-1), arginine 1 (Arg-1) and nitric oxide synthase 2 (NOS2) in macrophage were detected with immunofluorescence (IF). Results Autologous transfusion of long-time stored ("old") RBC promoted macrophage polarization to M2 phenotype and upregulated the expression of its surface proteins CD68 and CD200R. The pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1β, and IL-18 were inhibited, and the secretion of NOS isoforms (iNOS) in serum was reduced with blood transfusion; contrarily, the production of IL-10 and CCL22 was increased. Additionally, HO-1, Arg-1 and NOS2 proteins were located in the cytoplasm, and HO-1 and Arg-1 proteins were highly expressed in macrophage, while the expression of protein NOS2 was low. Moreover, Nrf2, HO-1 and Arg-1 proteins were upregulated in macrophage after receiving "old" RBC transfusion. Conclusions Autologous transfusion of "old" RBC drove the macrophage phenotype toward M2 macrophages and induced immunosuppressive effects through the IL-10-NRF2-HO-1 signals.

Transfusion of autologous blood is a timesaving, convenient, safe, and effective therapy from a clinical perspective, and often employed for the treatment of diabetic patients. Stabilization of HIF-1α has been widely reported to be a critical factor in the improvement of wound healing in diabetes. Therefore, our study reveals the roles of improved autologous blood in wound healing in diabetes, through autologous blood transfusion in a mouse model. Initially, BALB/c mice were subjected to streptozotocin for diabetic mouse model establishment. Diabetic mice were transfused with improved or standard autologous blood in perfusion culture system. Roles of improved autologous blood in mediating HIF-1α pathway were determined by measuring expression of VEGF, EGF, HIF-1α, and HSP-90. In order to assess the detailed regulatory mechanism of improved autologous blood in perspective of wound healing, cell proliferation, migration and cell cycle, fibroblasts isolated from diabetic mice were transfected with HIF-1α siRNA. Mice transfused with improved autologous blood exhibited increased levels of CD31 and α-SMA in skin tissues, and reduced TNF-α, IL-1β, and IL-6 levels, indicating that improved autologous blood promoted wound healing ability and reduced the release of inflammatory factors. Diabetic mice transfused with improved autologous blood presented activated HIF-1α pathway. The survival rate, proliferation, and migration of fibroblasts were elevated via activation of the HIF-1α pathway. Taken together, improved blood preservation solution could enhance the oxygen carrying capacity of red blood cells and wound healing in mice with diabetes, which is achieved through regulation of HIF-1α pathway.

Impaired wound healing is a debilitating complication of diabetes. The long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized to be differentially expressed in various diseases. However, its underlying mechanism in diabetes has not been fully understood. Notably, we aim to examine the expression of MALAT1 in diabetic mice and its role in wound healing involving the hypoxia-inducible factor-1α (HIF-1α) signaling pathway with a modified autologous blood preservative solution reported. A mouse model of diabetes was established. MALAT1 was identified to promote the activation of the HIF-1α signaling pathway and to be enriched in autologous blood through modified preservation, which might facilitate the improvement of physiological function of blood cells. Through gain- or loss-of-function approaches, viability of fibroblasts cultured in high glucose, wound healing of mice, and collagen expression in wound areas were enhanced by MALAT1 and HIF-1α. Taken together, the present study demonstrated that the physiological status of mouse blood was effectively improved by modified autologous blood preservation, which exhibited upregulated MALAT1, thereby accelerating the fibroblast activation and wound healing in diabetic mice via the activation of the HIF-1α signaling pathway. The upregulation of MALAT1 activating the HIF-1α signaling pathway provides a novel insight into drug targets against diabetes., Graphical Abstract

Background Sepsis is one of most common causes of death in the intensive care unit (ICU) due to infection and inflammation. The Duffy antigen receptor for chemokines (DARC) regulates pro-inflammatory cytokines, thus playing an important role in inflammation. Objectives This study aimed to elucidate the correlation among erythrocyte transfusion, macrophage pyroptosis and inflammation in the progression of sepsis. Material and methods Alanine aminotransferase (ALT/GPT) activity was measured with the ALT/GPT activity measurement kit (Jiancheng Bio, Nanjing, China) according to the kit manual. The ET-1 concentration was measured with enzyme-linked immunosorbent assay (ELISA) using the endothelin-1 (ET-1) measurement kit (Jiancheng Bio) according to the kit manual. Apoptosis was evaluated using flow cytometry-based Annexin V staining assay. The cells were collected using centrifugation and resuspended in binding buffer. Ultrastructural analysis of pyroptotic body, the levels of interleukin (IL)-1β, IL-18, IL-33, MIP-2, CXCL8, reactive oxygen species (ROS), and LTB4 were measured with ELISA. Results Our results showed that septic rats had impaired hepatic function and ET-1 levels. Erythrocyte transfusion upregulated DARC expression in the sepsis model. Erythrocyte transfusion also affected pyroptosis in macrophages, reduced the production of inflammatory cytokines, such as IL-1β, IL-18 and IL-33, and alleviated cytotoxicity in the sepsis model. Conclusions Erythrocyte transfusion may function as a therapeutic tool against sepsis by regulating pyroptosis, inflammation and cytotoxicity.

