UK5099 Inhibits the NLRP3 Inflammasome Independently of its Long-Established Target Mitochondrial Pyruvate Carrier.

Targeting NLRP3 inflammasome has been recognized as a promising therapeutic strategy for the treatment of numerous common diseases. UK5099, a long-established inhibitor of mitochondrial pyruvate carrier (MPC), is previously found to inhibit macrophage inflammatory responses independent of MPC expression. However, the mechanisms by which UK5099 inhibit inflammatory responses remain unclear. Here, it is shown that UK5099 is a potent inhibitor of the NLRP3 inflammasome in both mouse and human primary macrophages. UK5099 selectively suppresses the activation of the NLRP3 but not the NLRC4 or AIM2 inflammasomes. Of note, UK5099 retains activities on NLRP3 in macrophages devoid of MPC expression, indicating this inhibitory effect is MPC-independent. Mechanistically, UK5099 abrogates mitochondria-NLRP3 interaction and in turn inhibits the assembly of the NLRP3 inflammasome. Further, a single dose of UK5099 persistently reduces IL-1ß production in an endotoxemia mouse model. Importantly, structure modification reveals that the inhibitory activities of UK5099 on NLRP3 are unrelated to the existence of the activated double bond within the UK5099 molecule. Thus, this study uncovers a previously unknown molecular target for UK5099, which not only offers a new candidate for the treatment of NLRP3-driven diseases but also confounds its use as an MPC inhibitor in immunometabolism studies.

Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis.

Background: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders without available pharmacological therapies. Dynasore is a cell-permeable molecule that inhibits GTPase activity and exerts protective effects in several disease models. However, whether dynasore can alleviate lipopolysaccharide (LPS)-induced ALI is unknown. This study investigated the effect of dynasore on macrophage activation and explored its potential mechanisms in LPS-induced ALI in vitro and in vivo. Methods: Bone marrow-derived macrophages (BMDMs) were activated classically with LPS or subjected to NLRP3 inflammasome activation with LPS+ATP. A mouse ALI model was established by the intratracheal instillation (i.t.) of LPS. The expression of PYD domains-containing protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) protein was detected by Western blots. Inflammatory mediators were analyzed in the cell supernatant, in serum and bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assays. Morphological changes in lung tissues were evaluated by hematoxylin and eosin staining. F4/80, Caspase-1 and GSDMD distribution in lung tissue was detected by immunofluorescence. Results: Dynasore downregulated nuclear factor (NF)-κB signaling and reduced proinflammatory cytokine production in vitro and inhibited the production and release of interleukin (IL)-1ß, NLRP3 inflammasome activation, and macrophage pyroptosis through the Drp1/ROS/NLRP3 axis. Dynasore significantly reduced lung injury scores and proinflammatory cytokine levels in both BALF and serum in vivo, including IL-1ß and IL-6. Dynasore also downregulated the co-expression of F4/80, caspase-1 and GSDMD in lung tissue. Conclusion: Collectively, these findings demonstrated that dynasore could alleviate LPS-induced ALI by regulating macrophage pyroptosis, which might provide a new therapeutic strategy for ALI/ARDS.

MiniMelt: An instrument for real-time tracking of electron beam additive manufacturing using synchrotron x-ray techniques.

The development of a sample environment for in situ x-ray characterization during metal Electron Beam Powder Bed Fusion (PBF-EB), called MiniMelt, is presented. The design considerations, the features of the equipment, and its implementation at the synchrotron facility PETRA III at Deutsches Elektronen-Synchrotron, Hamburg, Germany, are described. The equipment is based on the commercially available Freemelt ONE PBF-EB system but has been customized with a unique process chamber to enable real-time synchrotron measurements during the additive manufacturing process. Furthermore, a new unconfined powder bed design to replicate the conditions of the full-scale PBF-EB process is introduced. The first radiography (15 kHz) and diffraction (1 kHz) measurements of PBF-EB with a hot-work tool steel and a Ni-base superalloy, as well as bulk metal melting with the CMSX-4 alloy, using the sample environment are presented. MiniMelt enables time-resolved investigations of the dynamic phenomena taking place during multi-layer PBF-EB, facilitating process understanding and development of advanced process strategies and materials for PBF-EB.

