METTL16 inhibits papillary thyroid cancer tumorigenicity through m6A/YTHDC2/SCD1-regulated lipid metabolism.

Papillary thyroid carcinoma (PTC) stands as the leading cancer type among endocrine malignancies, and there exists a strong correlation between thyroid cancer and obesity. However, the clinical significance and molecular mechanism of lipid metabolism in the development of PTC remain unclear. In this study, it was demonstrated that the downregulation of METTL16 enhanced lipid metabolism and promoted the malignant progression of PTC. METTL16 was expressed at lower levels in PTC tissues because of DNMT1-mediated hypermethylation of its promoter. Loss- and gain-of-function studies clarified the effects of METTL16 on PTC progression. METTL16 overexpression increased the abundance of m6A in SCD1 cells, increasing RNA decay via the m6A reader YTHDC2. The SCD1 inhibitor A939572 inhibited growth and slowed down lipid metabolism in PTC cells. These results confirm the crucial role of METTL16 in restraining PTC progression through SCD1-activated lipid metabolism in cooperation with YTHDC2. This suggests that the combination of METTL16 and anti-SCD1 blockade might constitute an effective therapy for PTC.

Analysis of sleep for the American population: Result from NHANES database.

OBJECTIVES: To assess the contemporary prevalence and decade-long trends of sleep duration, sleep disorders and trouble sleeping among adults in the United States, as well as their risk factors, from 2005 to 2018. MATERIALS AND METHODS: We used National Health and Nutrition Examination Survey data to calculate the sleep duration and weighted prevalence of sleep disorders and trouble sleeping in adults aged 20 years or older. Sleep duration, sleep disorders and trouble sleeping were assessed by questionnaire. RESULTS: A total of 27,399 people were included in the survey on sleep duration, with a weighted percentage of normal sleep (7-8 h/night) of 56.33 % (95 % CI, 53.06-59.60 %) and a weighted percentage of short sleep (5-6 h/night) of 31.73 %. In stratified descriptions, participants aged 40-49 years were more likely to sleep less than five hours, while women aged 80 years and older were more likely to sleep longer and blacks were more likely to sleep shorter. A total of 27,406 participants were included in the survey for sleep disorders. The weighted proportion of the population with sleep disorders was 8.44 % (95 % CI, 7.79-9.8 %). Independent risk factors for sleep disorders were being 40-69 years old, being white, having a high education level, smoking, having hypertension, diabetes, heart disease, and BMI ≥ 25. From 2005 to 2014, the prevalence of sleep disorders increased year by year, from 7.44 % in 2005-2006 to 10.40 % in 2013-2014 (P for Trend<0.001). A total of 38,165 participants were included in the survey on trouble sleeping. The weighted proportion of the population with troubled sleeping was 27.30 % (25.70-28.90 %). Independent risk factors for troubled sleeping were being 30-79 years old, being white, having a high education level, smoking, drinking, having hypertension, diabetes, heart disease and BMI ≥ 25. From 2005 to 2018, the prevalence of trouble sleeping increased annually, from 24.44 % in 2005-2006 to 30.58 % in 2017-2018 (P for trend<0.001). CONCLUSION: Adults in the United States are likely to have abnormal sleep durations, and the prevalence of sleep disorders and troubled sleeping is on the rise.

A Mammalian Conserved Circular RNA CircLARP1B Regulates Hepatocellular Carcinoma Metastasis and Lipid Metabolism.