Autotransfusion refers to a blood transfusion method in which the blood or blood components of the patient are collected under certain conditions, returned to himself when the patient needs surgery or emergency after a series of storing and processing. Although autotransfusion can avoid blood-borne diseases and adverse reactions related to allogeneic blood transfusion, a series of structural and functional changes of erythrocytes will occur during extension of storage time, thus affecting the efficacy of clinical blood transfusion. Our research was aimed to explore the change of erythrocyte oxygen-carrying capacity in different storage time, such as effective oxygen uptake (Q), P50, 2,3-DPG, Na+-K+-ATPase, to detect membrane potential, the change of Ca2+, and reactive oxygen species (ROS) change of erythrocytes. At the same time, Western blot was used to detect the expression of Mitofusin 1 (Mfn1) and Mitofusin 2 (Mfn2) proteins on the cytomembrane, from the perspective of oxidative stress to explore the function change of erythrocytes after different storage time. This study is expected to provide experimental data for further clarifying the functional status of erythrocytes with different preservation time in patients with autotransfusion, achieving accurate infusion of erythrocytes and improving the therapeutic effect of autologous blood transfusion, which has important clinical application value.

This study aimed to investigate the protective effects of erythrocyte-mediated endoplasmic reticulum (ER) stress in macrophages in hemorrhagic shock. An hemorrhagic shock model was established in male BALB/c mice. Animals were randomly divided into three groups ( n = 8): control group (A), erythrocyte reinfusion group (B), and TLR9 inhibition group (C). Eight healthy BALB/c mice were also included as group N ( n = 8). Mice in group A were not treated, while mice in groups B and C were transfused with red blood cells separated from the blood of mice in group N. Flow cytometry was used to detect the expression of erythrocyte surface protein TLR9 in each group. Immunofluorescence assay was used to analyze the distribution and relative expression of protein STING in macrophages. Flow cytometry was used to analyze the expression of STING, ATF6, and IRE1 in macrophages. Enzyme-linked immunosorbent assay was used to analyze the levels of inflammatory signal molecules, including IFN-α, IFN-β, IL-6, CCL4, CCL5, and IL-6. FITC-Annexin V was used to analyze the apoptosis of immune cells (macrophages) in mouse blood samples and to detect the concentration of calcium ions in erythrocyte cytoplasm. The results showed that the expression of erythrocyte surface protein TLR9; the distribution of STING-positive cells in macrophages; the expressions of STING, ATF6, and IRE1 in macrophages; the levels of inflammatory signal molecules; the apoptosis rate of macrophages; and the intracellular calcium concentration in erythrocytes in group B were higher than those in group A, followed by group C. These results suggest that TLR9 regulates ER stress in macrophages of mice with hemorrhagic shock through the TLR9-cGAS-STING-IFN signaling pathway. Increased expression of TLR9 enhanced macrophage activity, reduced apoptosis, enhanced inflammatory response and immune response, and restored electrolyte level, which might be a therapeutic option for the treatment of hemorrhagic shock.

Sepsis is a life-threatening disease that results in excessive stimulation of the host’s immune cells. In the animal study, the purpose was to investigate the roles of fresh frozen plasma (FFP) transfusion in shaping the CD4+ T lymphocytes immune response through modulating the secreted exosome protein Galectin-9 in mice with severe sepsis. By using Western blot analysis, we first identified that the protein Galectin-9 is highly accumulated in the blood plasma of severe sepsis mice, and with transmission electron microscopy (TEM) and protein analysis, we found that Galectin-9 is a secreted exosome protein. Thereafter, we treated the severe sepsis mice with the antibiotic Cefuroxime Axetil; one group of mice received FFP transfusion and the other group of mice received normal saline. Surprisingly, the FFP transfusion reduced the secretion of exosome protein Galectin-9 and there was crosstalking between the exosome protein Galectin-9 and CD4+ T lymphocytes in mice with severe sepsis. Results showed that the proliferation of T helper (Th) cells (Th1 and Th17) was promoted, and regulatory T (Treg) cells’ maintenance was inhibited in the sepsis mice after receiving FFP transfusion. Correspondingly, this immune reprogrammed activity shaped the inflammatory cytokine secretion with an increase in the interleukin (IL)-1β, IL-6, and interferon-gamma levels, while it decreased IL-10 levels. Taken together, it was suggested that FFP transfusion promoted reprogramming of CD4+ T lymphocytes’ immune response through inhibiting the secretion of exosome protein Galectin-9 in mice with severe sepsis to relieve immunosuppression.

