Six potential biomarkers in septic shock: a deep bioinformatics and prospective observational study.

Background: Septic shock occurs when sepsis is related to severe hypotension and leads to a remarkable high number of deaths. The early diagnosis of septic shock is essential to reduce mortality. High-quality biomarkers can be objectively measured and evaluated as indicators to accurately predict disease diagnosis. However, single-gene prediction efficiency is inadequate; therefore, we identified a risk-score model based on gene signature to elevate predictive efficiency. Methods: The gene expression profiles of GSE33118 and GSE26440 were downloaded from the Gene Expression Omnibus (GEO) database. These two datasets were merged, and the differentially expressed genes (DEGs) were identified using the limma package in R software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments of DEGs were performed. Subsequently, Lasso regression and Boruta feature selection algorithm were combined to identify the hub genes of septic shock. GSE9692 was then subjected to weighted gene co-expression network analysis (WGCNA) to identify the septic shock-related gene modules. Subsequently, the genes within such modules that matched with septic shock-related DEGs were identified as the hub genes of septic shock. To further understand the function and signaling pathways of hub genes, we performed gene set variation analysis (GSVA) and then used the CIBERSORT tool to analyze the immune cell infiltration pattern of diseases. The diagnostic value of hub genes in septic shock was determined using receiver operating characteristic (ROC) analysis and verified using quantitative PCR (qPCR) and Western blotting in our hospital patients with septic shock. Results: A total of 975 DEGs in the GSE33118 and GSE26440 databases were obtained, of which 30 DEGs were remarkably upregulated. With the use of Lasso regression and Boruta feature selection algorithm, six hub genes (CD177, CLEC5A, CYSTM1, MCEMP1, MMP8, and RGL4) with expression differences in septic shock were screened as potential diagnostic markers for septic shock among the significant DEGs and were further validated in the GSE9692 dataset. WGCNA was used to identify the co-expression modules and module-trait correlation. Enrichment analysis showed significant enrichment in the reactive oxygen species pathway, hypoxia, phosphatidylinositol 3-kinases (PI3K)/Protein Kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling, nuclear factor-κß/tumor necrosis factor alpha (NF-κß/TNF-α), and interleukin-6 (IL-6)/Janus Kinase (JAK)/Signal Transducers and Activators of Transcription 3 (STAT3) signaling pathways. The receiver operating characteristic curve (ROC) of these signature genes was 0.938, 0.914, 0.939, 0.956, 0.932, and 0.914, respectively. In the immune cell infiltration analysis, the infiltration of M0 macrophages, activated mast cells, neutrophils, CD8 T cells, and naive B cells was more significant in the septic shock group. In addition, higher expression levels of CD177, CLEC5A, CYSTM1, MCEMP1, MMP8, and RGL4 messenger RNA (mRNA) were observed in peripheral blood mononuclear cells (PBMCs) isolated from septic shock patients than from healthy donors. Higher expression levels of CD177 and MMP8 proteins were also observed in the PBMCs isolated from septic shock patients than from control participants. Conclusions: CD177, CLEC5A, CYSTM1, MCEMP1, MMP8, and RGL4 were identified as hub genes, which were of considerable value in the early diagnosis of septic shock patients. These preliminary findings are of great significance for studying immune cell infiltration in the pathogenesis of septic shock, which should be further validated in clinical studies and basic studies.

SILENCING M 6 A READER YTHDC1 REDUCES INFLAMMATORY RESPONSE IN SEPSIS-INDUCED CARDIOMYOPATHY BY INHIBITING SERPINA3N EXPRESSION.