Circular RNAs (circRNAs) have emerged as crucial regulators in physiology and human diseases. However, evolutionarily conserved circRNAs with potent functions in cancers are rarely reported. In this study, a mammalian conserved circRNA circLARP1B is identified to play critical roles in hepatocellular carcinoma (HCC). Patients with high circLARP1B levels have advanced prognostic stage and poor overall survival. CircLARP1B facilitates cellular metastatic properties and lipid accumulation through promoting fatty acid synthesis in HCC. CircLARP1B deficient mice exhibit reduced metastasis and less lipid accumulation in an induced HCC model. Multiple lines of evidence demonstrate that circLARP1B binds to heterogeneous nuclear ribonucleoprotein D (HNRNPD) in the cytoplasm, and thus affects the binding of HNRNPD to sensitive transcripts including liver kinase B1 (LKB1) mRNA. This regulation causes decreased LKB1 mRNA stability and lower LKB1 protein levels. Antisense oligodeoxynucleotide complementary to theHNRNPD binding sites in circLARP1B increases the HNRNPD binding to LKB1 mRNA. Through the HNRNPD-LKB1-AMPK pathway, circLARP1B promotes HCC metastasis and lipid accumulation. Results from AAV8-mediated hepatocyte-directed knockdown of circLARP1B or Lkb1 in mouse models also demonstrate critical roles of hepatocytic circLARP1B regulatory pathway in HCC metastasis and lipid accumulation, and indicate that circLARP1B may be potential target of HCC treatment.

Regulation of the stem­like properties of estrogen receptor­positive breast cancer cells through NR2E3/NR2C2 signaling.

Cancer stem cells (CSCs) are major drivers of metastasis, drug resistance and recurrence in numerous cancers. However, critical factors that can modulate CSC stemness have not been clearly identified. Nuclear receptor subfamily 2 group E member 3 (nr2e3) expression has been previously reported to be positively associated with drug sensitivity and favorable clinical outcomes in patients with estrogen receptor (ER)+ breast cancer. This suggests that nr2e3 expression may be inversely associated with CSC stemness in this type of tumor cells. The present study aimed to investigate the regulatory roles of NR2E3 in the stem-like properties of ER+ breast cancer cells and to identify the underlying mechanisms. Bioinformatics analysis was performed using the data derived from the Cancer Genome Atlas database. Nr2e3-specific shRNA and nuclear receptor subfamily 2 group C member 2 (nr2c2) overexpressed plasmids were constructed to silence and enhance the expression of nr2e3 and nr2c2, respectively. Transwell and wound healing experiments were conducted to evaluate the migration and invasion ability of MCF7 cells, while colony formation tests were used to evaluate the clonality. Flow cytometry was used to detect the percentage of CD44+CD24-/low cells. Reverse transcription-quantitative PCR and western blotting were performed to detect expression at the mRNA and protein levels. The results showed that compared with normal breast tissues and MCF10A cells, the expression of nr2e3 was increased in ER+ breast tumor tissues and cell lines. Nr2e3 silencing promoted the migration, invasion and colony-forming ability of the ER+ MCF7 cells. It also increased the expression of epithelial-mesenchymal transition markers and stem cell-related transcription factors, in addition to the percentage of CD44+CD24-/low cells. The expression of nr2e3 and nr2c2 was found to be positively correlated. Nr2e3 knockdown decreased the mRNA and protein expression levels of nr2c2, whereas nr2c2 overexpression reversed the elevated CD44+CD24-/low cell ratio and the increased migratory activity caused by nr2e3 silencing. The results of the present study suggest that NR2E3 may serve an important role in modulating the stem-like properties of ER+ breast cancer cells, where NR2E3/NR2C2 signaling may be a therapeutic target in ER+ breast cancer.

Identification of microRNA hsa-miR-30c-5p as an inhibitory factor in the progression of hepatocellular carcinoma and investigation of its regulatory network via comprehensive analysis.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high morbidity and mortality. An increasing number of abnormal gene expressions were identified to be associated with the progression of HCC. Previous studies showed that the hsa-miR-30 c-5p (miR-30 c), one of the miR-30 family members, might play a role in suppressing tumor progression in a variety of tumors. The present study aims to examine miR-30 c effects in the development of HCC. The role of miR-30 c in HCC was comprehensively investigated by using bioinformatics and experiments in vitro. The multiple databases were combined to predict and screen the target genes and upstream lncRNAs of miR-30 c, and then constructed a competitive endogenous RNA (ceRNA) regulatory network with miR-30 c as the central miRNA. The miR-30 c-related ceRNA regulatory network was also initially validated in vitro. The results showed that miR-30 c over-expression could inhibit proliferation, migration, invasion, induce apoptosis, and increase G0/G1 phase ratio of HCC cells. Three miR-30 c upstream lncRNAs and 12 miR-30 c target genes were expressed in HCC cells with increased expression and poor prognosis, and a miR-30 C-related ceRNA regulatory network was constructed. This study verified miR-30 c as an inhibitory factor in the progression of HCC and performed analyses on the miR-30 c regulatory network, which might provide potential target information for HCC prognoses and therapies. However, further experiments in vivo and studies including clinical trials will be conducted to validate our results.