Background: Pre-operative autologous blood donation (PABD) is one of the most widely distributed autologous blood donation means, which has positive effects on erythropoiesis. However, whether PABD can stimulate the bone marrow hematopoiesis after hepatectomy has not been reported. Methods: Totally 80 New Zealand rabbits were randomly divided into 4 groups that included control group, surgery group, hemodilutional autotransfusion (HA) group and PABD group. Automatic reticulocyte examination was performed to detect the content of reticulocyte and immature reticulocyte fractions (IRF). Flow cytometric analysis was employed to monitor the level of CD34+ cells and the cell cycle status. Southern blotting was conducted to determine the telomere length of CD34+ cells. Results: The content of high fluorescence reticulocytes (HFR) and IRF was decreased at 6 h and 24 h after autotransfusion. However, the level of CD34+ cells was upregulated after PABD. Cell cycle status analysis revealed that the majority of the CD34+ cells in HA and PABD group were maintained in G0/G1 phase. The telomere length in HA and PABD group was shortened than that of the control group and surgery group. Conclusion: PABD could promote the bone marrow hematopoietic functions in rabbits after hepatectomy via stimulating proliferation of CD34+ cells and shortening the telomere length of CD34+ cells, but the content of HFR was not increased immediately because of the stuck of CD34+ cells in the G0/G1 phase.

Objective By observing the changes in the number and activity of CD34+ cells in bone marrow after predeposit autotransfusion (PAT) to patients with femoral shaft fracture (FSF), to evaluate the effects of PAT on hematopoietic function and hematopoietic stem cells in bone marrow. Methods Selected FSF patients were randomly divided into 2 groups: the control group (patients did not receive blood transfusion after surgery) and PAT group (patients received PAT after surgery). The content of RBC and Plt in blood samples were counted by blood routine. The cell cycle and proportion of CD34+ myelinated cells in blood samples was analyzed by flow cytometry. The telomere DNA length of hematopoietic stem cells (HSCs) in the control groups and PAT group at postoperation 24 was analyzed by southern blot. Results The content of RBC and Plt in postoperation 6 h and 24 h in the control group was evidently higher compared to that in PAT group, while Hb content in control group was significantly lower compared to that in PAT group. The proportion of CD34+ myelinated cells in post-transfusion 6 h and postoperation 24 h in PAT group was evidently higher compared to that in the control group. In PAT group, S phase at postoperation 24 h was significantly larger compared to that at post-transfusion 6 h. The telomere DNA length of HSCs in PAT group was longer than that in the control group. Conclusion PAT can increase the number of HSC, while does not cause the abnormal aging of HSCs. PAT is suitable for postoperative blood transfusion of patients with FSF.

Erythrocyte membrane is crucial to maintain the stability of erythrocyte structure. The membrane protein on the surface of erythrocyte membrane enables erythrocyte to have plasticity and pass through the microcirculation without being blocked or destroyed. Decreased deformability of erythrocyte membrane protein will lead to a series of pathological and physiological changes such as tissue and organ ischemia and hypoxia. Therefore, this research collected 30 cases of healthy blood donors, and explored erythrocyte stored at different times relating indicators including effective oxygen uptake (Q), P50, 2,3-DPG, Na+-k+-ATP. Erythrocyte morphology was observed by electron microscopy. Western blot and immunofluorescence assay were used to detect membrane protein EPB41, S1P, GLTP, SPPL2A expression changes of erythrocyte. To explore the effective carry oxygen capacity of erythrocyte at different storage time resulting in the expression change of erythrocyte surface membrane protein.

Sepsis is a life-threatening disease resulting in excessive stimulation of the host’s immune cells. In the study, the purpose is to investigate the roles of fresh frozen plasma (FFP) transfusion in shaping the CD4+ T lymphocytes immune response through modulating the secreted exosome protein Galectin 9 in mice with severe sepsis. We firstly identified the protein Galectin 9 highly accumulated in the blood plasma of severe sepsis mice with western blot, then with transmission electron microscopy (TEM) and protein analysis, we found protein Galectin 9 was a secreted exosome protein in sepsis mice. Thereafter, we treated the severe sepsis mice with antibiotic-Cefuroxime Axetil, meanwhile, one group mice received FFP transfusion, the other one group mice received normal saline. Surprisingly, the FFP transfusion reduced the secretion of exosome protein Galectin 9 and there was crosstalking between the exosome protein Galectin 9 and CD4+ T lymphocytes in mice with severe sepsis. Results showed that the proliferation of Th1, Th17 were promoted and obiviously inhibited Treg cells maintenance in the sepsis mice receiving FFP transfusion. Correspondingly, this immune reprogrammed activity shaped the inflammatory cytokines secretion with increase of IL-1β, IL-6 and IFN-γ, whilst, decrease of IL-10. Taken together, it was suggested that FFP transfusion promoted reprogramming CD4+ T lymphocytes immune response through inhibiting the secretion of exosome protein Galectin 9 in mice with severe sepsis to relieve immunosuppression.

