Nucleotide-binding Protein Research Articles

Bortezomib inhibits NLRP3 inflammasome activation and NF-κB pathway to reduce psoriatic inflammation

The abnormal activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the pathogenesis of psoriasis. Accordingly, the inhibition of NLRP3 inflammasome may be an effective strategy for psoriasis treatment. However, the NLRP3 inflammasome inhibitors are not available in the clinic. Repurposing FDA-approved drugs is a highly attractive way for identifying new drugs. Here, proteasome inhibitor bortezomib, a marketed drug for treating multiple myeloma, was found to specifically inhibit NLRP3 inflammasome activation at nanomolar concentrations. Mechanistically, bortezomib did not inhibit reactive oxygen species generation, ion efflux, NLRP3 oligomerization, and NLRP3-ASC interactions. Bortezomib reduced ASC oligomerization and ASC speck formation. In addition, bortezomib inhibited the activity of the core subunit β5i in the immunoproteasome and reduced β5i binding to NLRP3. Bortezomib reduced the production of interleukin-1β and attenuated the severity of skin lesions in the imiquimod-induced psoriatic mouse model. Thus, bortezomib is a potential therapeutic drug for psoriasis. Our study also revealed that β5i may be an indirect target for regulating NLRP3 inflammasome activation and treating psoriasis and other NLRP3 inflammasome-related diseases.

Clinical and genetic characteristics for 4 patients with Type Ib pseudohypoparathyroidism.

Pseudohypoparayhyroidism (PHP) is a rare autosomal dominant or recessive genetic disorder characterized by low calcium, high phosphorus, and target organ resistance to parathyroid. The clinical characteristics and genetic features in 4 patients with Type Ib PHP in the Third Xiangya Hospital, Central South University, have been reviewed. All 4 patients had low calcium, high phosphorus, and parathyroid resistance. Among them, 2 patients had slightly elevated thyroid stimulating hormone and mild features of Albright's hereditary osteodystrophy, and one patient had hypokalemia. No guanine nucleotide-binding protein alpha-stimulating activity polypeptide 1 (GNAS) and gene variant associated with hypokalemia were identified using the whole exome sequencing. The results of the methylation-specific multiple ligation-dependent probe amplification showed that there were abnormal methylation of the upstream differentially methylated regions of GNAS in the 4 patients. There were phenotype overlap among the various subtypes of PHP. Detection of GNAS gene methylation in patients with clinical suspicion of Type Ib PHP is helpful for the diagnosis and treatment of PHP.

PM2b, a CC-NBS-LRR protein, interacts with TaWRKY76-D to regulate powdery mildew resistance in common wheat.

Powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive disease of wheat throughout the world. Host resistance is considered the most sustainable way to control this disease. Powdery mildew resistance gene Pm2b was mapped to the same genetic interval with Pm2a and PmCH1357 cloned previously, but showed different resistance spectra from them, indicating that they might be caused by different resistance genes or alleles. In this study, Pm2b was delimited to a 1.64 Mb physical interval using a large segregating population containing 4,354 F2:3 families of resistant parent KM2939 and susceptible cultivar Shimai 15. In this interval, TraesCS5D03G0111700 encoding the coiled-coil nucleotide-binding site leucine-rich repeat protein (CC-NBS-LRR) was determined as the candidate gene of Pm2b. Silencing by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technology and two independent mutants analysis in KM2939 confirmed the candidate gene TraesCS5D03G0111700 was Pm2b. The sequence of Pm2b was consistent with Pm2a/PmCH1357. Subcellular localization showed Pm2b was located on the cell nucleus and plasma membrane. Pm2b had the highest expression level in leaves and was rapidly up-regulated after inoculating with Bgt isolate E09. The yeast two-hybrid (Y2H) and luciferase complementation imaging assays (LCI) showed that PM2b could self-associate through the NB domain. Notably, we identified PM2b interacting with the transcription factor TaWRKY76-D, which depended on the NB domain of PM2b and WRKY domain of TaWRKY76-D. TaWRKY76-D negatively regulated the resistance to powdery mildew in wheat. The specific KASP marker K529 could take the advantage of high-throughput and high-efficiency for detecting Pm2b and be useful in molecular marker assisted-selection breeding. In conclusion, cloning and disease resistance mechanism analysis of Pm2b provided an example to emphasize a need of the molecular isolation of resistance genes, which has implications in marker assisted wheat breeding.

