Bacterial heat shock protein: A new crosstalk between T lymphocyte and macrophage via JAK2/STAT1 pathway in bloodstream infection.

Bloodstream infection (BSI) refers to the infection of blood by pathogens. Severe immune response to BSI can lead to sepsis, a systemic infection leading to multiple organ dysfunction, coupled with drug resistance, mortality, and limited clinical treatment options. This work aims to further investigate the new interplay between bacterial exocrine regulatory protein and host immune cells in the context of highly drug-resistant malignant BSI. Whether interfering with related regulatory signaling pathways can reverse the inflammatory disorder of immune cells. In-depth analysis of single-cell sequencing results in Septic patients for potential immunodeficiency factors. Analysis of key proteins enriched by host cells and key pathways using proteomics. Cell models and animal models validate the pathological effects of DnaK on T cells, MAITs, macrophages, and osteoclasts. The blood of patients was analyzed for the immunosuppression of T cells and MAITs. We identified that S. maltophilia-DnaK was enriched in immunodeficient T cells. The activation of the JAK2/STAT1 axis initiated the exhaustion of T cells. Septic patients with Gram-negative bacterial infections exhibited deficiencies in MAITs, which correspond to IFN-γ. Cellular and animal experiments confirmed that DnaK could facilitate MAIT depletion and M1 polarization of macrophages. Additionally, Fludarabine mitigated M1 polarization of blood, liver, and spleen in mice. Interestingly, DnaK also repressed osteoclastogenesis of macrophages stimulated by RANKL. S.maltophilia-DnaK prompts the activation of the JAK2/STAT1 axis in T cells and the M1 polarization of macrophages. Targeting the DnaK's crosstalk can be a potentially effective approach for treating the inflammatory disorder in the broad-spectrum drug-resistant BSI.

The m6A reader YTHDF2 alleviates the inflammatory response by inhibiting IL-6R/JAK2/STAT1 pathway-mediated high-mobility group box-1 release.

Background: Sepsis is a common severe complication in major burn victims and is characterized by a dysregulated systemic response to inflammation. YTH domain family 2 (YTHDF2), a well-studied N6-methyladenosine (m6A) reader that specifically recognizes and binds to m6A-modified transcripts to mediate their degradation, is connected to pathogenic and physiological processes in eukaryotes, but its effect on sepsis is still unknown. We aimed to discover the effects and mechanisms of YTHDF2 in sepsis. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analyses were used to measure the expression of YTHDF2, the interleukin 6 receptor (IL-6R), high-mobility group box-1 (HMGB1), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1) under different in vitro conditions. Enzyme-linked immunosorbent assays were utilized to evaluate the expression of HMGB1, IL-6, IL-1ß and tumor necrosis factor-α. To confirm that YTHDF2 specifically targets IL-6R mRNA, RNA immunoprecipitation and dual-luciferase reporter assays were performed. Finally, we utilized a mouse model of lipopolysaccharide (LPS)-induced sepsis to verify the effects of YTHDF2 in vivo. Results: According to our findings, YTHDF2 was expressed at a low level in peripheral blood mononuclear cells from septic mice and patients as well as in LPS-induced RAW264.7 cells. Overexpression of YTHDF2 alleviated the inflammatory response by inhibiting HMGB1 release and JAK2/STAT1 signalling in LPS-stimulated cells. Mechanistically, YTHDF2 suppressed JAK2/STAT1 signalling by directly recognizing the m6A-modified site in IL-6R and decreasing the stability of IL-6R mRNA, thereby inhibiting HMGB1 release. In vivo experiments showed that YTHDF2 played a protective role in septic mice by suppressing the IL-6R/JAK2/STAT1/HMGB1 axis. Conclusions: In summary, these findings demonstrate that YTHDF2 plays an essential role as an inhibitor of inflammation to reduce the release of HMGB1 by inhibiting the IL-6R/JAK2/STAT1 pathway, indicating that YTHDF2 is a novel target for therapeutic interventions in sepsis.

LncRNA GAS5 suppresses inflammatory responses by inhibiting HMGB1 release via miR-155-5p/SIRT1 axis in sepsis.