BackgroundTo explore the clinical effect of predeposit, salvage and hemodilution autotransfusion on patients with femoral shaft fractdure (FSF) surgery.MethodsSelected FSF patients were randomly divided into 3 groups: intraoperative blood salvage autotransfusion group, preoperative hemodilution autohemotransfusion group and predeposit autotransfusion. After the operation for 5 days, body temperature, heart rate, blood platelet (PLT) and hemoglobin (Hb) of patients were determined; the concentrations of EPO and G-MSF in the 3 groups were calculated by ELISA assay; the content of CD14 + monocytes were calculated by FCM assay. The growth time and condition of the patient’s callus were determined at the 30th, 45th and 60th days after operation. Cox regression analysis was used to analyze the correlation between EP0, G-MSF, CD14 + mononuclear content, callus growth and autotransfusion methods.ResultsThere were no statistically significant differences in body temperature and heart rate between the 3 groups (P > 0.05). PLT and Hb in Predeposit group were markedly increased compared to that in Salvage group and Hemodilution group. The concentrations of EPO and G-MSF in Predeposit group were markedly increased compared to that in Salvage group and Hemodilution group. The content of CD14 + monocytes in Predeposit group was significantly higher compared to that in Salvage group and Hemodilution group. Predeposit autotransfusion promotes callus growth more quickly. The concentration of EP0 and G-MSF were positively correlated with callus growth in the three groups, and the content of CD14 + monocytes was not correlated in any of the three groups.ConclusionPredeposit autotransfusion promoted the recovery of FSF patients after operation more quickly compared to salvage autotransfusion and hemodilution autotransfusion.# These authors contributed equally to this work.

Diabetes-associated dysfunction in angiogenesis predominantly contributes to impairment of wound closure, but a role of hypoxia-inducible factor 1 alpha (HIF-1a) in the process remain poorly understood. Here, we examined whether expression of HIF-1a in re-infused blood cells may improve the diabetic wound closure in mice. We found that that expression of HIF-1a in re-infused isogeneic blood cells significantly improved diabetic wound healing in mice, seemingly through augmentation of wound-associated angiogenesis. Mechanistically, expression of HIF-1a in re-infused blood cells significantly increased macrophage infiltration at the wound site, and macrophages produced vascular endothelial growth factor A (VEGF-A) to promote wound-associated angiogenesis. Together, our data suggest that augmentation of HIF-1a in reinfused blood cells may enhance diabetic ischemic wound closure.

Background: There remains a lack of a systematic summary of the efficacy and safety of various medicines for sciatica, and discrepancies among these exist. Objective: The aim of this study is to comprehensively assess the efficacy of and tolerance to several medical options for the treatment of sciatica. Methods: We performed a network meta-analysis and illustrated the results by the mean difference or odds ratio. The surface under the cumulative ranking curve (SUCRA) was used for indicating the preferable treatments. All data analyses and graphs were achieved via R 3.3.2 and Stata 13.0. Results: The subcutaneous anti–tumor necrosis factor–α (anti-TNF-α) was superior to the epidural steroid + anesthetic in reducing lumbar pain in both acute + chronic sciatica patients and acute sciatica patients. The epidural steroid demonstrated a better ability regarding the Oswestry disability score (ODI) compared to the subcutaneous anti-TNF-α. In addition, for total pain relief, the use of nonsteroidal antiinflammatory drugs was inferior to the epidural steroid + anesthetic. The epidural anesthetic and epidural steroid + anesthetic both demonstrated superiority over the epidural steroid and intramuscular steroid. The intravenous anti-TNF-α ranked first in leg pain relief, while the subcutaneous anti-TNF-α ranked first in lumbar pain relief, and the epidural steroid ranked first in the ODI on the basis of SUCRA. In addition, their safety outcome (withdrawal) rankings were all medium to high. Conclusions: Intravenous and subcutaneous anti-TNF-α were identified as the optimal treatments for both acute + chronic sciatica patients and acute sciatica patients. In addition, the epidural steroid was also recommended as a good intervention due to its superiority in reducing ODI.

// Jian-Rong Guo 1 , Huan Wang 1 , Xiao-Ju Jin 2 , Dong-Lin Jia 3 , Xun Zhou 1 and Qiang Tao 1 1 Department of Anesthesiology, Gongli Hospital, Second Military Medical University, Shanghai 200135, P.R. China 2 Department of Anesthesiology, Yijishan Hospital, Wannan Medical College, Wuhu 241001, P.R. China 3 Pain Department, Peking University Third Hospital, Beijing, 100191, P.R. China Correspondence to: Jian-Rong Guo, email: jr_guo@yeah.net Keywords: PI3K/Akt/mTOR signal pathway, chronic neuropathic pain, spinal microglia, chronic constriction injury, wortmannin Received: July 26, 2016 Accepted: January 03, 2017 Published: May 05, 2017 ABSTRACT Objective: To explore the effects of inhibition of PI3K/Akt/mTOR signal pathway on chronic neuropathic pain (CNP) and spinal microglia in a rat model of chronic constriction injury (CCI). Methods: Male SD rats were assigned into control, sham, CCI, wortmannin, dimethyl sulfoxide (DMSO) and wortmannin-positive control groups. Paw withdrawal mechanical threshold (PWMT) and thermal withdrawal latency (TWL) were recorded. qRT-PCR and Western blotting were used to detect PI3K, Akt and mTOR expressions and their phosphorylation. OX-4 expression was detected by immunohistochemistry and glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) expressions by immunofluorescence. Results: PWMT and TWL decreased in the CCI group than in the sham group on the 7 th and 14 th day after operation. Compared with the control and sham groups, the CCI group showed increased PI3K, Akt and mTOR mRNA expressions and elevated PI3K, p-Akt, p-mTOR and P70S6K protein expressions. More OX-42-positive cells and higher integrated optical density (IOD) of GFAP and NGF were found in the CCI group than the sham group at the 14 th day after operation. Compared with the DMSO group, the wortmannin group had higher PWMT and TWL, decreased PI3K, Akt and mTOR mRNA expressions and reduced PI3K, p-Akt, p-mTOR and P70S6K protein expressions. Less OX-42-positive cells and lower IOD of GFAP and NGF were found in the wortmannin group than the DMSO group 14 th day after operation. Conclusion: Inhibition of PI3K/Akt/mTOR signal pathway may alleviate CNP and reduce microglia and GFAP and NGF expressions in marrow in a rat model of CCI.

