Resistin as a potential diagnostic biomarker for sepsis: insights from DIA and ELISA analyses.

PURPOSE: The primary objective of this investigation is to systematically screen and identify differentially expressed proteins (DEPs) within the plasma of individuals afflicted with sepsis. This endeavor employs both Data-Independent Acquisition (DIA) and enzyme-linked immunosorbent assay (ELISA) methodologies. The overarching goal is to furnish accessible and precise serum biomarkers conducive to the diagnostic discernment of sepsis. METHOD: The study encompasses 53 sepsis patients admitted to the Affiliated Hospital of Southwest Medical University between January 2019 and December 2020, alongside a control cohort consisting of 16 individuals devoid of sepsis pathology. Subsequently, a subset comprising 10 randomly selected subjects from the control group and 22 from the sepsis group undergoes quantitative proteomic analysis via DIA. The acquired data undergoes Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses, facilitating the construction of a Protein-Protein Interaction (PPI) network to discern potential markers. Validation of core proteins is then accomplished through ELISA. Comparative analysis between the normal and sepsis groups ensues, characterized by Receiver Operating Characteristic (ROC) curve construction to evaluate diagnostic efficacy. RESULT: A total of 187 DEPs were identified through bioinformatic methodologies. Examination reveals their predominant involvement in biological processes such as wound healing, coagulation, and blood coagulation. Functional pathway analysis further elucidates their engagement in the complement pathway and malaria. Resistin emerges as a candidate plasma biomarker, subsequently validated through ELISA. Notably, the protein exhibits significantly elevated levels in the serum of sepsis patients compared to the normal control group. ROC curve analysis underscores the robust diagnostic capacity of these biomarkers for sepsis. CONCLUSION: Data-Independent Acquisition (DIA) and Enzyme-Linked Immunosorbent Assay (ELISA) show increased Resistin levels in sepsis patients, suggesting diagnostic potential, warranting further research.

Systemic lupus erythematosus with high disease activity identification based on machine learning.

OBJECTIVE: Clinical evaluation of systemic lupus erythematosus (SLE) disease activity is limited and inconsistent, and high disease activity significantly, seriously impacts on SLE patients. This study aims to generate a machine learning model to identify SLE patients with high disease activity. METHOD: A total of 1014 SLE patients with low disease activity and 453 SLE patients with high disease activity were included. A total of 94 clinical, laboratory data and 17 meteorological indicators were collected. After data preprocessing, we use mutual information and multisurf to evaluate and select the importance of features. The selected features are used for machine learning modeling. Performance of the model is evaluated and verified by a series of binary classification indicators. RESULTS: We screened out hematuria, proteinuria, pyuria, low complement, precipitation, sunlight and other features for model construction by integrated feature selection. After hyperparameter optimization, the LGB has the best performance (ROC: AUC = 0.930; PRC: AUC = 0.911, APS = 0.913; balance accuracy: 0.856), and the worst is the naive bayes (ROC: AUC = 0.849; PRC: AUC = 0.719, APS = 0.714; balance accuracy: 0.705). Finally, the selection of features has good consistency in the composite feature importance bar plot. CONCLUSION: We identify SLE patients with high disease activity by a simple machine learning pipeline, especially the LGB model based on the characteristics of proteinuria, hematuria, pyuria and other feathers screened out by collective feature selection.

SHP2: its association and roles in systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease. Src homology 2 domain containing protein tyrosine phosphatase (SHP2) is a member of the protein tyrosine phosphatases (PTPs) family. To date, relationship between SHP2 and SLE pathogenesis is not elucidated. METHOD: We measured plasma levels of SHP2 in 328 SLE patients, 78 RA patients, 80 SS patients and 79 healthy controls by ELISA, and discussed association of SHP2 in SLE patients, potential of plasma SHP2 as a SLE biomarker. Moreover, histological and serological changes were evaluated by flow cytometry, HE/Masson examination, immunofluorescence test in pristane-induced lupus mice after SHP2 inhibitor injection to reveal role of SHP2 in lupus development. RESULTS: Results indicated that SHP2 plasma levels were upregulated in SLE patients and correlated with some clinical, laboratory characteristics such as proteinuria, pyuria, and may be a potential biomarker for SLE. After SHP2 inhibitor treatment, hepatosplenomegaly and histological severity of the kidney in lupus mice were improved. SHP2 inhibitor reversed DCs, Th1, and Th17 cells differentiation and downregulated inflammatory cytokines (IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α) and autoantibodies (ANA, anti-dsDNA) production in pristane-lupus mice. CONCLUSION: In summary, SHP2 correlated with SLE pathogenesis and promoted the development of lupus.

