OBJECTIVE: To investigate the role and mechanism of the cyclic guanosine monophosphate-adenosine monophosphate synthase/stimulator of interferon gene (cGAS/STING) pathway in oleic acid-induced acute lung injury (ALI) in mice. METHODS: Male wild-type C57BL/6J mice were randomly divided into five groups (each n = 10): normal control group, ALI model group, and 5, 50, 500 μg/kg inhibitor pretreatment groups. The ALI model was established by tail vein injection of oleic acid (7 mL/kg), while the normal control group received no intervention. The inhibitor pretreatment groups were intraperitoneally injected with the corresponding doses of cGAS inhibitor RU.521 respectively 1 hour before modeling. At 24 hours post-modeling, blood was collected, and mice were sacrificed. Lung tissue pathological changes were observed under light microscopy after hematoxylin-eosin (HE) staining, and pathological scores were assessed. Western blotting was used to detect the protein expressions of cGAS, STING, phosphorylated TANK-binding kinase 1 (p-TBK1), phosphorylated interferon regulatory factor 3 (p-IRF3), and phosphorylated nuclear factor-κB p65 (p-NF-κB p65) in lung tissue. Immunohistochemistry was performed to observe STING and p-NF-κB positive expressions in lung tissue. Serum interferon-β (IFN-β) levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with the normal control group, the ALI model group exhibited significant focal alveolar thickening, intra-alveolar hemorrhage, pulmonary capillary congestion, and neutrophil infiltration in the pulmonary interstitium and alveoli, along with markedly increased pathological scores (10.33±0.58 vs. 1.33±0.58, P < 0.05). Protein expressions of cGAS, STING, p-TBK1, p-IRF3, and p-NF-κB p65 in lung tissue significantly increased [cGAS protein (cGAS/β-actin): 1.24±0.02 vs. 0.56±0.02, STING protein (STING/β-actin): 1.27±0.01 vs. 0.55±0.01, p-TBK1 protin (p-TBK1/β-actin): 1.34±0.03 vs. 0.22±0.01, p-IRF3 protein (p-IRF3/β-actin): 1.23±0.02 vs. 0.36±0.01, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 1.30±0.02 vs. 0.53±0.02, all P < 0.05], positive expressions of STING and p-NF-κB in lung tissue were significantly elevated [STING (A value): 0.51±0.03 vs. 0.30±0.07, p-NF-κB (A value): 0.57±0.05 vs. 0.31±0.03, both P < 0.05], and serum IFN-β levels were also significantly higher (ng/L: 256.02±3.84 vs. 64.15±1.17, P < 0.05). The cGAS inhibitor pretreatment groups showed restored alveolar structural integrity, reduced inflammatory cell infiltration, and decreased hemorrhage area, along with dose-dependent lower pathological scores as well as the protein expressions of cGAS, STING, p-TBK1, p-IRF3 and p-NF-κB p65 in lung tissue, with significant differences between the 500 μg/kg inhibitor group and ALI model group [pathological score: 2.67±0.58 vs. 10.33±0.58, cGAS protein (cGAS/β-actin): 0.56±0.03 vs. 1.24±0.02, STING protein (STING/β-actin): 0.67±0.03 vs. 1.27±0.01, p-TBK1 protein (p-TBK1/β-actin): 0.28±0.01 vs. 1.34±0.03, p-IRF3 protein (p-IRF3/β-actin): 0.32±0.01 vs. 1.23±0.02, p-NF-κB p65 protein (p-NF-κB p65/β-actin): 0.63±0.01 vs. 1.30±0.02, all P < 0.05]. Compared with the ALI model group, positive expressions of STING and p-NF-κB in lung tissue were significantly reduced in the 500 μg/kg inhibitor group [STING (A value): 0.40±0.01 vs. 0.51±0.03, p-NF-κB (A value): 0.43±0.02 vs. 0.57±0.05, both P < 0.05], and serum IFN-β levels were also markedly reduced (ng/L: 150.03±6.19 vs. 256.02±3.84, P < 0.05). CONCLUSIONS The cGAS/STING pathway is activated in oleic acid-induced ALI, leading to exacerbated inflammatory responses and increased lung damage. RU.521 can inhibit cGAS, thereby down-regulating the expression of pathway proteins and cytokines, and providing protection to lung tissue.