ABSTRACT: Sepsis-induced cardiomyopathy (SIC) is one of the most common complications of infection-induced sepsis. An imbalance in inflammatory mediators is the main factor leading to SIC . N 6 -methyladenosine (m 6 A) is closely related to the occurrence and development of sepsis. N 6 -methyladenosine reader YTH domain containing 1 (YTHDC1) is an m 6 A N 6 -methyladenosine recognition protein. However, the role of YTHDC1 in SIC remains unclear. Herein, we demonstrated that YTHDC1-shRNA inhibits inflammation, reduces inflammatory mediators, and improves cardiac function in a LPS-induced SIC mouse model. Based on the Gene Expression Omnibus database analysis, serine protease inhibitor A3N is a differential gene of SIC. Furthermore, RNA immunoprecipitation indicated that serine protease inhibitor A3N (SERPINA3N) mRNA can bind to YTHDC1, which regulates the expression of SERPINA3N. Serine protease inhibitor A3N-siRNA reduced LPS-induced inflammation of cardiac myocytes. In conclusion, the m 6 A reader YTHDC1 regulates SERPINA3N mRNA expression to mediate the levels of inflammation in SIC. Such findings add to the relationship between m 6 A reader YTHDC1 and SIC, providing a new research avenue for the therapeutic mechanism of SIC.

The PICK1/TLR4 complex on microglia is involved in the regulation of LPS-induced sepsis-associated encephalopathy.

The treatment options for sepsis-associated encephalopathy caused by systemic inflammation are still not sufficient. Protein kinase C interaction protein 1 (PICK1) has attracted much attention because of its important physiological functions in many tissues. However, its role in sepsis-associated encephalopathy remains elusive. Our study results revealed that the expression levels of PICK1 protein in mice with lipopolysaccharide-induced sepsis-associated encephalopathy were not significantly changed, but PICK1 deficiency led to excessive activation of microglia and Toll-like receptor (TLR)4 pathways, which aggravated the sepsis- associated encephalopathy. We also observed that PICK1 and TLR4 form a complex in microglial cells, thereby providing brain protection. These findings contribute to our understanding of the important role of PICK1 in sepsis and may provide novel therapeutic targets to treat sepsis-associated encephalopathy.

Red cell index: A novel biomarker for 3-month mortality in acute ischemic stroke patients treated with intravenous thrombolysis.

BACKGROUND: The red cell index (RCI) was described as a biomarker for evaluating respiratory function in previous studies, but the relationship between RCI and stroke, remained a mystery. The present study aimed to probe the association between RCI at 24-hr and 3-month mortality and functional outcomes among acute ischemic stroke (AIS) patients treated with recombinant tissue plasminogen activator (r-tPA). METHODS: A total of 217 AIS patients between January 2016 and January 2019 were recruited in this retrospective study. AIS patients were grouped in terms of RCI tertiles. Predictive factors were confirmed via multivariate logistic regression analysis. The receiver operating characteristic (ROC) was used to assess the ability of RCI in predicting mortality. In addition, the risk of 3-month all-cause mortality was evaluated by Cox proportional hazard model. RESULTS: We grouped AIS patients into tertiles with the purpose of comparing clinical factors and RCI levels. Multivariate logistic regression analysis presented that RCI (odds ratio [OR] = 1.443, 95% confidence interval [CI] [1.167-1.786], p = 0.001) was an independent biomarker for 3-month all-cause mortality. The best cutoff value of RCI was 2.41 (area under the curve [AUC] = 0.639, 95% CI [0.501-0.778], p = .032), with a sensitivity of 40.9% and a specificity of 89.7%. Cox survival analysis demonstrated a positive significant correlation between RCI (hazard ratio [HR] = 1.332, 95% CI [1.148-1.545], p < .001) and mortality risk. CONCLUSION: RCI, a potential predictor, was significantly associated with 3-month mortality in AIS patients with r-tPA.

Islet-cell autoantigen 69 mediates the antihyperalgesic effects of electroacupuncture on inflammatory pain by regulating spinal glutamate receptor subunit 2 phosphorylation through protein interacting with C-kinase 1 in mice.