Sepsis comprises a lethal immunologic response due to infection. Increasingly, evidence has demonstrated the important role of long non-coding RNA growth arrest-specific transcript 5 (GAS5) in the regulation of sepsis. Nevertheless, the mechanisms by which GAS5 participates in the progression of sepsis remain unclear. Our study demonstrated the role and underlying mechanism of GAS5 in regulating lipopolysaccharide (LPS)-induced inflammation. In this study, GAS5 expression was found to be markedly decreased in serum samples of sepsis patients and a sepsis mouse model, and was negatively related with HMGB1 expression. GAS5 overexpression inhibited cell inflammatory responses by decreasing HMGB1 release. Furthermore, GAS5 inhibited LPS-mediated hyperacetylation and the release of HMGB1 by increasing the expression of sirtuin1 (SIRT1). Additionally, upregulated GAS5 attenuated inflammatory responses in vitro and vivo, and the knockdown of a miR-155-5p mimic and SIRT1 rescued the effects of GAS5 upregulation. Mechanistically, GAS5 sponged miR-155-5p to upregulate SIRT1, thereby inhibiting HMGB1 acetylation and release. In conclusion, our findings indicate that GAS5 suppresses inflammatory responses by modulating the miR-155-5p/SIRT1/HMGB1 axis in sepsis, providing a novel therapeutic target for inflammation in sepsis.

Late-Onset Acute Kidney Injury is a Poor Prognostic Sign for Severe Burn Patients.

Background: Acute kidney injury (AKI) is a morbid complication and the main cause of multiple organ failure and death in severely burned patients. The objective of this study was to explore epidemiology, risk factors, and outcomes of AKI for severely burned patients. Methods: This retrospective study was performed with prospectively collected data of severely burned patients from the Institute of Burn Research in Southwest Hospital during 2011-2017. AKI was diagnosed according to Kidney Disease Improving Global Outcomes (KDIGO) criteria (2012), and it was divided into early and late AKIs depending on its onset time (within the first 3 days or >3 days post burn). The baseline characteristics, clinical data, and outcomes of the three groups (early AKI, late AKI and non-AKI) were compared using logistic regression analysis. Mortality predictors of patients with AKI were assessed. Results: A total of 637 adult patients were included in analysis. The incidence of AKI was 36.9% (early AKI 29.4%, late AKI 10.0%). Multiple logistic regression analysis revealed that age, gender, total burn surface area (TBSA), full-thickness burns of TBSA, chronic comorbidities (hypertension or/and diabetes), hypovolemic shock of early burn, and tracheotomy were independent risk factors for both early and late AKIs. However, sepsis was only an independent risk factor for late AKI. Decompression escharotomy was a protective factor for both AKIs. The mortality of patients with AKI was 32.3% (early AKI 25.7%, late AKI 56.3%), and that of patients without AKI was 2.5%. AKI was independently associated with obviously increased mortality of severely burned patients [early AKI, OR = 12.98 (6.08-27.72); late AKI, OR = 34.02 (15.69-73.75)]. Compared with patients with early AKI, patients with late AKI had higher 28-day mortality (34.9% vs. 19.4%, p = 0.007), 90-day mortality (57.1% vs. 27.4%, p < 0.0001). Conclusions: AKI remains prevalent and is associated with high mortality in severely burned patients. Late-onset acute kidney injury had greater severity and worse prognosis.

Nucleation and growth mechanisms of an electrodeposited Ni-Se-Cu coating on nickel foam.

Electrocatalysts for water splitting have been widely explored among recent years. In this study, nickel-selenium-copper (Ni-Se-Cu) coating was synthesized on nickel foam through potentiostatic electrodeposition. The electrochemical kinetics and nucleation mechanisms of the deposition were investigated, and the diffusion coefficient D from different deposition potentials and temperatures was calculated. Results reveal that the electrodeposition of Ni-Se-Cu follows an instantaneous nucleation and diffusion-controlled three-dimensional (3D) growth mechanism. Deposition potential and bath temperature slightly effect the nucleation mechanism of electrodeposition. The apparent activation energy Ea of the hydrogen evolution reaction (HER) in 1.0 M KOH electrolyte of Ni-Se-Cu is 21.1 kJ·mol-1, which is lower than that of Ni-Se (37.7 kJ·mol-1). The majority phase formed by nickel and selenium is Ni3Se2, and a Ni(Cu) solid solution forms after the incorporation of Cu atoms into a Ni lattice.