Animals ; Acute Lung Injury/chemically induced* ; Male ; Nucleotidyltransferases/metabolism* ; Mice ; Signal Transduction ; Mice, Inbred C57BL ; Membrane Proteins/metabolism* ; Oleic Acid/adverse effects* ; Transcription Factor RelA/metabolism* ; Lung/pathology* ; Interferon Regulatory Factor-3/metabolism* ; Disease Models, Animal

OBJECTIVE To investigate the ameliorative effect of Pinelliae Rhizoma Praeparatum Cum Alumine and its effective components on inflammatory response in mice with acute lung injury(ALI).METHODS A mice model of acute lung injury induced by lipopolysaccharide(LPS)was used as the in vivo animal model.The levels of inflammatory factors TNF-α,IL-6,and IL-1β,the total protein concentration,the number of inflammatory cells in bronchoalveolar lavage fluid,the ratio of lung wet weight to dry weight,and the pathological damage of lung tissues were taken as the evaluation indexes.RAW264.7 macrophages induced by LPS were used as the in vitro experimental model,with the mRNA levels of intracellular inflammatory factors TNF-α,IL-1β,and IL-6 as indicators,systematic solvent extraction and silica gel column chromatography were used to separate and screen the effective parts and components of Pinelliae Rhizoma Praeparatum Cum Alumine for treating acute lung injury.The component composition of the effective fractions was analyzed by mass spectrometry,the content of cyclic dipeptide components in the effective fraction was determined,and the anti-in-flammatory activity of cyclic dipeptides was verified in vitro.RESULTS The ethyl acetate extraction fraction obtained by extracting the supernatant of water extraction and ethanol precipitation of Pinelliae Rhizoma Praeparatum Cum Alumine(referred to as the ethyl acetate fraction for short)significantly improved the inflammatory response in mice with acute lung injury and significantly reduced the mRNA levels of TNF-α,IL-1β,and IL-6 in RAW264.7 cells induced by LPS.The Fr.1 fraction isolated from the ethyl acetate frac-tion by silica gel column chromatography had anti-inflammatory effects in vitro.Mass spectrometry determined the content of two main cyclic dipeptide components,cyclo-(Pro-Phe)and cyclo-(Pro-Val),in the Fr.1 fraction.After mixing these two cyclic dipeptide components in the molar ratio in the Fr.1 fraction and administering them in vitro,the results showed that the combination of these two cyclic dipeptide components could significantly inhibit the mRNA expression levels of TNF-α,IL-1β,and IL-6 in RAW264.7 cells induced by LPS.CONCLUSION Cyclic dipeptide component is one of the main material basis categories for the anti-inflammatory effect of Pinelliae Rhizoma Praeparatum Cum Alumine.

BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

OBJECTIVE To investigate the ameliorative effect of Pinelliae Rhizoma Praeparatum Cum Alumine and its effective components on inflammatory response in mice with acute lung injury(ALI).METHODS A mice model of acute lung injury induced by lipopolysaccharide(LPS)was used as the in vivo animal model.The levels of inflammatory factors TNF-α,IL-6,and IL-1β,the total protein concentration,the number of inflammatory cells in bronchoalveolar lavage fluid,the ratio of lung wet weight to dry weight,and the pathological damage of lung tissues were taken as the evaluation indexes.RAW264.7 macrophages induced by LPS were used as the in vitro experimental model,with the mRNA levels of intracellular inflammatory factors TNF-α,IL-1β,and IL-6 as indicators,systematic solvent extraction and silica gel column chromatography were used to separate and screen the effective parts and components of Pinelliae Rhizoma Praeparatum Cum Alumine for treating acute lung injury.The component composition of the effective fractions was analyzed by mass spectrometry,the content of cyclic dipeptide components in the effective fraction was determined,and the anti-in-flammatory activity of cyclic dipeptides was verified in vitro.RESULTS The ethyl acetate extraction fraction obtained by extracting the supernatant of water extraction and ethanol precipitation of Pinelliae Rhizoma Praeparatum Cum Alumine(referred to as the ethyl acetate fraction for short)significantly improved the inflammatory response in mice with acute lung injury and significantly reduced the mRNA levels of TNF-α,IL-1β,and IL-6 in RAW264.7 cells induced by LPS.The Fr.1 fraction isolated from the ethyl acetate frac-tion by silica gel column chromatography had anti-inflammatory effects in vitro.Mass spectrometry determined the content of two main cyclic dipeptide components,cyclo-(Pro-Phe)and cyclo-(Pro-Val),in the Fr.1 fraction.After mixing these two cyclic dipeptide components in the molar ratio in the Fr.1 fraction and administering them in vitro,the results showed that the combination of these two cyclic dipeptide components could significantly inhibit the mRNA expression levels of TNF-α,IL-1β,and IL-6 in RAW264.7 cells induced by LPS.CONCLUSION Cyclic dipeptide component is one of the main material basis categories for the anti-inflammatory effect of Pinelliae Rhizoma Praeparatum Cum Alumine.

BACKGROUND:Degeneration of nucleus pulposus cells is a key component of intervertebral disc degeneration.Ferroptosis,a novel form of programmed cell death,is closely associated with the onset and progression of intervertebral disc degeneration;however,its precise mechanisms remain unclear.OBJECTIVE:To establish an oxidative stress model in vitro by inducing ferroptosis in nucleus pulposus cells using tert-butyl hydroperoxide and to investigate the mechanisms of tert-butyl hydroperoxide-induced ferroptosis in nucleus pulposus cells,thereby elucidating the role of ferroptosis in the pathogenesis of intervertebral disc degeneration.METHODS:Nucleus pulposus cells were treated with varying concentrations of tert-butyl hydroperoxide(0,25,50,100,and 200 μmol/L),and cell morphology and viability were assessed using fluorescence microscopy and the cell counting kit-8 assay.Interventions with 100 μmol/L tert-butyl hydroperoxide,10 μmol/L RSL3,or dimethylsulfoxide were applied to nucleus pulposus cells,and cell proliferation was evaluated using the EdU assay.The expression levels of ferroptosis-related proteins(glutathione peroxidase 4,ferritin heavy chain 1,PTGS2,and ACSL4)and intervertebral disc degeneration marker proteins(matrix metalloproteinase 13 and Col2A)were analyzed via western blot and immunofluorescence.Additionally,reactive oxygen species and lipid peroxidation levels were quantified using the reactive oxygen species detection kit and C11-BODIPY probe.Mitochondrial morphological changes were observed under transmission electron microscopy.RESULTS AND CONCLUSION:(1)tert-Butyl hydroperoxide treatment significantly reduced the viability and proliferation of nucleus pulposus cells.(2)tert-Butyl hydroperoxide induced typical ferroptosis-related morphological changes in nucleus pulposus cells.(3)tert-Butyl hydroperoxide exposure led to a decrease in the expression of ferroptosis-suppressing proteins glutathione peroxidase 4 and ferritin heavy chain 1,while increasing the expression of ferroptosis-promoting factors ACSL4 and PTGS2.(4)tert-Butyl hydroperoxide elevated intracellular reactive oxygen species production and lipid peroxidation levels in nucleus pulposus cells.(5)Transmission electron microscopy revealed ferroptosis-specific mitochondrial changes in nucleus pulposus cells treated with tert-butyl hydroperoxide,including contraction,reduced cristae,and increased membrane density.(6)tert-Butyl hydroperoxide treatment also resulted in the increased expression of matrix metalloproteinase 13 and decreased expression of Col2A in nucleus pulposus cells.In conclusion,tert-butyl hydroperoxide induces ferroptosis in nucleus pulposus cells,contributing to the development of intervertebral disc degeneration.This process may represent a key pathological mechanism in intervertebral disc degeneration and offers potential targets for developing novel therapeutic strategies.