Betaine Inhibits NLRP3 Inflammasome Hyperactivation and Regulates Microglial M1/M2 Phenotypic Differentiation, Thereby Attenuating Lipopolysaccharide-Induced Depression-Like Behavior.

Depression is one of the most important mental illnesses and is closely related to inflammation. Betaine is a natural product with an anti-inflammatory and antioxidant activities. However, the mechanism by which betaine ameliorates depression-like behaviors induced by lipopolysaccharide (LPS) is poorly understood. The purpose of this study was to investigate the neuroprotective effect of betaine on LPS-induced depression-like behavior in mice and its mechanism of action. ICR mice were randomly divided into four groups: the control group, the LPS model group (0.83 mg/kg), the positive drug group (MIDO, 50 mg/kg), and the betaine group (5% and 1% in drinking water). The betaine group was administered for 21 days, and on the 22nd day, except for the blank group, LPS (0.83 mg/kg) was intraperitoneally injected to establish a lipopolysaccharide-induced mice depression-like model. Twenty-four hours after LPS injection, the tail suspension test (TST), open field test (OFT), and sucrose preference test (SPT) were performed to evaluate the effect of betaine on LPS-induced depressive behavior in mice. After the behavioral study, the mouse brain, hippocampus, and serum were taken for detection. The expressions of cytokines and inflammatory mediators were detected by ELISA, HE staining, immunofluorescence, immunohistochemistry, and western blotting. Western blotting was used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, and ASC, the protein expression levels of the microglial polarization markers COX-2, inducible nitric oxide synthase (iNOS), and CD206. The results showed that betaine significantly ameliorated the depression-like behavior in LPS-induced mice, significantly attenuated the production of proinflammatory cytokines and increased the release of an anti-inflammatory cytokines. Betaine decreased the expression of the NLRP3 inflammasome, decreased the expression of M1 polarization markers, tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), COX-2, and iNOS and promoted the expression of M2 polarization marker CD206. Our study suggests that betaine may promote the transition of microglia from the M1 to the M2 phenotype by inhibiting NLRP3 inflammasome activation, thereby attenuating lipopolysaccharide-induced depression-like behavior.

Emergence of the ABC Transporter Antibiotic Resistant Gene in Pathogenic Fungi Using In Silico Analysis

Adenosine triphosphate binding cassette proteins (ATP-ABC) transporters belong to a broad superfamily of proteins in all living organisms that have essential physiological functions. They are necessary for transporting a wide range of substrates across lipid membranes. ABC transport proteins share two conserved domain architectures: the multiple transmembrane helices (TMD) nucleotide binding protein domain (NBD). Fungi have evolved active transport pathways through endogenous and exogenous toxicants. Drug efflux from ABC transporters lowers the concentration of intracellular drugs, in many pathogens and cancer cells; therefore, efflux is subjected to extensive studies in pathogens. ABC transporter gene of aspergillus versicolor as reference and evaluated metadata of ABC transporter gene in comparison from dermatophyte and non-dermatophyte fungi using fungi DB and NCBI databases. ABC transporters gene was studied using gene homology via phylogenetic analysis. Study evaluated ABC transporters sub families, number of transcripts, exon, intron numbers sizes and position using gene sequence analysis by NCBI. The evolutionary history of ABC gene analyzed by using 12 proteins of both dermatophytes. Study also explored the single nucleotide polymorphism (SNPs) homology in dermatophytes using the NCBI SNP tool. ABC protein examined five sub-families which include ABC-B, ABC-C, ABC-D, ABC-F. Phylogenetic studies on ABC transporter genes showed its emergence in three major groups of dermatophytes and non- dermatophyte. Exon number of ABC transporter gene varies from 2 to 18 and one transcript number in dermatophytes and non-dermatophytes. The evolutionary history of ABC-B was analyzed by using 12 proteins of both dermatophytes and non-dermatophytes. ABC genes in dermatophytes were found in five subfamilies ABC-B, ABC-C, ABC-D, ABC-F and ABC-G. However, ABC transporter gene family survey suggested that ABC- C family is a highly emerged sub family in dermatophytes and non-dermatophytes. Study revealed that ABC protein has been found in five sub-families of ABC transporter gene in dermatophytes. It has one transcript number in dermatophytes and non-dermatophytes. Whereas, the exon numbers are varied from 2 to 18. Moreover, ABC transporter gene phylogenetic family survey suggested that ABC- C family is highly emerged in both fungal types.