Lytic Bacteriophage Screening Strategies for Multidrug-Resistant Bloodstream Infections in a Burn Intensive Care Unit.

BACKGROUND Increasing antibiotic resistance and multidrug resistance (MDR) in patients with bloodstream infection (BSI) has resulted in treatment using bacteriophage. This study aimed to identify Gram-negative bacilli and Gram-positive cocci and antibiotic resistance in patients with BSI in a burn intensive care unit (BICU). The environment, including sewage systems, were investigated for the presence of lytic bacteriophage. MATERIAL AND METHODS Between January 2011 to December 2017, 486 patients with BSI were admitted to the BICU. Blood culture identified the main infectious organisms. Bacterial screening tests for antibiotic resistance included the D test and the modified Hodge test (MHT). Lytic bacteriophage was isolated from the environment. RESULTS In 486 patients with BSI, the main causative organisms were Gram-negative bacilli (64.6%), Gram-positive cocci (27.7%), and fungi (7.7%). The main pathogenic organisms that showed multidrug resistance (MDR) were Acinetobacter baumannii (26.0%), Staphylococcus aureus (16.8%), and Pseudomonas aeruginosa (14.2%). Bacteriophage was mainly isolated from Gram-negative bacilli. Screening of hospital and residential sewage systems identified increased levels of bacteriophage in hospital sewage. CONCLUSIONS The causative organisms of BSI and the presence of MDR in a hospital BICU were not typical, which supports the need for routine bacterial monitoring. Hospital sewage provides a potential source of bacteriophage for the treatment of MDR pathogenic bacteria.

High-Mobility Group Box 1 (HMGB1) Induces Migration of Endothelial Progenitor Cell via Receptor for Advanced Glycation End-Products (RAGE)-Dependent PI3K/Akt/eNOS Signaling Pathway.

BACKGROUND High-mobility group box1 (HMGB1) is a cytokine that has been demonstrated to have an important role in inducing migration and homing of endothelial progenitor cells (EPCs) in the process of neovascularization during wound healing, but its specific mechanism remains elusive. The aim of this study was to investigate the effects of the HMGB-RAGE axis in EPC migration, as well as the underlying molecular mechanism responsible for these effects. MATERIAL AND METHODS EPCs were isolated from the mice and identified using flow cytometry and fluorescence staining. The effect of HMGB1 on the activity of EPCs was detected using the Cell Counting Kit-8 (CCK-8). Then, the migration of EPCs was detected by scratch wound-healing and cell migration assay. NO levels were analyzed by ELISA. The expression of p-PI3K, p-Akt, and p-eNOS was determined by Western blot analysis. RAGE expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. F-actin was assessed by fluorescent staining. RESULTS The results showed that HMGB1 induced a concentration-dependent migration of EPCs, and the migration was RAGE-dependent. The migration could be almost completely blocked by PI3K inhibitors and eNOS inhibitor. HMGB1-RAGE upregulated the expression of p-Akt, p-eNOS, and p-ERK. We also demonstrated that the MEK/ERK signaling pathway is not involved in the EPC migration induced by HMGB1-RAGE. CONCLUSIONS These data demonstrate that HMGB1 activates RAGE and induces PI3K/Akt/eNOS signaling transduction pathway activation to promote EPC migration. Therefore, the HMGB1-RAGE axis plays an important role in the EPC migration process and may become a potential target in wound healing.

The Interaction of N-Acetylcysteine and Serum Transferrin Promotes Bacterial Biofilm Formation.

BACKGROUND/AIMS: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. METHODS: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. RESULTS: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. CONCLUSIONS: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Astragaloside-IV Protects Against Heat-induced Apoptosis by Inhibiting Excessive Activation of Mitochondrial Ca2+ Uniporter.