Mechanisms of Panax Ginseng on Treating Sepsis by RNA-Seq Technology.

Purpose: To explore the potential active targets and mechanisms of Panax Ginseng in the treatment of sepsis using network pharmacology and RNA-seq technology. Patients and Methods: Patients with sepsis and healthy volunteers were collected according to SEPSIS 3.0, and their peripheral blood was used for RNA-seq analysis. The active ingredients and targets of Panax Ginseng were obtained using the TCMSP database, PPI and GO analysis were performed for disease-drug intersection targets. Then, we used Meta-analysis to screen core genes. Finally, single-cell RNA-seq was used to perform cell localization analysis on core genes. Results: RNA-seq analysis collected 4521 sepsis-related genes, TCMSP database obtained 86 Panax Ginseng active ingredients and their 294 active targets. PPI and GO analysis showed intersection targets were closely linked, and mainly involved in cellular response to chemical stress, response to drug and molecule of bacterial origin, etc. Then, core targets, IL1B, ALOX5, BCL2 and IL4R, were sorted by Meta-analysis, and all four genes have high expression in the sepsis survivor group compared to the sepsis non-survivor group; single-cell RNA-seq revealed that IL1B was mainly localized in macrophages, ALOX5 was mainly localized in macrophages and B cells, BCL2 was mainly localized in natural killer cells, T cells and B cells, IL4R was widely distributed in immune cells. Finally, according to the correspondence between the active ingredients and targets of Panax Ginseng in TCMSP database, we found that Ginsenoside rh2 regulates the expression of IL1B, Ginsenoside rf regulates the expression of IL1B and IL4R, Kaempferol regulates the expression of ALOX5 and BCL2, and ß-sitosterol regulates the expression of BCL2. Conclusion: Ginsenoside rh2, Ginsenoside rf, Kaempferol and ß-sitosterol may produce anti-sepsis effects by regulating the expression of IL1B, ALOX5, BCL2 and IL4R, thus improving the survival rate of sepsis patients.

IL-38, a potential therapeutic agent for lupus, inhibits lupus progression.

BACKGROUND: Previous studies reported that IL-38 was abnormally expressed in patients with systemic lupus erythematosus (SLE). However, the involvement of IL-38 in the pathophysiology of SLE remains unknown. METHODS: The therapeutic potential of IL-38 was tested in pristane-treated wild-type (WT) and IL-38-/- mice. Thus, SLE was induced via pristane in WT and IL-38-/- mice. Afterwards, the liver, spleen, and kidney of each mouse were obtained. The flow cytometric analysis of the immune cells, serologic expression of inflammatory cytokines and autoantibodies, renal histopathology, and inflammatory signaling were evaluated. RESULTS: WT mice with pristane-induced lupus exhibited hepatomegaly, splenomegaly, severe kidney damages, increased lymphoproliferation, enhanced lymphoproliferation, and upregulated inflammatory cytokines, such as IL-6, IL-13, IL-17A, MIP-3α, IL-12p70, and IFNγ, and elevated levels of autoantibodies, such as ANA IgG, anti-dsDNA IgG, and total IgG. IL-38-/- mice whose lupus progressed, had elevated cells of CD14+, CD19+, CD3+, and Th1, upregulated inflammatory cytokines and autoantibodies, and severe pathological changes in kidney. Administration of recombinant murine IL-38 to pristane-treated IL-38-/- mice improved their renal histopathology, which depended on ERK1/2, JNK1/2, p38, NF-κB p65, and STAT5 signaling pathways. CONCLUSION: IL-38 regulates SLE pathogenesis. Furthermore, targeting IL-38 is critical in the treatment of SLE.

Association between IL-37 and Systemic Lupus Erythematosus Risk.