De-ubiquitination of p300 by USP12 Critically Enhances METTL3 Expression and Ang II-induced cardiac hypertrophy.

Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N6-methyladenosine (m6A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy.

[Effects of chitinase-3-like protein 1 on the expression of inflammatory damage-related molecules in mouse skeletal muscle satellite cells induced by lipopolysaccharide].

OBJECTIVE: To explore the potential mechanism of chitinase-3-like protein 1 (CHI3L1) involved in skeletal muscle stem cell injury induced by sepsis. METHODS: Six different concentrations of lipopolysaccharide (LPS) were used to stimulate mouse skeletal muscle satellite cells cultured in vitro. Enzyme linked immunosorbent assay (ELISA) and cell counting kit-8 (CCK-8) were used to determine the optimal concentration. The overexpression and interference vectors of CHI3L1 were constructed to transfect skeletal muscle satellite cells, and the transfection efficiency was verified by polymerase chain reaction (PCR) and Western blotting. The cells were randomly divided into blank control group (cells without any intervention), model group (LPS-stimulated untransfected cells), overexpressing CHI3L1 group (LPS-stimulated cells transfected with CHI3L1 plasmid), overexpressing CHI3L1 control group [LPS-stimulated cells transfected with negative control (NC) plasmid], CHI3L1 interference group [LPS-stimulated cells transfected with CHI3L1 small interfering RNA (siRNA)], CHI3L1 interference control group (LPS-stimulated cells transfected with CHI3L1-siRNA NC). The levels of extracellular caspase-1 and interleukin-1ß (IL-1ß) were detected by ELISA. The protein expressions of intracellular IL-1ß, signal transducters and activator of transcription 3 (STAT3), protein kinase B (Akt) and phosphorylated Akt (p-Akt) were detected by Western blotting. RESULTS: According to the results of CCK-8 and ELISA, the best concentration of 5 mg/L LPS was selected for the subsequent experiment. The transfection was validated by PCR and Western blotting. Compared with the blank control group, the levels of extracellular IL-1ß, caspase-1 and the protein expressions of intracellular Akt, p-Akt, and IL-1ß were significantly increased in the model group [IL-1ß (ng/L): 11.22±0.55 vs. 8.63±0.63, caspase-1 (pmol/L): 9.47±0.22 vs. 8.65±0.15, Akt/GAPDH: 1.36±0.12 vs. 1.06±0.15, p-Akt/GAPDH: 0.78±0.07 vs. 0.09±0.01, IL-1ß/GAPDH: 1.38±0.12 vs. 0.18±0.03, all P < 0.05]. Compared with the model group and the overexpressing CHI3L1 control group, the levels of extracellular IL-1ß, caspase-1 and the protein expressions of intracellular p-Akt and IL-1ß were significantly increased in the overexpressing CHI3L1 group [IL-1ß (ng/L): 14.93±0.97 vs. 11.22±0.55, 9.38±0.40, caspase-1 (pmol/L): 10.35±0.03 vs. 9.47±0.22, 8.46±0.24, p-Akt/GAPDH: 1.21±0.04 vs. 0.78±0.07, 0.63±0.04, IL-1ß/GAPDH: 1.87±0.08 vs. 1.38±0.12, 1.51±0.17, all P < 0.05]. Compared with the model group and the CHI3L1 interference control group, the levels of extracellular IL-1ß, caspase-1 and the protein expressions of intracellular p-Akt and IL-1ß were significantly decreased in the CHI3L1 interference group [IL-1ß (ng/L): 8.98±0.73 vs. 11.22±0.55, 10.44±0.65, caspase-1 (pmol/L): 7.61±0.63 vs. 9.47±0.22, 8.37±0.38, p-Akt/GAPDH: 0.50±0.04 vs. 0.78±0.07, 0.94±0.06, IL-1ß/GAPDH: 0.77±0.02 vs. 1.38±0.12, 1.13±0.07, all P < 0.05]. CONCLUSIONS: CHI3L1 may mediate the damage of skeletal muscle stem cells in sepsis by increasing the expression of caspase-1 and IL-1ß. CHI3L1 may be involved in the regulation of Akt signaling pathway in skeletal muscle stem cells, but has no significant effect on STAT3 signaling pathway.