BACKGROUND: Heat causes airway damage during inhalation injury because of bronchial epithelial cell damage. Accumulating evidence shows that mitochondrial uniporter (MCU) is involved in cell damage. We investigated the MCU activity after heat treatment and assessed whether Astragaloside-IV (AS-IV) suppresses heat-induced apoptosis in bronchial epithelial cells by inhibiting the activation of the mitochondrial Ca2+ uniporter (MCU), mitochondrial depolarisation and reactive oxygen species (ROS) production. METHODS: The bronchial epithelial cell line 16HBE14o- was heat treated, and cell apoptosis was induced in vitro and in vivo. AS-IV was inorganically administered to Wistar rats twice a day after thermal inhalation injury, and 16HBE140- cells were treated with AS-IV after incubation at 47°C for 5 min. Protein expression was determined using Western blotting and commercial kits, apoptosis with TUNEL staining, mitochondrial channel activity by patch clamp, reactive oxygen species by MitoSOXTM fluorescence, ATP levels and enzyme activities by commercial kits as well as mitochondrial respiration and calcium by fluorescence. RESULTS: AS-IV markedly inhibited heat-induced apoptosis, as indicated by the increased expression of the pro-apoptotic genes Bak, Bik and Bmf and increased expression of the apoptosis markers Bax, cleaved parp, cleaved caspase3 and cytochrome C. We found that MCU activation promoted mitochondrial Ca2+ overload, ATP depletion, mitochondrial ROS production and cytochrome c release and rapidly induced apoptosis. However, AS-IV treatment reduced excessive MCU activation and led to resistance against mitochondrial Ca2+ overload and excessive cytochrome C release; these effects were blocked by the MCU activator spermine. AS-IV treatment elevated ATP production and decreased ROS activity. CONCLUSIONS: MCU plays crucial roles in heat-induced mitochondrial apoptosis in 16HBE140- cells, suggesting a potential target for AS-IV treatment.

Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

BACKGROUND/AIMS: Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. METHODS & RESULTS: We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. CONCLUSION: Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection.

Mitochondrial Ca2+ Uniporter Is a Mitochondrial Luminal Redox Sensor that Augments MCU Channel Activity.

Ca2+ dynamics and oxidative signaling are fundamental mechanisms for mitochondrial bioenergetics and cell function. The MCU complex is the major pathway by which these signals are integrated in mitochondria. Whether and how these coactive elements interact with MCU have not been established. As an approach toward understanding the regulation of MCU channel by oxidative milieu, we adapted inflammatory and hypoxia models. We identified the conserved cysteine 97 (Cys-97) to be the only reactive thiol in human MCU that undergoes S-glutathionylation. Furthermore, biochemical, structural, and superresolution imaging analysis revealed that MCU oxidation promotes MCU higher order oligomer formation. Both oxidation and mutation of MCU Cys-97 exhibited persistent MCU channel activity with higher [Ca2+]m uptake rate, elevated mROS, and enhanced [Ca2+]m overload-induced cell death. In contrast, these effects were largely independent of MCU interaction with its regulators. These findings reveal a distinct functional role for Cys-97 in ROS sensing and regulation of MCU activity.

Epidermal CFTR Suppresses MAPK/NF-κB to Promote Cutaneous Wound Healing.

BACKGROUND: CFTR is implicated in cutaneous wound healing although the underlying mechanisms are not fully understood. In other cell types, CFTR is reported to regulate MAPK/ NF-κB signaling. We undertook the present study to explore a possible role of CFTR in regulating MAPK/NF-κB during cutaneous wound healing. Methods& Results: The splint-excisional and incisional wound healing models were used in CFTR mutant (DF508) mice. The cell-scratch model was used in a human keratinocyte line, HaCaT, in conjunction with CFTR knockdown or overexpression. The epidermal inflammation, keratinocyte proliferation and differentiation, as well as MAPK/NF-κB signaling were examined. Inhibitors of MAPK/NF-κB were also used. RESULTS: Both DF508 mice and HaCaT cells with CFTR knockdown exhibited delayed cutaneous wound healing with exuberant inflammation, increased proliferation and aberrant differentiation. Knockdown of CFTR in HaCaT cells resulted in phosphorylation of ERK, p38 and IκBα. The disturbance of inflammation, proliferation and differentiation in HaCaT cells were reversed by CFTR overexpression or inhibition of MAPK or NF-κB. CONCLUSION: CFTR plays a role in suppressing MAPK/NF-κB to relieve inflammation, reduce proliferation and promote differentiation of keratinocytes, and thus promotes cutaneous wound healing.