Polysaccharide of Lactobacillus casei SB27 reduced colon cancer cell prognosis through mitochondrial damage by upregulation of HINT2.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. This study aimed to explore the effects of Polysaccharide of Lactobacilluscasei SB27 in colon cancer and its possible mechanisms. Colon cancer was induced by giving dextran sulfate sodium and Azoxymethane. Uman Colon Cancer Cell Line (HCT)-116 cells were used to vitro model in this experiment. Polysaccharide of L. casei SB27 reduced colon cancer in azoxymethane-dextran sulfate sodium (AOM+DSS)-induced mice. Polysaccharide of L. casei SB27 reduced colon cancer prognosis in vitro model. Polysaccharide of L. casei SB27 reduced short chain fatty acids by Bacillus coli. Polysaccharide of L. casei promoted mitochondrial damage by Calcium ion entry. Polysaccharide of L. casei induced histidine nucleotide binding protein 2/mitochondrial calcium uniporter (HINT2/MCU) signaling pathway. Immunocoprecipitation (IP) showed that HINT2 protein interlinked MCU protein. Polysaccharide of L. casei suppressed HINT2 ubiquitination. The regulation of HINT2 affected the effects of L. casei polysaccharide on colon cancer prognosis and mitochondrial damage by Calcium ion entry in vitro model. In conclusion, the present report demonstrated that polysaccharide of L. casei SB27 reduced colon cancer cell prognosis through mitochondrial damage by upregulation of HINT2. Polysaccharide of L. casei SB27 might be used for colon cancer treatment and could be helpful for personalized treatment.

Electroacupuncture alleviates cognitive dysfunction and neuronal pyroptosis in septic mice.

Sepsis is defined as organ dysfunction caused by an uncontrolled response to infection and is followed by a high incidence of cognitive dysfunction, which can severely affect patients' quality of life. Previous studies have suggested that electroacupuncture (EA) is protective against sepsis-associated cognitive dysfunction and that pyroptosis plays a vital role in cognitive function. The aim of this study was to investigate the effect of EA on cognition and neuronal pyroptosis in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) surgery. Mice were randomly divided into three groups (control, CLP and CLP + EA). EA was performed at bilateral ST36 for three consecutive days after the surgery. The 7-day survival rate of each group was observed on the seventh day after the surgery. The Morris water maze (MWM) was used to test cognitive function from the 8th to 12th day after the surgery. We used transmission electron microscopy (TEM) and transferase dUTP nick-end labeling (TUNEL) staining to determine the structural integrity of hippocampal neuronal membranes and the number of surviving neurons in the hippocampal tissues, respectively. Expression of nucleotide-binding domain-like receptor protein 1 (NLRP1), caspase-1 and gasdermin-D (GSDM D) in hippocampal CA1 neurons was detected by Western blotting and real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), and caspase-1 concentrations were measured by enzyme-linked immunosorbent assay. Compared with the CLP group, 7-day survival rates and cognitive function were significantly improved in the CLP + EA group. After EA treatment, the integrity of the hippocampal CA1 neuronal membrane and mortality of hippocampal neurons were significantly decreased, and expression of NLRP1, caspase-1 and GSDM D was downregulated. EA can alleviate cognitive dysfunction and neuronal pyroptosis in septic mice.

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1β (T. nigroviridis IL-1β). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1β after activation.

Pentoxifylline decreases the activity of the nucleotide-binding oligomerization domain-like receptor protein 3 pathway: potential role for preventing arteriovenous fistula stenosis.