Immunomodulatory property and its regulatory mechanism of double network hydrogel on dendritic cells.

Modulation of the key immune cell subsets by biomaterial has emerged as a potential target to promote tissue repair and regeneration. Based on calcium alginate (Alg) and glycol chitosan (GC), an injectable double-network (DN) hydrogel has been developed as a scaffold for cell delivery and cell cocultured system. Previous studies have documented the interaction between dendritic cells (DCs) and GC or Alg hydrogel, but the potential effect of DN hydrogel on activation of DCs still remains unclear. This research was conducted to explore the immunomodulatory influence and underlying mechanisms of GC/Alg DN hydrogel on DCs in vitro and in vivo. Stimulation of DCs with DN hydrogel obviously induced the maturation of DCs in vitro. In vivo, DN hydrogel did not have obvious influence on the maturation of splenic DCs on postimplantation days 3, 10, and 30. Mechanistically, we found that DN hydrogel induced the maturation of DCs via phosphorylation of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin in vitro. It provides a novel understanding of the immunomodulatory property of DN hydrogel on DCs, which may serve as potential target for designing immune-mediated regenerative strategies.

Sevoflurane-induced cognitive decline in aged mice: Involvement of toll-like receptors 4.

Toll-like receptors 4 (TLR4) contributes to the pathogenesis of some neurodegenerative diseases. However, little is known about whether TLR4 is associated with sevoflurane-induced cognitive decline. This investigation aims to address the effect of global TLR4 gene knockout on cognitive decline following sevoflurane exposure to mice. Wild-type and TLR4-/- mice were exposed to 3% sevoflurane. Novel object recognition test and Y-maze test were used to analyze cognitive function. Tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in plasma and hippocampus were measured by ELISA. Peripheral administration of recombinant TNF-α to TLR4-/- mice was used to observed the role of TNF-α in cognitive function following sevoflurane. Our results showed that, in contrast to wild-type mice, TLR4 deficiency protected against the cognitive function impairment following sevoflurane exposure, and abrogated IL-1ß, IL-6 and TNF-α response to sevoflurane in the system and the hippocampus. Subcutaneous administration of recombinant TNF-α elevated these cytokine levels in the hippocampus, and resulted in cognitive decline in TLR4-/- mice exposed to sevoflurane. Taken together, our results identify the crucial role of TLR4 in sevoflurane-induced cognitive decline, and showed that TLR4 mediated pro-inflammatory cytokine response to sevoflurane, and consequent cognitive decline in aged mice exposed to sevoflurane, and imply a novel target for improvement and therapy of sevoflurane-associated cognitive decline.

[Update in immune regulatory pathway of tumor necrosis factor-α-induced protein 8-like 2].