AIM:To explore the effects of CD38 on lysosome reformation and cholesterol efflux in macro-phages.METHODS:Bone marrow-derived macrophages from low-density lipoprotein(LDL)receptor knockout(LDLr-/-)mice were cultured as cell model.Live cell imaging system was applied to evaluate the effect of nicotinic acid adenine di-nucleotide phosphate(NAADP)on lysosome number.ELISA was conducted to measure NAADP level in macrophages.After the cells were treated with nicotinic acid(NA),RT-qPCR was conducted to detect CD38 mRNA expression,and Western blot was conducted to observe CD38 protein expression and phosphorylated transcription factor EB(TFEB)level.Laser scanning confocal microscopy was applied to evaluate the influence of CD38/NAADP signaling on lysosome number and cholesterol egression.RESULTS:NAADP remarkably increased lysosome number(P<0.05),and this effect was significantly inhibited by NAADP antagonist NED-19,Ca2+ chelator BAPTA,and calcineurin inhibitor CsA(P<0.05).CD38 markedly enhanced NAADP synthesis in macrophages(P<0.05).NAADP synthetic substrate NA prominently ele-vated the expression of CD38 mRNA and protein(P<0.05).NA significantly decreased the phosphorylated TFEB level;this effect was also attenuated by NED-19,BAPTA and CsA(P<0.05).Disrupting CD38/NAADP signaling pathway markedly inhibited NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux in macrophages(P<0.05).NA-induced enhancement of lysosome number,lysosomal free cholesterol and cytosol cholesterol ester efflux abolished in LDLr/CD38 DKO macrophages(P<0.05),whereas these effects induced by NA were recovered after CD38 gene rescue.CONCLUSION:CD38 triggers lysosome reformation via TFEB and consequently pro-motes the efflux of lysosomal free cholesterol and cytosol cholesterol ester.

Objective:To explore the risk factors of intensive care unit-acquired weakness (ICU-AW) and the characteristics of Medical Research Council (MRC) score and electromyogram.Methods:A case control study was conducted. Patients with mechanical ventilation ≥ 7 days and MRC score admitted to department of respiratory and critical care medicine of China-Japan Friendship Hospital from September 2018 to January 2020 were enrolled, and they were divided into ICU-AW group (MRC score < 48) and non-ICU-AW group (MRC score ≥ 48) according to MRC score. The general situation, past medical history, related risk factors, MRC score, respiratory support mode, laboratory examination results, electromyogram examination results, ICU-AW related treatment, outcome and length of ICU stay were collected, and the differences between the two groups were compared. The risk factors of ICU-AW were analyzed by binary multivariate Logistic regression, and the characteristics of MRC score and electromyogram were analyzed.Results:A total of 60 patients were enrolled in the analysis, including 17 patients in ICU-AW group and 43 patients in non-ICU-AW group. Univariate analysis showed that there were significant differences in acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score, sequential organ failure assessment (SOFA) score, brain natriuretic peptide (BNP), blood urea nitrogen (BUN) on the first day of ICU admission and the ratio of invasive mechanical ventilation between ICU-AW group and non-ICU-AW group [APACHEⅡ score: 21 (18, 25) vs. 18 (15, 22), SOFA score: 7 (5, 12) vs. 5 (3, 8), BNP (ng/L): 364.3 (210.1, 551.2) vs. 160.1 (66.8, 357.8), BUN (mmol/L): 9.9 (6.2, 17.0) vs. 6.0 (4.8, 9.8), invasive mechanical ventilation ratio: 88.2% vs. 46.5%, all P < 0.05]. Binary multivariate Logistic regression analysis showed no independent risk factor for ICU-AW. The average MRC score of 17 ICU-AW patients was 33±11. The limb weakness was symmetrical, and the proximal limb weakness was the main manifestation. Electromyography examination