Electroacupuncture (EA) is widely used in clinical settings to reduce inflammatory pain. Islet-cell autoantigen 69 (ICA69) has been reported to regulate long-lasting hyperalgesia in mice. ICA69 knockout led to reduced protein interacting with C-kinase 1 (PICK1) expression and increased glutamate receptor subunit 2 (GluR2) phosphorylation at Ser880 in spinal dorsal horn. In this study, we evaluated the role of ICA69 in the antihyperalgesic effects of EA and the underlying mechanism through regulation of GluR2 and PICK1 in spinal dorsal horn. Hyperalgesia was induced in mice with subcutaneous plantar injection of complete Freund adjuvant (CFA) to cause inflammatory pain. Electroacupuncture was then applied for 30 minutes every other day after CFA injection. When compared with CFA group, paw withdrawal frequency of CFA+EA group was significantly decreased. Remarkable increases in Ica1 mRNA expression and ICA69 protein levels on the ipsilateral side were detected in the CFA+EA group. ICA69 expression reached the peak value around day 3. More importantly, ICA69 deletion impaired the antihyperalgesic effects of EA on GluR2-p, but PICK1 deletion could not. Injecting ICA69 peptide into the intrathecal space of ICA69-knockout mice mimicked the effects of EA analgesic and inhibited GluR2-p. Electroacupuncture had no effects on the total protein of PICK1 and GluR2. And, EA could increase the formation of ICA69-PICK1 complexes and decrease the amount of PICK1-GluR2 complexes. Our findings indicate that ICA69 mediates the antihyperalgesic effects of EA on CFA-induced inflammatory pain by regulating spinal GluR2 through PICK1 in mice.

PICK1 deficiency exacerbates sepsis-associated acute lung injury and impairs glutathione synthesis via reduction of xCT.

The role of oxidative stress has been well documented in the development of sepsis-induced acute lung injury (ALI). Protein interaction with C-kinase 1 (PICK1) participates in oxidative stress-related neuronal diseases. However, its function in lung infections and inflammatory diseases is not known. We therefore sought to investigate whether PICK1 is involved in sepsis-induced ALI. Cecal ligation and puncture (CLP) was performed in anesthetized wild type (WT) and PICK1 knock out (KO, PICK1-/-) mice with C57BL/6 background. At the time of CLP, mice were given fluid resuscitation. Mouse lungs were harvested at 24 and 72 h for Western blot analysis, qRT-PCR, BALF analysis, Hematoxylin and Eosin staining, TUNEL staining, maleimide staining, flow cytometry analysis, GCL, GSH, GSSG and cysteine levels measurement. A marked elevation of PICK1 mRNA and protein level were demonstrated in lung tissue, which was accompanied by increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and consumption of glutathione (GSH). N-acetylcysteine (NAC), buthionine sulfoximine (BSO) and GSH-monoethyl ester (GSH-MEE) were injected into mice via caudal vein to regulate glutathione (GSH) level in lung. Alterations of lung GSH content induced PICK1 level change after CLP challenge. In PICK1-/- underwent with CLP, lung injury and survival were significantly aggravated compared with wild-type mice underwent with CLP. Concomitantly, CLP-induced lung cell apoptosis was exacerbated in PICK1-/- mice. The level of xCT, other than PKCα, in lung tissue was significantly lowered in PICK1-/- but not in wild type that underwent CLP surgery. Meanwhile, Nrf2 activation, which dominating xCT expression, was inhibited in PICK1-/- but not in wild type mice that underwent CLP surgery, as well. Moreover, higher level of PICK1 was detected in PBMCs of septic patients than healthy controls. Taken together, PICK1 plays a pivotal role in sepsis-induced ALI by regulating GSH synthesis via affecting the substrate-specific subunit of lung cystine/glutamate transporter, xCT.

The correlation of neutrophil-to-lymphocyte ratio with the presence and activity of myasthenia gravis.