Objective To investigate the pharmacokinetics and pharmacodynamics of cisatracurium in patients undergoing surgery under acute normovolemic hemodilution (ANH) and acute hypervolemic hemodilution (AHH). Methods Ninety patients with orthopedic surgery were divided into ANH, AHH and control groups, which received hemodilution by hydroxyethyl starch 130/0.4 transfusion, Voluven transfusion and regular transfusion and infusion during surgery, respectively. Each group was divided into 3 sub-groups, administrated with cisatracurium at dosage of 0.1, 0.2 and 0.3mg/kg respectively. The changes in plasma protein, pH and electrolytes from the hemodilution beginning to surgery finish were monitored. Before and after cisatracurium administration, the phamocodynamic indicators of muscle relaxant were observed, and the plasma drug concentration was measured. Results After hemodilution or regular transfusion, all three groups experienced a distinct drop in total plasma protein, albumin and pH. Compared with control group, the plasma concentrations of both K + and Ca 2+ in ANH and AHH groups significantly dropped ( P P P P >0.05). In the same hemodilution or regular infusion, with increase of drug dosage, the onset time of muscle relaxation was shortened, and the period of no response to train-of-four stimulation, muscle relaxation blockade duration and action time of muscle relaxation blockade in body were extended ( P Conclusion When using cisatracurium under AHH, the dosage should be increased appropriately, while it need not be adjusted under ANH.

Background: Blood is a fluid tissue composed of blood cells and plasma. It mainly transports oxygen, nutrients and metabolites for various tissues and organs of the body. Among them, red blood cells are the important means of oxygen transport. After the blood is stored for a period of time, the structure and properties of the red blood cells will change, which results in a decrease in the oxygen carrying capacity, and the change in the oxygen carrying capacity will also have a certain impact on their exosomes. Methods: Therefore, our research is to detect some carry oxygen function related indicators of red blood cells in different storage times, including effective oxygen uptake (Q), P50, 2, 3-DPG, Na+-K+-ATP. Electron microscopy was used to observe the morphology of red blood cells and the characteristics of secreting exosomes. Western Blot was used to detect the expression of phenotypes CD63 and CD81 of exosomes, and the expression of mitochondrial riboprotein MRPS35 of exosomes was also detected to explore the mechanism of decreased function of red blood cells with the extension of preservation time. Results: Changes in the rheological properties and oxygen-carrying functions of erythrocytes during the preservation process are all appearances, underlying which lies the damage mechanism at the molecular level of erythrocytes. Conclusion:Erythrocytes secrete exosomes, which resulted in abnormal expression of related proteins in mitochondria to increase ROS, mitochondrial apoptosis, and finally changes or damage to erythrocytes. Funding Statement: This work is supported by Key Disciplines Group Construction Project of Pudong Health Bureau of Shanghai (No.PWZxq2017-10), and National Natural Science Foundation of China (No. 81671919, 81870147). Declaration of Interests: All of the authors have no conflict of interest in this research. Ethics Approval Statement: Ethical approval was given by Gongli hospital, The Second Military Medical University.

Background: Delirium is commonly observed among elderly surgical subjects during the postoperative period, and the incidence of postoperative delirium (POD) in elderly patients (≥65 years) ranges widely from 10% to 70%. This study aimed at investigating potential serum predicative factors for POD in elderly patients after open abdominal surgery. Methods: 140 subjects scheduled to undergo elective gastrointestinal tumor resection via laparotomy from March, 2013 to May, 2015 were enrolled in this study. Participants enrolled were evaluated for delirium and delirium severity on preoperative day, postoperative days 2 and 3. Non-fasting blood samples were collected in the morning on the day before surgery for the detection of serum cytokines expressions by enzyme-linked immunesorbent assays (ELISA). Results: A total of 140 patients were finally enrolled in this study and 36 of them occurred POD, with a POD incidence of 25.7%. In comparison with non-POD group, the serum levels of insulin-like growth factor-1 (IGF-1) demonstrated significantly decreased of patients in POD group (P