This study aimed to determine the effect of pentoxifylline (PTX) on the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome pathway and its role in preventing arteriovenous fistula (AVF) failure. Vein samples were collected from AVF failure patients and from patients who underwent surgical AVF as a control. The expressions of CD34 and NLRP3 in AVF tissues were detected by immunohistochemistry and Western blotting. Arteriovenous fistula rat models were established by the end-to-end anastomosis of the common carotid artery and external jugular vein. The AVF models were divided into the following groups: AVF, AVF + PTX, AVF + uraemia and AVF + uraemia + PTX. Six weeks after surgery, the AVF tissues in each group were collected to detect the expressions of CD34, NLRP3, caspase-1 and interleukin (IL)-1β by immunohistochemistry, Western blotting and real-time polymerase chain reaction. The expressions of NLRP3 and CD34 in human AVF failure tissues were significantly higher than those in normal veins (p < 0.001), indicating that NLRP3 was upregulated in patients with AVF failure. In our animal study, the veins in the AVF + uraemia group exhibited heavy hyperplasia, and the boundary between the media and the adventitia was not clear. However, PTX alleviated this hyperplasia. Compared with the AVF models, the AVF + uraemia models had much higher expressions of NLRP3, caspase-1, IL-1β and CD34 (p < 0.001). However, PTX had the opposite effect against uraemia on the NLRP3 inflammasome pathway at both the gene and protein levels. Our findings provide new insights that show that PTX can decrease the activity of the NLRP3 inflammasome pathway in AVF models. Pentoxifylline has the potential as a drug for preventing intimal hyperplasia and AVF failure.

Effect of Bushen Antai recipe on pyroptosis mechanism of subclinical hypothyroidism decidual cells in early pregnancy.

Subclinical hypothyroidism (SH) is common during pregnancy. It is not clear whether decidual cells in SH undergo pyroptosis during pregnancy. This study aimed to investigate the possible mechanism of Bushen Antai recipe (BAR) in the treatment of SH in early pregnancy and the relationship between SH during pregnancy and decidual cell pyroptosis through a rat model. A total of 60 female rats were divided into control group, model group, levothyroxine (L-T4) group, low-dose BAR group (6 g/kg), medium-dose BAR group (12 g/kg), and high-dose BAR group (24 g/kg). The control group underwent pseudothyroidectomy, while the remaining groups established nonpregnant SH rat models. Except for the blank control group, rats were successfully established with SH models during pregnancy. The control group and the model group were treated with saline or BAR. The animals were sacrificed 12 hours after the last administration. The levels of serum thyroid-stimulating hormone (TSH), free thyroxine (FT4), interleukin-1β (IL-1β), and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot was used to detect the expression of decidual nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, Caspase-1, and porin family proteins. There was no significant difference in serum FT4 among groups (P>0.05). Compared with the control group, serum TSH, IL-1β, IL-18, and NLRP3, Caspase-1, and gasdermin D (GSDMD) proteins in the decidua of the model group were significantly increased (P<0.05). Compared with the model group, the L-T4 group and the high-dose BAR group could significantly decrease the levels of serum TSH, IL-1β, IL-18, and NLRP3, Caspase-1, and GSDMD in decidual tissue (P<0.05). The medium dose of BAR could significantly decrease the levels of TSH, NLRP3, Caspase-1, and GSDMD (P<0.05), and the low dose group of BAR significantly decreased the levels of TSH, NLRP3, and GSDMD (P<0.05). Among them, the high-dose group of BAR had the best reducing effect on IL-18, NLRP3, Caspase-1, and GSDMD. The decidual cells of SH rats in early pregnancy underwent pyroptosis with a high inflammatory response. BAR could improve TSH level in SH during pregnancy, inhibit decidual cell pyroptosis, and reduce the expression of inflammatory factors.