Subtilisin-like protease-1 secreted through type IV secretion system contributes to high virulence of Streptococcus suis 2.

Streptococcus suis serotype 2 is an emerging zoonotic pathogen that triggered two outbreaks of streptococcal toxic shock syndrome (STSS) in China. Our previous research demonstrated that a type IV secretion system (T4SS) harbored in the 89K pathogenicity island contributes to the pathogenicity of S. suis 2. In the present study, a shotgun proteomics approach was employed to identify the effectors secreted by T4SS in S. suis 2, and surface-associated subtilisin-like protease-1 (SspA-1) was identified as a potential virulence effector. Western blot analysis and pull-down assay revealed that SspA-1 secretion depends on T4SS. Knockout mutations affecting sspA-1 attenuated S. suis 2 and impaired the pathogen's ability to trigger inflammatory response in mice. And purified SspA-1 induced the secretion of IL-6, TNF-α, and IL-12p70 in THP-1 cells directly. SspA-1 is the first T4SS virulence effector reported in Gram-positive bacteria. Overall, these findings allow us to gain further insights into the pathogenesis of T4SS and STSS.

[Effects of estrogen on epidermis growth of mice and proliferation of human epidermal cell line HaCaT and its mechanism].

OBJECTIVE: To observe the effects of estrogen on epidermis growth of mice and proliferation of keratinocytes (human epidermal cell line HaCaT), and to explore its mechanism. METHODS: (1) Five adult C57BL/6 mice in estrus cycle were identified by vaginal exfoliative cytology diagnosis and set as estrus group, while another 5 adult C57BL/6 mice with ovary resected before sexual development were set as ovariectomized group. The full-thickness skin from the tail root of mice in two groups were collected. The thickness of epidermis was observed and measured after HE staining. The distribution of proliferating cell nuclear antigen (PCNA)-positive cells in epidermis was observed by immunohistochemical staining, the number of which was counted. (2) HaCaT cells in logarithmic growth phase were cultured with RPMI 1640 nutrient solution containing 10% fetal bovine serum, and they were divided into negative control group (NC), pure estradiol group (PE), protein kinase B (Akt) inhibitor group (AI), and extracellular signal-regulated kinase (ERK) inhibitor group (EI) according to the random number table, with 20 wells in each group. To nutrient solution of each group, 1 µL dimethyl sulfoxide, 1 µL 17ß-estradiol (100 nmol/L), 1 µL LY294002 (10 µmol/L), and 1 µL PD98059 (30 µmol/L) were added in group NC, group PE, group AI, and group EI respectively, and the last two groups were added with 1 µL 17ß-estradiol (100 nmol/L) in addition. At post culture hour (PCH) 0 (immediately after culture), 24, 48, 72, 5 wells of cells from each group were collected to detect the proliferation activity of cells by cell counting kit 8 and microplate reader. (3) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 wells in each group. At PCH 72, cell cycle distribution was detected by flow cytometer to calculate proliferation index (PI) of cells. (4) HaCaT cells in logarithmic growth phase were collected, grouped, and treated with the above-mentioned methods, with 3 dishes in each group. At PCH 72, the protein levels of phosphorylated Akt (p-Akt), phosphorylated ERK (p-ERK), and PCNA were determined with Western blotting. The cell experiments were repeated for 3 times. Data were processed with t test, one-way analysis of variance, analysis of variance of factorial design, and LSD test. RESULTS: (1) The epidermis thickness of mice in ovariectomized group was (33.5±3.0) µm, which was obviously thinner than that in estrus group [(51.4±3.1) µm, t=20.7, P<0.01]. The PCNA-positive cells mainly aggregated in the basal layer of epidermis of mice in two groups. The number of PCNA-positive cells in epidermis of mice in ovariectomized group was 37±12 per 200 fold visual field, obviously fewer than that in estrus group (96±15 per 200 fold visual field, t=15.3, P<0.01). (2) During PCH 0 to 48, there were no significant differences in the proliferation activity of cells between group PE and group NC (with P values above 0.05). At PCH 72, compared with that in group NC, the proliferation activity of cells in group PE was obviously increased (P<0.01). The proliferation activity of cells in groups AI and EI was obviously lower than that in the previous two groups (with P values below 0.01). (3) Compared with that in group NC [(51.6±1.1)%], the PI of cells in group PE was obviously increased [(58.5±0.8)%, P<0.05]. The PI values of cells in groups AI and EI were (34.9±0.8)% and (48.2±0.4)% respectively, both obviously lower than those in the previous two groups (with P values below 0.01). (4) Compared with that of group NC (0.566±0.034), the protein level of p-Akt in cells of group PE was significantly increased (1.048±0.077, P<0.01). Compared with that of group PE, the protein level of p-Akt was obviously decreased in cells of groups AI and EI (respectively 0.682±0.095 and 0.672±0.019, with P values below 0.01). Compared with that of group NC (0.469±0.013), the protein level of p-ERK obviously increased in cells of groups PE, AI, and EI (respectively 1.064±0.089, 1.010±0.038, 0.778±0.065, with P values below 0.01). The protein level of p-ERK in cells of group EI was obviously lower than that in group PE (P<0.01). Compared with that of group NC (0.386±0.053), the protein level of PCNA was obviously increased in cells of group PE (0.743±0.043, P<0.01). The protein levels of PCNA in cells of groups AI and EI were 0.264±0.019 and 0.223±0.065 respectively, both obviously lower than those in the previous two groups (with P values below 0.01). CONCLUSIONS: Lack of estrogen damages the growth ability of epidermis of mice. Estrogen (17ß-estradiol) can promote the proliferation of HaCaT cells by increasing the expression of PCNA via activating ERK/Akt signaling pathway.