OBJECTIVE: The tumor necrosis factor-α-induced protein 8-like (TNFAIP8, TIPE) family is recently identified proteins consisting of four highly homologous mammalian proteins: TIPE, TIPE1, TIPE2, and TIPE3. Although the four members share similar molecular structure and function, involving effects in pathophysiological processes of inflammation, immunity, tumors, stroke, angiogenesis, and other diseases, they have individual characteristics. Many studies have shown that TIPE2 is an essential negative regulator of both innate and adaptive immunity. Up-regulation of TIPE2 expression can alleviate excessive inflammation during septic shock and maintain hemostasis of macrophages, neutrophils, dendritic cells, T cells, and B cells. In this review, we summarize the current literature for structure feature, immune function, and regulatory mechanism of TIPE2, together with its clinical significance in the pathogenesis of immune disorders of a wide array of human diseases. Understanding the basic biology of this new molecule might help us to seek novel strategies for the immunomodulation of human diseases.

Analyses of a Panel of Transcripts Identified From a Small Sample Size and Construction of RNA Networks in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world. Dysregulation of mRNAs and non-coding RNAs (ncRNAs) plays critical roles in the progression of HCC. Here, we investigated HCC samples by RNA-seq and identified a series of dysregulated RNAs in HCC. Various bioinformatics analyses established long non-coding RNA (lncRNA)-mRNA co-expression and competing endogenous RNA (ceRNA) networks in circRNA-miRNA-mRNA axis, indicating the potential cis and/or trans regulatory roles of lncRNAs and circRNAs. Moreover, GO pathway analysis showed that these identified RNAs were associated with many biological processes that were related to tumorigenesis and tumor progression. In conclusion, we systematically established functional networks of lncRNA-mRNA, circRNA-miRNA-mRNA to further unveil the potential interactions and biological processes in HCC. These results provide further insights into gene expression network of HCC and may assist future diagnosis of HCC.

Role of dendritic cells in the host response to biomaterials and their signaling pathways.

Strategies to enhance, inhibit, or qualitatively modulate immune responses are important for diverse biomedical applications such as vaccine adjuvant, drug delivery, immunotherapy, cell transplant, tissue engineering, and regenerative medicine. However, the clinical efficiency of these biomaterial systems is affected by the limited understanding of their interaction with complex host microenvironments, for example, excessive foreign body reaction and immunotoxicity. Biomaterials and biomedical devices implanted in the body may induce a highly complicated and orchestrated series of host responses. As macrophages are among the first cells to infiltrate and respond to implanted biomaterials, the macrophage-mediated host response to biomaterials has been well studied. Dendritic cells (DCs) are the most potent antigen-presenting cells that activate naive T cells and bridge innate and adaptive immunity. The potential interaction of DCs with biomaterials appears to be critical for exerting the function of biomaterials and has become an important, developing area of investigation. Herein, we summarize the effects of the physicochemical properties of biomaterials on the immune function of DCs together with their receptors and signaling pathways. This review might provide a complete understanding of the interaction of DCs with biomaterials and serve as a reference for the design and selection of biomaterials with particular effects on targeted cells. STATEMENT OF SIGNIFICANCE: Biomaterials implanted in the body are increasingly applied in clinical practice. The performance of these implanted biomaterials is largely dependent on their interaction with the host immune system. As antigen-presenting cells, dendritic cells (DCs) directly interact with biomaterials through pattern recognition receptors (PRRs) recognizing "biomaterial-associated molecular patterns" and generate a battery of immune responses. In this review, the physicochemical properties of biomaterials that regulate the immune function of DCs together with their receptors and signaling pathways of biomaterial-DC interactions are summarized and discussed. We believe that knowledge of the interplay of DC and biomaterials may spur clinical translation by guiding the design and selection of biomaterials with particular effects on targeted cell for tissue engineering, vaccine delivery, and cancer therapy.

Effect of tumor necrosis factor-α induced protein 8 like-2 on immune function of dendritic cells in mice following acute insults.