showed that the results of nerve conduction examination in ICU-AW patients mainly revealed that the amplitude of compound muscle action potential (CMAP) and sensory nerve action potentials (SNAP) were decreased, and the conduction velocity was slowed down; needle electromyography showed increased area of motor unit potential (MUP), prolonged time limit and a large number of spontaneous potentials. Prognosis evaluation showed that compared with non-ICU-AW group, patients in ICU-AW group underwent more tracheotomy (70.6% vs. 11.6%), longer length of ICU stay (days: 57±52 vs. 16±8), and more rehabilitation treatment (58.8% vs. 14.0%), and the differences were statistically significant (all P < 0.01). Conclusions:The occurrence of ICU-AW may be related to high APACHEⅡ score and SOFA score, high levels of BNP and BUN on the first day of ICU admission and the proportion of invasive mechanical ventilation, but the above factors are not independent risk factors for ICU-AW. The MRC score of ICU-AW patients was characterized by symmetrical limb weakness, mainly proximal limb weakness; in electromyography examination, the nerve conduction examination results mainly showed that CMAP and SNAP amplitude were decreased, and conduction velocity was slowed down; needle electromyography examination showed increased MUP area, prolonged duration and a large number of spontaneous potentials.

Extracorporeal membrane oxygenation (ECMO) is a viable ultimate support therapy for patients with severe cardiorespiratory failure. Antimicrobial agents are commonly prescribed in critically ill patients with ECMO. However, an increasing number of studies have shown that ECMO circuit is associated with significant pharmacokinetic (PK) alterations, including the increased volume of distribution and reduced the clearance. In addition, the critical illness pathophysiology can also influence PK of antimicrobial, such as systemic inflammation, excessive fluid resuscitation, hypo-albuminemia, worsening hepatic or renal function. These PK alterations may increase the possibility of therapeutic failure or toxicity. Therefore, this study reviews published studies of the effects of ECMO on PK of antimicrobial agents in adults and makes preliminary recommendations on possible dosing regimen.

Rv2742 is a novel gene identified from Mycobacterium tuberculosis H37Rv by the proteogenomics strategy. The aim of this study was to establish a system of soluble expression and purification of the missing protein Rv2742 in M. tuberculosis H37Rv, to provide reference for further research on the biological function of Rv2742. The soluble protein was not successfully induced by prokaryotic expression vectors pGEX-4T-2-Rv2742, pET-32a-Rv2742, pET-28a-Rv2742 and pMAL-c2X-Rv2742. After the codon of novel gene Rv2742 was optimized according to E. coli codon usage frequency, only the recombinant strain containing plasmid pMAL-c2X-Rv2742 could produce soluble products of Rv2742 encoding gene. In addition, the expression effects of the desired fusion protein were also analyzed under different conditions including hosts, culture temperatures and IPTG concentrations. The optimum expression conditions were as follows: Rosetta (DE3) host, 16 °C culture temperature and 0.5 mmol/L IPTG. After being purified by affinity chromatography with amylose resin, the fusion protein sequence was confirmed by LC-MS/MS. These results indicated that the novel gene Rv2742 product could be successfully induced and expressed in a soluble form by the expression system pMAL-c2X with MBP tag. Our findings provide reference for studies on potential interaction and immunogenicity.
Chromatography, Liquid ; Cloning, Molecular ; Escherichia coli ; Mycobacterium tuberculosis ; genetics ; Recombinant Fusion Proteins ; Tandem Mass Spectrometry