Estrogen Accelerates Cutaneous Wound Healing by Promoting Proliferation of Epidermal Keratinocytes via Erk/Akt Signaling Pathway.

BACKGROUND: Previous studies have established that estrogen is capable of accelerating cutaneous wound healing through multiple mechanisms, one of which involves affecting keratinocytes biological properties, such as migration, proliferation, etc. This study aims to reveal the underlying molecular mechanisms of estrogen promoting epidermal keratinocytes proliferation. Method & RESULTS: We found that compared with female mice with a normal estrous cycle, female mice with their ovaries removed before puberty exhibited a delayed cutaneous wound healing, thinner epidermis, and significantly fewer proliferating cell nuclear antigen (PCNA)-positive keratinocytes. Moreover, a significant increase in HaCaT proliferation was detected by a CCK8 assay when treated with 17 ß-estradiol compared with those treated with control vehicle. Consistent with the results of the CCK8 assay, flow cytometry indicated a high proportion of 17 ß-estradiol-treated HaCaT cells in S phase compared with vehicle-treated cells. Western blot analysis demonstrated the activation of Akt, Erk and upregulation of PCNA in HaCaT cells treated with 17 ß-estradiol. Interestingly, Erk activation occurred prior to Akt activation. Upregulation of PCNA expression, elevated proliferation and high S phase fraction of HaCaT cell by 17 ß-estradiol could be reversed by an Akt or Erk inhibitor. Moreover, Erk inhibition reversed 17 ß-estradiol-induced Akt activation, whereas an Akt inhibitor exhibited no effect on Erk, further suggesting that Erk was on the upstream while Akt on the downstream of the signaling pathway. CONCLUSION: This study demonstrates that one of the critical mechanisms underlying 17 ß-estradiol promoting skin wound healing is through regulation of keratinocyte proliferation via Erk/Akt signaling pathway.

PPARγ inhibits HMGB1 expression through upregulation of miR-142-3p in vitro and in vivo.