Clinical features of Chilean patients with Fibrous Dysplasia/McCune-Albright Syndrome

Fibrous Dysplasia/McCune-Albright Syndrome (FD/MAS) is characterized by a spectrum of manifestations that may include fibrous dysplasia of bone and multiple endocrinopathies. To describe the clinical spectrum, the study and follow-up of patients with FD/MAS cared at our institution. Review of medical records of 12 pediatric and adult patients (11 women) who met the clinical and genetic diagnostic criteria for FD/ MAS. The patients' mean age at diagnosis was 4.9 ± 5.5 years. The most common initial clinical manifestation was peripheral precocious puberty (PPP) in 67% of patients and 75% had café-au-lait spots. Fibrous dysplasia was present in 75% of patients and the mean age at diagnosis was 7.9 ± 4.7 years. Ten patients had a bone scintigraphy, with an age at the first examination that varied between 2 and 38 years of age. The most frequent location of dysplasia was craniofacial and appendicular. No patient had a recorded history of cholestasis, hepatitis, or pancreatitis. In four patients, a genetic study was performed that was positive for the pathogenic variant of guanine nucleotide binding protein, alpha stimulating (GNAS). These patients demonstrate the variable nature of the clinical presentation and study of FD/MAS. It is essential to increase the index of diagnostic suspicion and adherence to international recommendations.

Multi-Omics Analysis of GNL3L Expression, Prognosis, and Immune Value in Pan-Cancer.

Simple SummaryGuanine nucleotide-binding protein-like 3-like (GNL3L) is a novel GTP-binding nucleolar protein. In this study, we analyzed the expression, prognosis, and immune roles of GNL3L in pan-cancer from multiple omics analyses. The final results showed that GNL3L is differentially expressed in a variety of cancers, plays a prognostic role, and has good immune value. Moreover, GNL3L may affect the occurrence of cancer through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, we established an esophageal cancer (ESCA) prediction model with strong predictive ability and proved that GNL3L can significantly affect the proliferation ability of esophageal cancer cells through clone formation assays. In conclusion, GNL3L is an important biomarker.Guanine nucleotide-binding protein-like 3-like protein (GNL3L) is a novel, evolutionarily conserved, GTP-binding nucleolar protein. This study aimed to investigate the expression, prognosis, and immune value of GNL3L in pan-cancer from multiple omics analyses. Firstly, the expression and prognostic value of GNL3L in pan-cancer were discussed using the TIMER2 database, the GEPIA database, the cBioportal database, COX regression analysis, and enrichment analysis. The association of GNL3L with tumor mutational burden (TMB), tumor microsatellite instability (MSI), mismatch repair (MMR) genes, and immune cells was then analyzed. Finally, an esophageal cancer (ESCA) prediction model was established, and GNL3L clone formation assays were performed. The final results showed that GNL3L is differentially expressed in the vast majority of cancers, is associated with the prognosis of various cancers, and may affect cancer occurrence through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, it was found that the correlation between GNL3L and TMB, MSI, MMR, and various immune cells is significant. The established ESCA prediction model had a strong predictive ability, and GNL3L could significantly affect the proliferation of esophageal cancer cells. In conclusion, GNL3L may serve as an important prognostic biomarker and play an immunomodulatory role in tumors.

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria.

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Cilostazol Alleviates NLRP3 Inflammasome–Induced Allodynia/Hyperalgesia in Murine Cerebral Cortex Following Transient Ischemia: Focus on TRPA1/Glutamate and Akt/Dopamine/BDNF/Nrf2 Trajectories