Tumor necrosis factor-α induced protein 8 like-2 (TNFAIP8L2, TIPE2) is a lately discovered negative regulator of innate immunity and cellular immunity. The present study was designed to investigate whether naturally occurring dendritic cells (DCs) could express TIPE2 mRNA/protein and its potential significance. Expressions of co-stimulatory molecules on DC surface and cytokines were analyzed to assess the functional role of TIPE2 in controlling DC maturation as well as activation. The activated DCs were assessed for their capacity to stimulate the proliferation and differentiation of T cells. It was found that TIPE2 was a cytoplasmic protein expressed in DCs, and the percentage of DCs which expressed co-stimulatory molecules and cytokines were obviously up-regulated when TIPE2 gene silenced by siRNA in vitro and in vivo. DCs undergone TIPE2 knockdown were found to promote the maturation of DCs, T-cell proliferation as well as differentiation, and they were significantly elevated IL-2 level and intranuclear NF-AT activation. Conversely, in over-expressing TIPE2 DC cells, it could inhibit T-cell proliferation and differentiation, and markedly down-regulate IL-2 expression and intranuclear NF-AT activation after scald injury. The results suggested that TIPE2 appeared to be a critical immunoregulatory molecule which affected DC maturation and subsequent T-cell mediated immunity.

Expression and clinical significance of Nectin-4 in hepatocellular carcinoma.

OBJECTIVE: Nectin-4 is a member of the Nectin family of four Ca(+)-independent immunoglobulin-like cell adhesion molecules and implicated in cell adhesion, movement, proliferation, differentiation, polarization, and survival. The aberrant expression of Nectin-4 has been found in a variety of tumors; however, its expression in hepatocellular carcinoma (HCC) is still poorly understood. This study was to investigate the expression of Nectin-4 and its clinical significance in the patients with HCC. METHODS: The expression of Nectin-4 was assessed at mRNA and protein levels by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting assays in 20 HCC specimens and adjacent non-tumor live tissues. Furthermore, the clinical significance of Nectin-4 in 87 cases of HCC was confirmed by immunohistochemistry. RESULTS: The mRNA and protein levels of Nectin-4 were higher in HCC tumor tissues than in the matched non-tumor tissues. Nectin-4 was located in the cytoplasm of tumor cells and over-expressed in 67.82% (59/87) HCC tissues by immunohistochemical staining. Positive Nectin-4 expression was significantly correlated with tumor size (P=0.029), status of metastasis (P=0.023), vascular invasion (P=0.018) and tumor-node-metastasis stage (P=0.003). In addition, Kaplan-Meier survival analysis indicated that positive Nectin-4 expression was associated with worse recurrence-free survival (RFS) and overall survival (OS) (P=0.006 and P=0.005, respectively). In multivariate analysis, Nectin-4 was an independent prognostic factor for RFS and OS in the patients with HCC. CONCLUSION: Nectin-4 is upregulated in HCC and may be a novel prognostic biomarker for the patients after surgical resection.

Effect of Regulatory T Cells on Promoting Apoptosis of T Lymphocyte and Its Regulatory Mechanism in Sepsis.

With both in vivo and in vitro experiments, the present study was conducted to investigate the effect of regulatory T cell (Treg) on promoting T-lymphocyte apoptosis and its regulatory mechanism through transforming growth factor-beta (TGF-ß1) signaling in mice. A murine model of polymicrobial sepsis was reproduced by cecal ligation and puncture (CLP); PC61 and anti-TGF-ß antibodies were used to decrease counts of CD4(+)CD25(+) Tregs and inhibit TGF-ß activity, respectively. Splenic CD4(+)CD25(+) Tregs and CD4(+)CD25(-) T cells were isolated. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), and TGFß1(m+), as well as the apoptotic rate of CD4(+)CD25(-) T cell, were analyzed by flow cytometry. Real-time reverse transcription-polymerase chain reaction was performed to determine mRNA expression of TGF-ß1, and the expressions of Smad2/Smad3, Bcl-2 superfamily members of Bcl-2/Bim, cytochrome C, the mitochondrial membrane potential, and caspases in CD4(+)CD25(-) T cells were simultaneously determined. After treatment with PC61 or anti-TGF-ß antibody, CTLA-4, Foxp3, and TGFß1(m+) expressions of CD4(+)CD25(+) Tregs were markedly decreased in comparison to that of the CLP group and the apoptosis rate of CD4(+)CD25(-) T cells was significantly positively correlated with the expression of TGF-ß1. Meanwhile, levels of P-Smad2/P-Smad3, proapoptotic protein Bim, cytochrome C, and activity of caspase-3, -8, -9 were downregulated, whereas the mitochondrial membrane potential and antiapoptotic protein Bcl-2 expression were restored. Taken together, our data indicated that the TGF-ß1 signal could be partly involved in the apoptosis of CD4(+)CD25(-) T cells promoted by CD4(+)CD25(+) Tregs, therefore inhibition of TGF-ß1 expression may provide a novel strategy for the improvement of host immunosuppression following sepsis.