Interleukin-37 (IL-37) is an anti-inflammatory cytokine. In our former study, we found increased plasma IL-37 levels in systemic lupus erythematosus (SLE) patients. However, relationship between IL-37 levels and clinical laboratory characteristics of SLE patients has not been elucidated. In addition, association of IL37 gene polymorphism with SLE risk needs to be discussed. A group of 580 individuals (220 SLE patients and 360 healthy controls) in a Southern Chinese Han population were recruited. Plasma IL-37 levels were evaluated using enzyme-linked immunosorbent assay (ELISA). Four single-nucleotide polymorphisms (rs3811047, rs2723186, rs2723176 and rs4364030) of IL37 gene were genotyped. Relationship of IL-37 expression, IL37 gene polymorphisms and clinical characteristics was discussed. We found that plasma levels of IL-37 were negatively associated with SLE disease activity index (SLEDAI) (rs = -0.352, P = .001), and were higher in less active patients compared with active patients (P = .003). Decreased levels of IL-37 were found in SLE patients with discoid rash when compared to patients who did not have this symptom (P < .001). Plasma IL-37 levels were significantly lower in patients with hypocomplementemia comparing to those without this feature (P = .009). Levels of IL-37 in SLE with positive proteinuria were lower than patients with negative proteinuria (P = .046). Furthermore, allele distribution of rs2723186, rs4364030 between SLE cases and healthy individuals was significantly different (P = .001, P = .010, respectively). Genotype of rs4364030 was different between SLE cases and controls (P = .015). Haplotype analysis revealed that the frequency of haplotype CG (rs2723176 (C) +rs2723186 (G)) was higher in SLE, as compared with healthy individuals (P = .002). In conclusion, the plasma levels of IL-37 were related to SLE severity, and IL37 gene polymorphisms (rs2723186, rs2723176 and rs4364030) may associate with SLE susceptibility.

Gene polymorphisms of SIRT1 in patients with rheumatoid arthritis.

AIM: Previous studies have shown that silent information regulator 1 (SIRT1) expression is elevated in rheumatoid arthritis (RA) patients. However, whether gene polymorphisms in SIRT1 gene associated with RA in a Chinese Han population remains to be discussed. METHOD: In this case-control study, 529 RA patients and 700 healthy controls were selected, and association of 11 SIRT1 gene polymorphisms (rs12415800, rs3740051, rs932658, rs3740053, rs7895833, rs10509291, rs33957861, rs7069102, rs2273773, rs3818292, rs1467568) with RA susceptibility was evaluated. RESULTS: Frequency of GA+GG genotype of rs3740051 in RA patients was significantly lower than that in healthy controls (P = .037). Frequencies of GC and GC+GG genotypes of rs7069102 were significantly lower than those in healthy controls (P = .036, P = .047). Frequencies of GA and GA+GG genotypes of rs1467568 were lower in RA patients as compared to those in healthy controls (P = .011, P = .013). For rs2273773, RA patients who carried the T allele had higher number of tender joints than patients who carried the C allele (P = .033). Other polymorphisms did not associate with RA risk. CONCLUSION: The findings suggest that rs3740051, rs7069102 and rs1467568 variants in SIRT1 gene are related to RA susceptibility in a Chinese Han population.

Gene polymorphisms of LGALS2, LGALS3 and LGALS9 in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complicated rheumatic autoimmune disease. Lectin, galactoside-binding soluble, 2 (LGALS2), LGALS3 and LGALS9, three members of the galectin family, play potential roles in autoimmune diseases, including RA. However, association of genetic polymorphisms of LGALS2, LGALS3 and LGALS9 with RA risk in a Southern Chinese Han population has not been elucidated. A case-control study was conducted herein, including 500 RA patients and 650 healthy individuals of Southern Chinese Han origin. Twelve single nucleotide polymorphisms (SNPs), including rs7291467 for the LGALS2 gene, rs4644, rs4652, rs1009977, rs2274273 and rs17128183 for the LGALS3 gene, and rs4795835, rs3763959, rs4239242, rs3751093, rs732222 and rs4794976 for the LGALS9 gene, were genotyped. Polymorphisms were genotyped using the KASP method. Frequencies of rs1009977 genotype TG and rs3751093 genotype GA of LGALS3 gene were significantly different between RA patients and healthy controls (P = 0.049, P = 0.033). Allele T and genotypes TT and TT + TG of rs4794976 for LGALS9 gene were significantly correlated with RA risk (P = 0.017, P = 0.012, P = 0.041). Subgroup analysis revealed that rs1009977, rs2274273 and rs17128183 polymorphisms of LGALS3 gene and rs4795835 polymorphism of LGALS9 gene were correlated with several RA clinical manifestations (all P < 0.05). In addition, haplotype GCGTT showed an increased risk for RA (OR = 1.216, 95% CI: 1.028-1.438, P = 0.023), whereas haplotype GCGTG showed a reduced risk for RA susceptibility (OR = 0.779, 95% CI: 0.625-0.971, P = 0.026). In conclusion, LGALS3 and LGALS9 gene polymorphisms may associate with RA predisposition in a Southern Chinese Han population.