Global cerebral ischemia/reperfusion (I/R) provokes inflammation that augments neuropathic pain. Cilostazol (CLZ) has pleiotropic effects including neuroprotection in several ravaging central disorders; nonetheless, its potential role in transient central ischemic-induced allodynia and hyperalgesia has not been asserted before. Rats were allocated into 4 groups; sham, sham + CLZ, and 45 min-bilateral carotid occlusion followed by a 48 h-reperfusion period either with or without CLZ (50 mg/kg; p.o) post-treatment. CLZ prolonged latency of hindlimb withdrawal following von Frey filaments, 4 °C cold, and noxious mechanical stimulations. Histopathological alterations and the immunoexpression of glial fibrillary acidic protein induced by I/R were reduced by CLZ in the anterior cingulate cortex (ACC) area, while, CLZ enhanced intact neuronal count. Meanwhile, CLZ modulated cerebral cortical glutamate, dopamine neurotransmission, and transient receptor potential ankyrin 1 (TRPA1). CLZ anti-inflammatory potential was mediated by the downregulated p65 NF-κB and sirtuin-1 enhancement to reduce nucleotide-binding domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), active caspase-1, and interleukin-1β, indicative of inflammasome deactivation. It also revealed an antioxidant capacity via boosting nuclear factor E2-related factor (Nrf2) enhancing glutathione through forkhead box protein O3a (FOXO3a) reduction. Additionally, CLZ triggered neuronal survival by promoting the p-content of Akt, TrkB, and CREB as well as BDNF content. A novel approach of CLZ in hindering global cerebral I/R–mediated neuropathy is firstly documented herein to forward its adjunct action via deactivating the NLRP3 inflammasome, besides enhancing Nrf2 axis, neuronal survival, and dopamine neurotransmission as well as inhibiting TRPA1 and excitotoxicity.Graphical abstract

GNB2L1 gene expression and clinical value in hepatocellular carcinoma based on bioinformatics

Objective: To analyze guanine nucleotide-binding protein subunit beta-2-like 1 (GNB2L1) expression based on bioinformatics, so as to evaluate its role and its relationship with survival rate during the occurrence and development of hepatocellular carcinoma. Methods: GEPIA, UALCAN and HPA databases were used to analyze the expression level of GNB2L1 and its relationship with HCC survival rate. Mutations in the GNB2L1 gene and their impact on survival were analyzed using the cBioPortal database. LinkedOmics database was used to analyze GNB2L1-related genes in HCC. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were performed simultaneously. STEING database was used to construct the GNB2L1 protein interaction network. TIMER database was used to analyze the relationship between GNB2L1 gene expression and immune infiltration in hepatocellular carcinoma. Differential expression of GNB2L1 in plasma platelets of HCC patients and healthy controls was analyzed using mRNA-based sequencing technology. Data between groups were compared using an independent-samples t-test. Results: GNB2L1 expression level was significantly increased in HCC tissues (P<0.05), and its expression was significantly correlated with body weight, classification and stage (P<0.05). The overall survival rate was higher in GNB2L1 low expression group (P<0.001). GNB2L1 and its related genes were related to biological process regulation, metabolic process, protein binding, oxidative phosphorylation, JAK-STAT signaling pathway, Ras signaling pathway and so on. GNB2L1 had interaction with RPS12, RPS11 and RPL19, and participated in multiple biological processes such as liver regeneration and positive regulation of endogenous apoptotic signaling pathway. GNB2L1 expression was significantly positively correlated with the infiltration degree of various immune cells in HCC (P<0.05). Cox regression analysis showed that GNB2L1 was an independent risk factor for lower survival rate in patients with HCC [Hazard ratio (95% confidence interval)=1.456 (1.034~2.051), P=0.031]. GNB2L1expression levels were significantly higher in platelets of HCC patients than that of healthy controls (10.40±1.36 vs. 9.58±0.51, t=2.194, P=0.037). Conclusion: GNB2L1 has high expression and close relationship to survival rate in HCC. Therefore, GNB2L1 may be a potential biomarker of HCC.

Berberine ameliorates pulmonary inflammation in mice with influenza viral pneumonia by inhibiting NLRP3 inflammasome activation and gasdermin D-mediated pyroptosis.