Effects of enteral nutrition with parenteral glutamine supplementation on the immunological function in septic rats.

The aim of the present study was to investigate the effects of enteral nutrition (EN) with parenteral glutamine (GLN) supplementation on inflammatory response, lymphatic organ apoptosis, immunological function and survival in septic rats by caecal ligation and puncture (CLP). Male rats were randomly assigned into two experimental groups and two sham CLP control groups (n 10 per group). After CLP or sham CLP model and nutrition programme were completed, the GLN concentrations of plasma and tissues and several indices of immunological function including serum Ig content, circulating lymphocyte number, the CD4:CD8 ratio, the neutrophil phagocytosis index (NPI), the organ index and apoptosis of thymus and spleen, and plasma cytokine levels were determined. Moreover, the survival in septic rats was observed. The results revealed that EN with parenteral GLN supplementation remarkably increased the GLN concentrations of plasma and tissues, serum Ig content, the circulating lymphocyte number, the CD4:CD8 ratio, the indexes of thymus and spleen, NPI and survival compared with the control group (P< 0·05). In contrast, the apoptosis of thymus and spleen and the levels of TNF-α, IL-1ß and IL-6 in plasma were obviously decreased compared with the control group (P< 0·05). These results show that EN with parenteral GLN supplementation diminished the release of inflammatory cytokines, attenuated lymphatic organ apoptosis, enhanced the immunological function and improved survival in septic rats.

Pathophysiological aspects of sepsis: an overview.

Sepsis is defined as severe systemic inflammation in response to invading pathogens, or an uncontrolled hyperinflammatory response, as mediated by the release of various proinflammatory mediators. Although some patients may die rapidly from septic shock accompanied by an overwhelming systemic inflammatory response syndrome (SIRS) triggered by a highly virulent pathogen, most patients survive the initial phase of sepsis, showing multiple organ damage days or weeks later. These patients often demonstrate signs of immune suppression accompanied by enhanced inflammation. Sepsis is a result of a complex process; there is interaction of various pathways, such as inflammation, immunity, coagulation, as well as the neuroendocrine system. This treatise is an attempt to provide a summary of several key regulatory mechanisms and to present the currently recognized molecular pathways that are involved in the pathogenesis of sepsis.

Insights into the apoptotic death of immune cells in sepsis.

Sepsis with subsequent multiple-organ dysfunction is a distinct systemic inflammatory response to concealed or obvious infection, and it is a leading cause of death in intensive care units. Thus, one of the key goals in critical care medicine is to develop novel therapeutic strategies that will affect favorably on outcome of septic patients. In addition to systemic response to infection, apoptosis is implicated to be an important mechanism of the death of immune cells, including neutrophils, macrophages, T lymphocytes, and dendritic cells, and it is usually followed by the development of multiple-organ failure in sepsis. The implication of apoptosis of immune cells is now highlighted by multiple studies that demonstrate that prevention of cell apoptosis can improve survival in relevant animal models of severe sepsis. In this review, we focus on major apoptotic death pathways and molecular mechanisms that regulate apoptosis of different immune cells, and advances in these areas that may be translated into more promising therapies for the prevention and treatment of severe sepsis.