Association of IL-35 expression and gene polymorphisms in rheumatoid arthritis.

Interleukin (IL)-35 is the newest member of the IL-12 family. It is expressed in many immune cells and has been recognized as a novel inflammatory cytokine that may have bifunctional properties. Recent findings have indicated that the expression of IL-35 is abnormal in rheumatoid arthritis (RA) patients. However, the results were inconsistent. In this study, 400 RA patients were recruited to evaluate serum levels of IL-35 in a Chinese Han population by enzyme-linked immunosorbent assay. The association of IL-35 gene polymorphisms and RA genetic susceptibility was investigated in 400 RA patients and 612 healthy controls. The results showed that serum levels of IL-35 were elevated in 100 RA patients compared to 51 healthy controls, relating to disease activity and synovial fluid IL-35 expression in the training cohort. Another independent 300 RA patients and 369 other rheumatic disease patients (98 lupus, 95 osteoarthritis, 95 gout, 42 Sjogren's syndrome and 39 ankylosing spondylitis patients) confirmed that serum levels of IL-35 were elevated in RA patients, and serum IL-35 has good diagnostic ability for differentiating RA from the other rheumatic diseases. The genotyping of 10 IL-35 polymorphisms, including rs2227314, rs2243115, rs2243123, rs2243131, rs568408, rs583911, rs428253, rs4740, rs9807813 and rs4905, revealed that rs2227314, rs2243131, rs9807813, and rs583911 were correlated with RA risk. Different genotypes (rs2227314, rs583911, and rs9807813) exhibited different expression of IL-35. These findings demonstrate that serum levels of IL-35 are increased in RA patients and that IL-35 polymorphisms are correlated with RA risk.

Association of MBL2 gene polymorphisms and systemic lupus erythematosus susceptibility: A meta-analysis.

OBJECTIVES: Mannose binding lectin (MBL) gene single nucleotide polymorphisms have been associated with systemic lupus erythematosus (SLE) risk with inconsistent results. This study aimed to explore whether MBL2 A\B, A\C, A\D, A\O, L\H and Y\X polymorphisms affected SLE susceptibility. METHODS: A meta-analysis was performed on 20 studies, containing allelic contrast, additive, dominant and recessive models. Odds ratio (OR) was calculated to reflect the effect of association. RESULTS: A total of 64 pooled comparisons were conducted, including 7194 SLE patients and 7401 healthy controls. The meta-analysis inducted a significant association between allele B and SLE (OR = 0.766, 95% CI = 0.681-0.862, P < .001). The genotype BB in the additive model and AB + BB in the recessive model both reduced the risk of SLE (OR = 0.611, 95% CI = 0.422-0.882, P = .009; OR = 0.806, 95% CI = 0.688-0.944, P = .008). Regarding A\O polymorphisms, results revealed statistical differences in allelic contrast, additive model and recessive models (OR = 0.826, 95% CI = 0.732-0.931, P = .002; OR = 0.737, 95% CI = 0.557-0.977, P = .034 and OR = 0.793, 95% CI = 0.683-0.921, P = .002, respectively). As for L\H, meta-analysis revealed that allele H and genotype HH both decreased SLE susceptibility in allelic contrast and dominant models (OR = 1.463, 95% CI = 1.097-2.007, P = .018; OR = 1.383, 95% CI = 1.124-1.701, P = .002). Stratification by ethnicity indicated that allele H related to SLE in European populations (OR = 0.736, 95% CI = 0.617-0.879, P = .001), and the recessive model correlated with SLE in Asians (OR = 0.808, 95% CI = 0.667-0.979, P = .03). CONCLUSION: The present study suggests that A\B and A\O polymorphisms were associated with SLE susceptibility, and the allele H may be a protective factor in SLE.

Down-regulation of microRNA-138 improves immunologic function via negatively targeting p53 by regulating liver macrophage in mice with acute liver failure.