Viral pneumonia is a common complication caused by Influenza A virus infection and is characterized by severe pulmonary inflammation. A previous study showed that berberine (BBR) significantly ameliorated the pulmonary inflammation in mice with influenza viral pneumonia but its underlying mechanism is not entirely understood. In this study, we reproduced the mouse model of influenza viral pneumonia through intranasal infection of A/Puerto Rico/8/34 (H1N1), to further investigate the anti-inflammatory mechanism of BBR based on nucleotide-binding oligomerization domain-like receptor protein (NLRP) 3 inflammasome activation and Gasdermin D (GSDMD)-mediated pyroptosis. Consistent with MCC950 (10 mg/kg, a specific NLRP3 inflammasome inhibitor), BBR (10 mg/kg) obviously improved the weight loss and survival rate of infected mice, alleviated their pulmonary inflammation, and suppressed the accumulation of tumor necrosis factor and interleukin (IL)-6 in lungs without obvious inhibition on viral multiplication (hemagglutinin titer and nucleoprotein messenger RNA). Moreover, BBR (10 mg/kg) reduced the expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cysteinyl aspartate-specific proteinase (Caspase)1 (Caspase1 precursor [Pro-caspase1] + Caspase1p20 subunit) and the ratio of Caspase1p20 subunit to Caspase1, thus inhibiting the NLRP3 inflammasome activation and resulting in the decreased contents of mature IL-1β and IL-18 in lungs. The GSDMD expression (GSDMD precursor [Pro-GSDMD] + GSDMD-N terminal [NT]) and the ratio of GSDMD-NT to GSDMD were also declined by BBR (10 mg/kg). These evidence indicate that BBR may ameliorate pulmonary inflammation in mice with influenza viral pneumonia through inhibiting NLRP3 inflammasome activation, as well as depressing GSDMD-mediated pyroptosis via declining GSDMD expression and restraining NLRP3 inflammasome-mediated GSDMD activation.

Low-intensity focused ultrasound attenuates early traumatic brain injury by OX-A/NF-κB/NLRP3 signaling pathway.

Background: Traumatic brain injury (TBI) is a serious hazard to human health and is characterized by high rates of disability and mortality. It is necessary to explore new effective treatment methods to reduce the impact of TBI on individuals and society. As an emerging neuromodulation technique, ultrasound is used to treat some neurological diseases, but the neuroprotective mechanism of low-intensity focused ultrasound (LIFUS) in TBI remains unclear. We aimed to investigate the protective effects and potential mechanisms of LIFUS in TBI.Methods: A rat model of TBI was established using the free-fall method. After establishing the TBI model, the hypothalamus region was covered with LIFUS radiation, and an orexin receptor 1 (OXR1) antagonist (SB334867) was injected intraperitoneally. Neurobehavioral examination, Nissl staining, hematoxylin and eosin staining of the brain tissue, and brain water content, were performed 3 days later. Western blotting, quantitative real-time polymerase chain reaction, immunofluorescence staining, and immunohistochemical staining, were used to evaluate the neuroprotective mechanisms of LIFUS.Results: LIFUS improved tissue damage, neurological deficits, and brain edema. LIFUS can increase the expression of orexin-A (OX-A) and OXR1, significantly inhibit the activation of nuclear factor-κB (NF-κB) protein and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome after TBI, and reduce the release of pro-inflammatory factors after TBI; however, SB334867 can reverse this effect.Conclusions: This study suggests that LIFUS may play a neuroprotective role by promoting the release of OX-A from the hypothalamus and inhibiting the inflammatory response after TBI through the OX-A /NF-κB/NLRP3 pathway.

Effect of α-helical domain of Gi/o α subunit on GDP/GTP turnover.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, β, and γ subunits, and Gα has a GDP/GTP-binding pocket. When a guanine nucleotide exchange factor (GEF) interacts with Gα, GDP is released, and GTP interacts to Gα. The GTP-bound activated Gα dissociates from GEF and Gβγ, mediating the induction of various intracellular signaling pathways. Depending on the sequence similarity and cellular function, Gα subunits are subcategorized into four subfamilies: Gαi/o, Gαs, Gαq/11, and Gα12/13. Although the Gαi/o subtype family proteins, Gαi3 and GαoA, share similar sequences and functions, they differ in their GDP/GTP turnover profiles, with GαoA possessing faster rates than Gαi3. The structural factors responsible for these differences remain unknown. In this study, we employed hydrogen/deuterium exchange mass spectrometry and mutational studies to investigate the factors responsible for these functional differences. The Gα subunit consists of a Ras-like domain (RD) and an α-helical domain (AHD). The RD has GTPase activity and receptor-binding and effector-binding regions; however, the function of the AHD has not yet been extensively studied. In this study, the chimeric construct containing the RD of Gαi3 and the AHD of GαoA showed a GDP/GTP turnover profile similar to that of GαoA, suggesting that the AHD is the major regulator of the GDP/GTP turnover profile. Additionally, site-directed mutagenesis revealed the importance of the N-terminal part of αA and αA/αB loops in the AHD for the GDP/GTP exchange. These results suggest that the AHD regulates the nucleotide exchange rate within the Gα subfamily.