Desflurane, halothane, isoflurane, propofol, and sevoflurane are widely used anesthetics in pediatric anesthesia. Adverse effect including emergence agitation, postoperative nausea and vomiting, and postoperative pain are common. Prolonged extubation time and emergency time are also troubling anesthesiologists. Previous studies have noted the characteristics of various anesthetics in pediatric anesthesia, while the results were inconclusive and conflicting. In this study, we aimed at performing a comprehensive network meta-analysis concerning the emergence and recovery characteristics of pediatric anesthetics. Relevant articles were retrieved and selected according to our inclusion criteria. Network meta-analysis was performed with a random-effect model within a Bayesian framework. ORs and corresponding 95 % credible intervals were calculated by Markov chain Monte Carlo methods. Node-splitting method was used to calculate the inconsistency. Rank probabilities were assessed by the surface under the cumulative ranking curve (SUCRA). Propofol was recommended as the most efficient and safe anesthetic in pediatric anesthesia with few adverse effects. Desflurane has the highest incidence of emergence agitation and worst recovery characteristics. Halothane was regarded as an efficient anesthetic with the best recovery characteristics, while postoperative nausea and vomiting is a common adverse effect. Isoflurane was reported to be the safest concerning postoperative pain, and cases using sevoflurane in pediatric anesthesia reported the highest incidence of analgesic requirement. Our network meta-analysis demonstrated that propofol was suggested as the first choice in the clinical practice for its efficiency and safe in pediatric anesthesia.

Objective To investigate the characteristics of the macrophage response to transfusion of erythrocytes kept at different storage times in the mouse model of haemorrhagic shock. Methods Erythrocytes were isolated from mice and stored for 7, 21 or 35 days and samples injected intravenously into haemorrhagic shock mice. Changes in macrophages, inflammatory cytokines and T cell differentiation were assessed using flow cytometry or enzyme-linked immunosorbent assay (ELISA). In a second experiment, haemorrhagic shock mice were injected with 21D-erythrocytes and the expression of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), arginine -1 (Arg-1) and inducible nitrous oxide (iNOS) determined. Results The proportion of M1-polarized macrophages was greatest in the 21D group while M2 macrophages tended to increase with the erythrocyte storage time. Levels of inflammatory cytokines and T helper 1 (Th1) cells increased in proportion to erythrocytes storage time. Most regulatory T cells (Treg) were found at 21D. Arg-1 expression was significantly increased in a group that received an heme oxygenase 1 (HO-1) agonist and significantly decreased in a group that received an HO-1 inhibitor but there were no differences in the expression of iNOS or Nrf2. Conclusion 21D storage time may be an important time point for erythrocyte storage and immunity response and Arg-1 may have a role in the macrophage response to erythrocyte infusion.

Background Impaired wound healing frequently occurs in diabetes mellitus (DM) and is implicated in impaired angiogenesis. Long non-coding RNA (lncRNA) H19 has been reported as being reduced in DM and played a critical role in inducing angiogenesis. Thus, we hypothesized that H19 may affect impaired wound healing in streptozotocin (STZ)-induced diabetic mice transfused with autologous blood preserved in standard preservative fluid or modified preservative fluid. Methods Fibroblasts in injured skin were isolated and cultured in vitro. After location of H19 in fibroblasts using fluorescence in situ hybridization (FISH), RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), Co immunoprecipitation (COIP) and dual luciferase reporter gene assay were used to verify the binding of H19 to HIF-1α. Results The modified preservative fluid preserved autologous blood increased the H19 expression in fibroblasts, and maintained better oxygen-carrying and oxygen release capacities as well as coagulation function. Furthermore, H19 promoted HIF-1α histone H3K4me3 methylation and increased HIF-1α expression by recruiting EZH2. H19 promoted fibroblast activation by activating HIF-1α signaling pathway in fibroblasts and enhanced wound healing in diabetic mice. Conclusions Taken together, H19 accelerated fibroblast activation by recruiting EZH2-mediated histone methylation and modulating the HIF-1α signaling pathway, whereby augmenting the process of modified preservative fluid preserved autologous blood enhancing the postoperative wound healing in diabetic mice.

Lodging is a serious problem in plant growth, especially in crops growth of the natural habitat. In order to determine the influence of lodging on the growth characters of sweet sorghum, plants grown in natural saline-alkali environment were used to investigate the fresh weight, dry weight, sugar content in the stalks and the photosynthesis index of salt tolerant crop sweet sorghum. Results showed that lodging significantly reduced the growth of sweet sorghum, the fresh weight and dry weight was only 28.3% and 22.5% of the normal plants when lodging occurred after 49 days. Lodging also reduced the stalks sugar content of sweet sorghum, the stalk sugar content of lodged plants was only 45.4% of that in the normal plants, when lodging occurred for 49 days. Lodging reduced the growth and sugar content by reducing the photosynthesis parameters of sweet sorghum grown in the saline-alkali field, thus, affected the accumulation of photosynthate. Interestingly, with the extension of the lodging time, lodging led to a decrease in photosynthetic rate of sweet sorghum mainly due to non-stomatal factors.