MicroRNAs (miRNAs) have been frequently identified as key mediators in almost all developmental and pathological processes, including those in the liver. The present study was conducted with aims of investigating the role of microRNA-138 (miR-138) in acute liver failure (ALF) via a mechanism involving p53 and liver macrophage in a mouse model. The ALF mouse model was established using C57BL/6 male mice via tail vein injection of Concanamycin A (Con A) solution. The relationship between miR-138 and p53 was tested. The mononuclear macrophages were infected with mimic and inhibitor of miR-138 in order to identify roles of miR-138 in p53 and levels of inflammatory factors. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and ELISA were conducted in order to determine the levels of miR-138, inflammatory factors, and p53 during ALF. The results showed an increase in the levels of miR-138 and inflammatory factors in ALF mice induced by the ConA as time progressed and reached the peak at 12 h following treatment with ConA, while it was on the contrary when it came to the level of p53. Dual-luciferase reporter gene assay revealed that p53 was a target gene of miR-138. Furthermore, the results from the in vitro transfection experiments in primary macrophages of ALF mouse showed that miR-138 down-regulated p53 and enhanced levels of inflammatory factors; thus, improving immune function in ALF mice. In conclusion, by negatively targeting p53, the decreased miR-138 improves immunologic function by regulating liver macrophage in mouse models of ALF.

CCR5 silencing reduces inflammatory response, inhibits viability, and promotes apoptosis of synovial cells in rat models of rheumatoid arthritis through the MAPK signaling pathway.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that can, in severe cases, lead to disability. CC chemokine receptor (CCR), an integral membrane protein, has been suggested to play a key role in the RA developmentThis study is to explore the role of CCR5 silencing in inflammatory response, viability, and apoptosis of synovial cells in RA rats by inactivating the mitogen-activated protein kinase (MAPK) signaling pathway. Microarray analysis was conducted to screen out differentially expressed genes from RA-related chips. The rat model was established by injection of siRNA-CCR5 and PD98059 (inhibitor of mitogen-activated protein kinase kinase 1) to evaluate the role of CCR5 silencing in RA, with the involvement of inflammatory response, synovial cell viability, apoptosis, and cycle. CCR5 was predicted to participate in RA by regulating the MARK pathway. In animal experiments, reduction was identified in arthritis index (AI), CCR5 positive expression rate, levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase (MMP)-1, and MMP-3 in serum of RA rats after CCR5 siRNA and PD98059 injections. RA rats treated with CCR5 siRNA, and PD98059 presented with inhibition in cell viability, promotion of apoptosis, increase in cell proportion in G0/G1 phase, and shortened the S phase. In addition, the treatment of CCR5 siRNA, and PD98059 resulted in downregulated JNK1, ERK1, p38, Cyclin D1, Cyclin E1, Cyclin B1, and Bcl-2 and upregulated Bax and Cas3. These findings reveal that CCR5 silencing suppresses inflammatory response, inhibits viability, and promotes apoptosis of synovial cells in RA rats by inhibiting MAPK pathway. Therefore, CCR5 silencing may provide a novel therapeutic target for RA.

99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin.

The aim of the present study was to examine the influence of technetium methylenediphosphonate (99Tc-MDP) on the proliferation and differentiation of human osteoblasts. Human iliac cancellous bone was isolated and cultured with either 99Tc­MDP, ß fibroblast growth factor (as a positive control) or medium only (as a negative control). Proliferation was assessed by direct cell counting, CCK­8 assay and bromodeoxyuridine staining. The cell cycle and rate of apoptosis was assessed by propidium iodide staining and flow cytometry. Alkaline phosphatase (ALP) activity was assessed by the p­nitrophenyl phosphate method and mineralized nodules were stained with Alizarin Red. Expression of osteocalcin (OCN) and bone morphogenetic protein­2 (BMP­2) was assessed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR), and expression levels of osteoprotegerin (OPG) and receptor activator of NF­κB ligand (RANKL) were assessed by RT­qPCR and ELISA. Isolated human osteoblasts stained positively for ALP and developed mineralized nodules. Treatment with 10­5­10­10 M 99Tc­MDP enhanced proliferation and 48 h incubation with 10­8 M 99Tc­MDP increased the proportion of cells in S­phase, decreased the proportion in G0/G1 phase, and increased the cell proliferation index. The rate of apoptosis also increased, but the increase was not significant. Cells incubated with 10­6­10­9 M 99Tc­MDP for 3­9 days exhibited increased ALP activity and mineralized nodule development. 10­8 M 99Tc­MDP increased BMP­2 and OPG expression levels and OPG secretion, but OCN mRNA expression levels and RANKL secretion were not significantly altered at 72 h. 99Tc­MDP treatment induced osteoblast proliferation and differentiation without affecting apoptosis. These findings provide proof of concept for the future use of 99Tc­MDP in the treatment of bone­destructive diseases.