PNU-282987 improves the hemodynamic parameters by alleviating vasopermeability and tissue edema in dogs subjected to a lethal burns shock.

Excessive inflammation and high vasopermeability can lead to blood volume loss and tissue edema, which can affect the resuscitation and prognosis for serious burn patients. In this experiment, we investigated the effect of PNU-282987, an α7 nicotine cholinergic receptor agonist on the hemodynamic parameters and survival rate by inhibiting vasopermeability and tissue edema during the fluid resuscitation for lethal burn shock. Forty Beagle dogs with intubation of the carotid artery and jugular vein 24 hours before the injury were subjected to 50% TBSA full-thickness burns, and were randomly divided into following four groups: no resuscitation group (group NR), venous fluid resuscitation group (group R), PNU-282987 treatment group (group P), and fluid resuscitation group plus PNU-282987 group (group RP), with 10 dogs in each group. Hemodynamic variables and biochemical parameters were determined with animals in a conscious and cooperative state. The plasma volume and the vasopermeability were determined by indocyanine green and fluorescein isothiocyanate-dextran, respectively. The level of tumor necrosis factor-α and interleukin-1ß in plasma, and the water content of different organs were also determined. The mean arterial pressure, cardiac output, and plasma volume of all dogs decreased significantly, and the lung extravascular water index and pulmonary vascular permeability index increased remarkably after burn. The hemodynamic parameters deteriorated continually in group N dogs, and then anuria, hyperlactacidemia, and multiple organ dysfunctions developed. The mean arterial pressure and cardiac output of dogs in group R and group RP returned to preinjury levels at 48 hours postburn. The lung extravascular water index and pulmonary vascular permeability in group R were higher than those before preinjury. The dogs in group RP were found to have a significant increase in plasma volume and urine output, and a remarkable decrease in the levels of tumor necrosis factor-α, interleukin-1α, lactic acid, and organ functions compared with those of group R (P <.05). The survival rate of RP group (100%; 10/10) was significantly higher than that of group N (0; 0/10), group P (20%; 2/10), and group R (60%; 6/10). PNU-282987 combined with intravenous fluid resuscitation significantly improved hemodynamics and the survival rate in the early period after this lethal burn shock. The mechanism may be attributable to the lowering of the level of proinflammatory mediators, amelioration of vasopermeability-induced visceral edema, less of blood volume loss, and protection of vital organs through activation of cholinergic anti-inflammatory pathway.

Pyruvate-enriched oral rehydration solution improved intestinal absorption of water and sodium during enteral resuscitation in burns.

AIM: To investigate alteration in intestinal absorption during enteral resuscitation with pyruvate-enriched oral rehydration solution (Pyr-ORS) in scalded rats. METHODS: To compare pyruvate-enriched oral rehydration solution (Pyr-ORS) with World Health Organisation oral rehydration solution (WHO-ORS), 120 rats were randomly divided into 6 groups and 2 subgroups. At 1.5 and 4.5 h after a 35% TBSA scald, the intestinal absorption rate, mucosal blood flow (IMBF), Na(+)-K(+)-ATPase activity and aquaporin-1 (AQP-1) expression were determined (n = 10), respectively. RESULTS: The intestinal Na(+)-K(+)-ATPase activity, AQP-1 expression and IMBF were markedly decreased in scald groups, but they were profoundly preserved by enteral resuscitation with WHO-ORS and further improved significantly with Pyr-ORS at both time points. Na(+)-K+-ATPase activities remained higher in enteral resuscitation with Pyr-ORS (Group SP) than those with WHO-ORS (Group SW) at 4.5 h. AQP-1 and IMBF were significantly greater in Group SP than in Group SW at both time points. Intestinal absorption rates of water and sodium were obviously inhibited in scald groups; however, rates were also significantly preserved in Group SP than in Group SW with an over 20% increment at both time points. CONCLUSION: The Pyr-ORS may be superior to the standard WHO-ORS in the promotion of intestinal absorption of water and sodium during enteral resuscitation.