In our work, we used an in vitro culture model to investigate whether antidepressant imipramine (Ip) may protect bupivacaine (Bv)-induced neurotoxicity in mouse dorsal root ganglion (DRG). Adult mouse DRG was treated with 5 mM Bv in vitro to induce neurotoxicity. DRG was then pre-treated with Ip, prior to Bv, to examine its effects on protecting Bv-induced DRG apoptosis and neurite degeneration. Ip-induced dynamic changes in Trk receptors, including TrkA/B/C and phosphor (p-)TrkA/B/C, were examined by qPCR and Western blot. TrkA and TrkB were inhibited by siRNAs to further investigate their functional role in Ip- and Bv-treated DRG. Ip protected Bv-induced apoptosis and neurite loss in DRG. Ip did not alter TrkA/B/C expressions, whereas significantly augmented protein productions of p-TrkA and p-TrkB, but not p-TrkC. SiRNA-mediated TrkA or TrkB downregulation inhibited Trk receptors, and reduced p-TrkA and p-TrkB in DRG. TrkA or TrkB downregulation alone had no effect on Ip-induced protection in Bv-injured DRG. However, co-inhibition of TrkA and TrkB significantly ameliorated the protective effect of Ip on Bv-induced apoptosis and neurite loss in DRG. Imipramine protected bupivacaine-induced neurotoxicity in DRG, likely via the co-activation of TrkA and TrkB signaling pathways. J. Cell. Biochem. 118: 3960-3967, 2017. © 2017 Wiley Periodicals, Inc.

Acute normovolemic hemodilution (ANH) has been widely used to prevent the massive blood loss during hepatic carcinoma. The influences of ANH on coagulation function are still controversy, especially in elderly patients. The study observed ANH effects on coagulation function and fibrinolysis in elderly patients undergoing the disease.Thirty elderly patients (aged 60-70 yr) with liver cancer (ASA I or II) taken hepatic carcinectomy from February 2007 to February 2008 were randomly divided into ANH group (n=15) and control group (n=15). After tracheal intubation, patients in ANH group and control group were infused with 6% hydroxyethyl starch (130/0.4) and Ringer's solution, respectively. Blood samples were drawn from patients in both groups at five different time points: before anesthesia induction (T1), 30 min after ANH (T2), 1 h after start of operation (T3), immediately after operation (T4), and 24 h after operation (T5). Then coagulation function, soluble fibrin monomer complex (SFMC), prothrombin fragment (F1+2), and platelet membrane glycoprotein (CD62P and activated GP IIb/GP IIIa) were measured.The perioperative blood loss and allogeneic blood transfusion were recorded during the surgery. The perioperative blood loss was not significantly different between two groups (p0.05), but the volume of allogeneic blood transfusion in ANH group was significantly less than in control group (350.0±70.7) mL vs. (457.0±181.3) mL (p0.01). Compared with the data of T1, the prothrombin time (PT) and activated partial thromboplastin time (APTT) measured after T3 were significantly longer (p0.05) in both groups, but within normal range. There were no significant changes of thrombin time (TT) and D-dimer between two groups at different time points (p0.05). SFMC and F1+2 increased in both groups, but were not statistically significant. PAC-1-positive cells and CD62P expressions in patients of ANH group were significantly lower than those at T1 (p0.05) and T2-T5 (p0.05).ANH has no obvious impact on fibrinolysis and coagulation function in elderly patients undergoing resection of liver cancer. The study suggested that ANH is safe to use in elderly patients and it could reduce allogeneic blood transfusion.

This study was carried out to investigate the suitable technique for increasing the stalk sugar content of two sweet sorghum cultivars (salt-tolerant cultivar Jitianza 2 and salt-sensitive cultivar Lvneng 1) in saline soils of Yellow River Delta. Stalk sugar content of sweet sorghum in saline soil was significantly increased using the following integrated cultivation technique: (1) increasing organic fertilizer level; (2) applying optimum phosphorus and potassium fertilizer; (3) surface fertilizer application; (4) cultivating salt-tolerant sweet sorghum cultivars; (5) planting with plastic mulching; (6) planting in the early of the optimum seedling time (sowing as early as possible); (7) removing tillers during seedling stage; (8) cutting spike by half-strength before flowering. Jitianza 2 had higher stalk Brix than Lvneng 1 under the same cultivation condition. Therefore, planting salt-tolerant sweet sorghum cultivars in saline soil combined with these integrated cultivation techniques can get higher stalk sugar content in Yellow River Delta of China, which was beneficial for energy alcohol production from sweet sorghum.

The aim of this study was to investigate the efficiency of intrathecal glial cell line-derived neurotrophic factor (GDNF) on nitric oxide (NO) and nitric oxide synthase (NOS) activity following a spinal nerve ligation (SNL) of male Sprague Dawley rats. The rats were randomly divided into four groups: normal (control), sham-operated, SNL (SNL followed by a physiological saline injection into the subarachnoid space), and GDNF (SNL followed by a GDNF injection into the subarachnoid space). Each group was divided into three subgroups (n = 10). The rats in each subgroup were euthanised 3, 7, and 14 days after the operation. Rat behaviour was evaluated before euthanising, and the ipsilateral spinal cords were harvested after euthanising to determine the NO content and NOS activity. Compared with the control and sham-operated groups, the NO content and NOS activity in the SNL group increased significantly 3 days after the operation; this increase was maintained until 14 days after the operation (P < 0.01 or 0.05). A significant decrease was observed in the NO content and NOS activity in the GDNF group compared with the SNL group. The decrease continued until 14 days after the operation (P < 0.01 or 0.05). The results indicated that the NO and NOS activity in the rat spinal cord are associated with SNL-induced neuropathic pain. The decreased neuropathic pain from the intrathecal GDNF is correlated with the decrease in NO content and NOS activity in the spinal cord. Key words: Glial cell line-derived neurotrophic factor, neuralgia, nitric oxide, nitric oxide synthase.