Though the pathogenesis of myasthenia gravis (MG) is not fully understood, the role of inflammation has been well appreciated in the development of MG. We aimed to investigate the role of neutrophil-to-lymphocyte ratio (NLR) in MG patients and the relationship between the NLR and the activity of the disease. A total number of 172 MG patients and 207 healthy controls (HC) were enrolled in this study. The MG patients were divided into tertiles according to NLR (low NLR < 1.58, n = 57; intermediate NLR 1.58-2.33, n = 57 and high NLR > 2.33, n = 58). The disease activity assessment was performed according to the standard criteria established by the Myasthenia Gravis Foundation of America. Patients with MG had significantly higher NLR when compared with the HC group (P < 0.0001). The NLR levels were higher in the MG patients with severe disease activity than those with mild disease activity (P < 0.001), meanwhile, median NLR was statistically higher in MG patients with myasthenic crisis (MC) than those without MC (P < 0.001). Incidences of severe disease activity and MC were both higher in the high NLR group, compared to low and intermediate NLR groups (both P < 0.001). Multivariate logistic regression analysis suggested that elevated NLR was an independent predictor of severe disease activity (odds ratio = 13.201, CI% = 1.418-122.938, P = 0.023). These results indicate that NLR may be a simple and useful potential marker in indicating disease activity in patients with MG.

Low serum albumin concentrations are associated with disease severity in patients with myasthenia gravis.

Serum albumin (S-Alb) is a widely used biomarker of nutritional status and disease severity in patients with autoimmune diseases. We investigated the correlation between S-Alb and the severity of myasthenia gravis (MG).A total number of 166 subjects were recruited in the study. Subjects were divided into 3 groups (T1 to T3) by S-Alb levels: T1: 21.1 to 38.4 g/L, T2: 38.5 to 41.5 g/L, T3: 41.6 to 48.9 g/L. Regression analysis was performed to determine the correlation of initial albumin concentrations and the severity of disease of MG.Lower levels of S-Alb were observed in subjects with increased disease severity than those with slight disease severity, meanwhile, incidence of myasthenia crisis increased in the lower albumin tertiles (P < 0.001). The disease severity assessment was performed according to the criteria established by the Myasthenia Gravis Foundation of America. After adjusting for age, sex, body mass index (BMI), and duration of disease, it showed that higher S-Alb concentrations were associated with lower disease severity. Odds ratios (ORs) of T2 to T3 were 0.241 (95% CI: 0.103-0.566, P < 0.001), 0.140 (95% CI: 0.054-0.367, P < 0.001) when compared with subjects in the T1, respectively. When subjects were stratified into hypoalbuminemia and normal albumin groups, we found that the association between S-Alb and MG remained significant in the hypoalbuminemia group only (OR: 0.693, 95% CI: 0.550-0.874, P = 0.002) after further adjustment for age, sex, BMI, and duration of disease.This is the first study to demonstrate that S-Alb was independently associated with MG severity. In patients with low S-Alb, S-Alb concentration could be a potential biomarker for MG disability.

Loss of ICA69 potentiates long-lasting hyperalgesia after subcutaneous formalin injection into the mouse hindpaw.

Islet-cell autoantigen 69 kDa (ICA69) plays an important role in many diseases and physiological activities by forming heteromeric complexes with protein interacts with C-kinase 1 (PICK1). PICK1 is critical for inflammatory pain hypersensitivity by regulating trafficking of AMPA receptor subunit GluA2 in spinal neurons. However, the role of ICA69 in inflammatory pain has not yet been investigated. Here we reported that expression of PICK1 in spinal cord was reduced largely in ICA69 knockout mice. The pain hypersensitivity was enhanced in the second phase 7 days after formalin administration. Meanwhile, increased Ser880 phosphorylation in GluA2 and decreased surface GluA2 were concordant with the pain. Furthermore, the number of activated microglia in spinal dorsal horn increased in line with pain hypersensitivity. Together, ICA69 deficiency promoted the internalization of GluA2 and FML-induced long-lasting pain hypersensitivity. In addition, microglia activation might be an important factor in the development of the pain hypersensitivity.