Research trends in ulcerative colitis: A bibliometric and visualized study from 2011 to 2021.

Background: Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease with repeated relapses and remissions. Despite decades of effort, numerous aspects, including the initiating event and pathogenesis of UC, still remain ambiguous, which requires ongoing investigation. Given the mass of publications on UC, there are multidimensional challenges to evaluating the scientific impact of relevant work and identifying the current foci of the multifaceted disease. Accordingly, herein, we aim to assess the global growth of UC research production, analyze patterns of research areas, and evaluate trends in this area. Methods: The Web of Science Core Collection of Clarivate Analytics was searched for articles related to UC published from 2011 to 2021. Microsoft Office Excel 2019 was used to visualize the number of publications over time. Knowledge maps were generated using CiteSpace and VOSviewer to analyze collaborations among countries, institutions, and authors and to present the journey of UC research as well as to reveal the current foci of UC research. Results: A total of 5,088 publications were evaluated in the present study. China had the most publications (1,099, 22.5%). Univ Calif San Diego was the most productive institution (126, 2.48%). William J Sandborn published the greatest number of articles (100, 1.97%). Toshifumi Hibi was the most influential author in the field with a betweenness centrality of 0.53. Inflammatory bowel diseases was identified as the most prolific journal (379, 7.45%). Gastroenterology was the most co-cited journal (3,730, 4.02%). “Vedolizumab,” “tofacitinib,” “Faecalibacterium prausnitzii,” “fecal microbiota transplantation (FMT),” “toll-like receptor 4,” and “nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome” were considered the hot topics. Conclusion: In UC research, manuscripts that had high impacts on the scientific community provided an evidence base. UC therapy has entered the era of personalized and precision therapy. As research on FMT, anti-integrin antibodies, Janus kinase inhibitors, and anti-tumor necrosis factor drugs continues to grow, their use in the clinical setting may also expand.

Probenecid induces the recovery of renal ischemia/reperfusion injury via the blockade of Pannexin 1/P2X7 receptor axis

Renal ischemia/reperfusion injury (RI/RI) is one of the main driving causes of acute kidney injury. However, effective treatment to limit injury and promote recovery and/or survival is still unavailable. Probenecid (PBN), a drug indicated for refractory gout, exhibits protective activities against several preclinical diseases including cerebral and myocardial I/RI via Pannexin 1 (Panx1) and P2X7 receptors' (P2X7R) inhibition. However, its protective role against RI/RI has not been previously addressed. Accordingly, we subjected rats to bilateral RI/RI with/or without PBN treatment. Twenty-four hours post-reperfusion, PBN showed mild tubular injury and reduced serum nephrotoxicity indices, gene and protein expression levels of Panx 1 and P2X7R, and ATP and pro-inflammatory cytokines' levels. The nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome signaling was also downregulated, as demonstrated by reduced gene and protein expression of NLRP3 and caspase-1, along with suppressed IL-1β maturation. Furthermore, PBN enhanced Tregs activity as indicated by elevated FoxP3 gene expression, IL-10, and TGF-β renal levels. On day 5 post-reperfusion, PBN noticeably enhanced renal recovery, as demonstrated by intact tubular epithelium and restored nephrotoxicity indices, Panx 1 and P2X7R gene and protein expression levels, ATP and pro-inflammatory cytokine levels, and NLRP3 inflammasome signaling. Besides, renal Tregs activity was also significantly increased. Our study elaborates for the first time the effectiveness of PBN in recovering post-ischemic renal injury through synergistic inhibition in Panx1/P2X7R axis leading to inactivation of NLRP3 inflammasome signaling and activation of Tregs in ischemic renal tissues. Therefore, PBN can be considered a promising drug for RI/RI treatment.