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS/ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than IGS/ITS-MP-PCR because of their higher redundancy in the fungal genome. The number of fragments generated by both techniques varied according to the fungal species and also with the primer combination used. Each primer used could differentiate all of the fungal isolates examined in this study. The profiles generated were identical and reproducible between repeated PCR experiments. PRACTICAL APPLICATIONS Combining the intergenic spacer/internal transcribed spacer-microsatellite-primed polymerase chain reaction technique with microsatellite–detection assay allows the rapid and specific detection of Rhizoctonia solani anastomosis groups and different phytopathogenic fungi. The utility of this approach stems from its simplicity and reproducibility, the high number of polymorphisms revealed, the very small amounts of DNA needed, rapidity, and ease of performance. The improved technique will present valuable information on the role of some phytopathogenic fungi and R. solani in agriculturally important plant diseases.

Disease severity (% of Fusarium-damaged spikelets), disease incidence (% of Fusarium-damaged wheat heads), Fusarium graminearurn DNA (pg per ng total DNA), deoxynivalenol (DON) content (mg DON per kg grain dry mass) and yield (t per ha) were recorded in a field experiment carried out in Northern Germany. Plants (susceptible cultivar Ritmo) were inoculated at anthesis by overhead application of a E graminearum mycelial suspension during a 6.7 mm precipitation event. Fungicides were applied around the time of wheat anthesis to generate different levels of Fusarium-damage. Infected heads were sampled in two-week-intervals post inoculation. Disease incidence increased almost linearly with time, reaching levels of about 80% at four weeks post inoculation. Disease severity increased in a slightly exponential manner with time, reaching levels of about 60% after four weeks. Six weeks after inoculation, Fusarium head blight symptoms could not be distinguished from blighting of wheat heads due to plant senescence anymore. The ratio of F. graminearum DNA and total DNA increased exponentially with time up to four weeks post inoculation. Relationships between fungal DNA and yield or DON were closer when DNA was extracted two weeks post anthesis in comparison with four weeks post anthesis. Hence, quantification of fungal DNA seems to be advantageous for assessing FHB in wheat at early stages of disease development. In the middle of disease development, disease severity allowed the most precise estimation of yield and DON contents. Towards the end of disease development, the DON content was the most useful variable to describe the intensity of Fusarium-damage, because it could be evaluated in senescent plant material. Yield of infected plants increased from about 6.5 to 10 t ha(-1) upon fungicide (Caramba, Folicur, or Input) application at two days before or two days after anthesis. Effects were smaller when fungicides were applied earlier or later. At equal application time, equal disease severity level, and equal fungal DNA concentration, effects of fungicides on yield were not significantly different (P = 0.734) whereas effects on the DON content were (P < 0.001).Folicur applied around the time of wheat anthesis decreased the DON content of the grain from about 14 mg kg(-1) to 9 mg kg(-1) whereas Caramba and Input decreased the DON content to about 5 mg kg(-1).

A real-time PCR assay was applied to evaluate the effects of two fungicide treatments on Mycosphaerella graminicola leaf blotch in the field and compared with two usual visual methods. The three fungicides Fandango, Input and Prosaro used in the two treatments showed high inhibitions against germ tube growth and conidial germination in vitro, suggesting that they had high curative and protective activities. In the field, the fungicide effect was assessed for two times on 17 May and 14 June, 2004. M. graminicola DNA, disease index and pycnidial number per leaf reduced obviously on most leaf layers after both treatments. According to the real-time PCR data, significant effects were still observed on leaf 3, 4 and 5 even after 25 days of treatment (17 May). And on 14 June, treatment A significantly decreased the DNA amount by 99.42, 87.68, 24.12 and 68.87% on leaf 1, 2, 3 and 4 and treatment B by 98.83, 98.12, 70.79 and 64.15% respectively. In contrast, effects could neither be assessed by disease classification on leaf 5 of treatment B on 17 May and leaf 3 and 4 of treatment A on 14 June and nor by pycnidia-counting method on leaf 3 and 5 of treatment B on 17 May and leaf 4 of both treatments on 14 June. The band intensity of final real-time PCR products could be hardly distinguished on agarose gel amongst those samples with disease index >2.7, although their initial DNA amounts quantified by real-time PCR were different, indicating the superiority of the real-time PCR assay over those PCR assays estimating initial DNA input with endpoint measurement. All this shows that the real-time PCR assay may serve as an alternative method for accurate assessment of the fungicide effects on M. graminicola leaf blotch.