Peroxisome proliferator-activated receptor gamma (PPARγ) belongs to the nuclear receptor superfamily and it has received much attention because of its anti-inflammatory activity. However, the underlying molecular mechanism is not completely understood. In the present study, we demonstrated that the level of PPARγ is inversely correlated with that of high mobility group box 1 (HMGB1, a late proinflammatory mediator) in patients with sepsis. Activation of PPARγ inhibits the basal and LPS-induced expression of HMGB1. The PPARγ-mediated inhibition of HMGB1 is associated with the upregulation of miR-142-3p, which can target the 3'-UTR of HMGB1, by directly binding to the PPRE in the miR-142-3p promoter region. Functional experiments reveal that the PPARγ-induced miR-142-3p suppresses inflammatory response in vivo. These results suggest that PPARγ-mediated upregulation of miR-142-3p inhibits the HMGB1 expression, which, in turn, is a novel anti-inflammatory mechanism of PPARγ and has an important role in the treatment of inflammatory diseases.

[Effects of blood purification in the treatment of patients with burn sepsis].

OBJECTIVE: To observe the effects of blood purification in the treatment of burn sepsis, in order to provide evidence for its application. METHODS: Twenty-seven patients with burn sepsis admitted to our burn ward from June 2012 to December 2013, conforming to the study criteria, were divided into conventional treatment group (CT, n = 15) and blood purification group (BP, n = 12) according to the random number table. After the diagnosis of sepsis was confirmed, patients in group CT received CT, while patients in group BP received both CT and continuous veno-venous hemodiafiltration for 48 hours. At the time of diagnosis of sepsis (before treatment) and post treatment hour (PTH) 24 and 48, levels of blood lactate and PaO2 were analyzed with blood gas analyzer, and the oxygenation index (OI) was calculated; blood sodium, blood glucose, blood urea nitrogen (BUN), creatinine, white blood cell count (WBC), procalcitonin (PCT) were determined; acute physiology and chronic health evaluation (APACHE) II score was estimated basing on the body temperature, respiratory rate, mean arterial pressure, PaO2, and blood pH values. The levels of TNF-α, IL-8, and IL-6 in serum were determined by ELISA. Data were processed with Fisher's exact test, t test, analysis of variance for repeated measurement, LSD- t test, and LSD test. RESULTS: (1) The levels of blood lactate of patients in group BP were significantly lower than those of group CT at PTH 24 and 48 (with t values respectively 1.62 and 2.44, P values below 0.05). Compared with that detected before treatment, the level of blood lactate in group BP was significantly decreased at PTH 48 (P < 0.05). The OI values of patients in group BP at PTH 24 and 48 [(247 ± 30), (288 ± 41) mmHg, 1 mmHg = 0.133 kPa] were significantly higher than those of group CT [(211 ± 32), (212 ± 30) mmHg, with t values respectively 3.02 and 5.63, P values below 0.01]. Compared with that detected before treatment, the OI values of patients in group BP at PTH 24 and 48 were significantly higher (P values below 0.01). (2) Compared with those of group CT at PTH 24 and 48, the levels of blood sodium, BUN, and creatinine were significantly lower (with t values from 1.74 to 6.75, P < 0.05 or P < 0.01), while the level of blood glucose was approximately the same (with t values respectively -0.92, -0.38, P values above 0.05) in group BP. Compared with those detected before treatment, the levels of blood sodium, BUN, and creatinine of group BP at PTH 24 and 48 were significantly lower (P < 0.05 or P < 0.01). (3) The levels of WBC and PCT of patients in group BP at PTH 24 and 48 were significantly lower than those of group CT (with t values from 2.11 to 6.63, P < 0.05 or P < 0.01). Compared with those detected before treatment, the levels of WBC and PCT of patients in group BP at PTH 24 and 48 were significantly lower (P < 0.05 or P < 0.01). (4) The APACHE II scores of patients in group BP at PTH 24 and 48 [(18.7 ± 2.6) and (16.7 ± 3.0) scores] were significantly lower than those of group CT [(23.1 ± 1.6) and (25.5 ± 1.6) scores, with t values respectively 5.44 and 9.87, P values below 0.01]. Compared with those calculated before treatment, the APACHE II scores of patients in group CT were significantly increased (P < 0.05 or P < 0.01), while those in group BP were decreased at PTH 24 and 48 (P < 0.05 or P < 0.01). (5) The levels of TNF-α, IL-6, and IL-8 in serum of patients in group BP at PTH 24 and 48 were significantly lower than those of group CT (with t values from 6.12 to 19.78, P values below 0.01). Compared with those detected before treatment, the levels of TNF-α, IL-6, and IL-8 in serum of group BP at PTH 24 and 48 were significantly decreased (with P values below 0.01). CONCLUSIONS: BP+CT is effective in improving organ function, correcting the disorder of internal environment, and controlling inflammation. Therefore, BP is an important method in the treatment of burn sepsis.

Blunted cardiac beta-adrenergic response as an early indication of cardiac dysfunction in Duchenne muscular dystrophy.

AIMS: To determine whether altered beta-adrenergic responses contribute to early cardiac dysfunction in mdx (X-linked muscular dystrophy) mice, an animal model for human Duchenne muscular dystrophy. METHODS AND RESULTS: Replacement fibrosis in mdx hearts gradually increased with age, suggesting a gradual loss of cardiomyocytes. Echocardiography and intra-left ventricular haemodynamic measurements detected baseline cardiac dysfunction in mdx mice at ≥8 months. However, a reduction of cardiac beta-adrenergic response to isoproterenol (ISO) was already present in mdx mice at 4 months. Ventricular myocytes (VMs) isolated from 4- and 8-month-old mdx mice had greater baseline contractile function {fractional shortening, [Ca(2+)]i, and sarcoplasmic reticulum (SR) Ca(2+) content} and ICa-L than age-matched control VMs and than myocytes isolated from 2-month-old mdx mice. ISO increased myocyte function in the VMs of 4- and 8-month-old mdx mice to the same level as in age-matched control VMs. In the VMs of 12-month-old mdx mice, ISO failed to increase myocyte function to the level in VMs of 12-month-old control mice and could not further increaseICa-L. No differences were observed in the expression of Cav1.2α1c, Cav1.2ß1, Cav1.2ß2, sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), and the Na(+)/Ca(2+) exchanger. In contrast, total ryanodine receptor 2 (RyR2) and basal phosphorylation of RyR2, phospholamban, and Cav1.2α1c were found to be increased in hearts of 4-month-old mdx mice; baseline protein kinase A activity was also increased. After ISO treatment, phosphorylation levels were the same in mdx and control hearts. VMs of 4-month-old mdx mice had reduced beta1-adrenergic receptor (ß1-AR) density and beta-adrenergic sensitivity. CONCLUSION: In young mdx mice, the myocyte increases its contractile function to compensate for myocyte loss. However, these myocytes with enhanced baseline function have reduced potential for stimulation, decreased ß1-AR density/sensitivity, leading to blunted cardiac beta-adrenergic response.

Recombinant adenovirus-mediated intestinal trefoil factor gene therapy for burn-induced intestinal mucosal injury.

Intestinal trefoil factor (ITF) is a small polypeptide with potential medical values whose main pharmacological effects are to alleviate gastrointestinal mucosal injury caused by various injury factors and promote the repair of damaged mucosa. However, its low yield limits its application. The purpose of our study was to construct a recombinant adenoviral vector containing the hITF gene and observe the therapeutic effect of burn-induced intestinal mucosal injury using in vitro and in vivo analysis. First, a recombinant shuttle plasmid was constructed by ligating a pAdTrack-CMV vector with a full-length hITF gene containing a signal peptide and the mature peptide, followed by the recombinant Ad-hITF adenovirus vector after linearization and homologous recombination with the backbone plasmid in the competent BJ5183 strain. Second, the hITF expression level was detected using reverse transcription polymerase chain reaction and western blotting after Ad-hITF infection of colon cancer HT-29 cells. The recombinant adenovirus significantly promoted cell migration in an in vitro wounding model. Finally, we confirmed that the recombinant adenovirus could significantly expedite the healing of intestinal mucosal injury after establishing a mouse model in which severe burns were stimulated and the recombinant adenovirus was delivered by intragastric injection. In summary, we constructed a recombinant adenoviral vector containing the hITF gene and confirmed its role in promoting repair of the intestinal mucosa. Our study provides a novel way to treat burn-induced intestinal mucosal injury.