MiR-361-5p Inhibits the Wnt Axis via Targeting Lgr4 and Promotes Sepsis-induced Myocardial Injury.

OBJECTIVE: A growing body of evidence demonstrated that microRNAs (miRNAs) play a key role in sepsis-induced organ dysfunction. However, the mechanism of miR-361-5p in sepsis-induced myocardial injury remains to be clarified. METHODS: A mouse model of sepsis-induced myocardial injury was established using lipopolysaccharide (LPS). MiR-361-5p expression level was determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). G protein-coupled receptor-4 (Lgr4), apoptosis-related proteins, and the Wnt signaling pathway-related proteins were determined by Western blotting. The relationship between miR-361-5p and Lgr4 was determined using dual-luciferase reporter (DLR) and RNA immunoprecipitation (RIP) assays. RESULTS: MiR-361-5p expression level was upregulated in the mouse model of sepsis-induced myocardial injury, while an opposite result was found for Lgr4 expression level. Knockdown of miR-361-5p protected the mouse model of sepsis-induced myocardial injury against inflammation and oxidative stress, and reduced cardiomyocyte (CM) apoptosis, which could be reversed by knockdown of Lgr4. The analysis of underlying mechanism revealed that miR-361-5p could target Lgr4 to modulate the activity of Wnt axis in CM apoptosis. CONCLUSION: MiR-361-5p could aggravate myocardial injury in LPS-induced septic mice by targeting Lgr4 to inhibit the Wnt axis.

Clinical and Pulmonary CT Characteristics of Patients Infected With the SARS-CoV-2 Omicron Variant Compared With Those of Patients Infected With the Alpha Viral Strain.

Background: Omicron has become the dominant variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) globally. We aimed to compare the clinical and pulmonary computed tomography (CT) characteristics of the patients infected with SARS-CoV-2 Omicron with those of patients infected with the Alpha viral strain. Methods: Clinical profiles and pulmonary CT images of 420 patients diagnosed with coronavirus disease-2019 (COVID-19) at Ningbo First Hospital between January 2020 and April 2022 were collected. Demographic characteristics, symptoms, and imaging manifestations of patients infected with the SARS-CoV-2 Omicron variant were compared with those of patients infected with the Alpha strain. Results: A total of 38 patients were diagnosed to be infected with the Alpha strain of SARS-CoV-2, whereas 382 patients were thought to be infected with the Omicron variant. Compared with patients infected with the Alpha strain, those infected with the Omicron variant were younger, and a higher proportion of men were infected (P < 0.001). Notably, 93 (24.3%) of the patients infected with Omicron were asymptomatic, whereas only two (5.3%) of the patients infected with the Alpha strain were asymptomatic. Fever (65.8%), cough (63.2%), shortness of breath (21.1%), and diarrhea (21.1%) were more common in patients infected with the SARS-CoV-2 Alpha strain, while runny nose (24.1%), sore throat (31.9%), body aches (13.6%), and headache (12.3%) were more common in patients with the Omicron variant. Compared with 33 (86.84%) of 38 patients infected with the Alpha strain, who had viral pneumonia on pulmonary CT images, only 5 (1.3%) of 382 patients infected with the Omicron variant had mild foci. In addition, the distribution of opacities in the five patients was unilateral and centrilobular, whereas most patients infected with the Alpha strain had bilateral involvement and multiple lesions in the peripheral zones of the lung. Conclusion: The SARS-CoV-2 Alpha strain mainly affects the lungs, while Omicron is confined to the upper respiratory tract in patients with COVID-19.

[Protective effect of mitochondria-targeted antioxidant peptide SS-31 in sepsis-induced acute kidney injury].

OBJECTIVE: To study the effect and mechanism of mitochondria-targeted antioxidant peptide SS-31 on sepsis-induced acute kidney injury (AKI). METHODS: Sixty adult male C57BL/6 mice were randomly divided into four groups according to the random number table method: sham group (10 mice), positive control group (10 mice), sepsis model group (20 mice), and SS-31 peptide group (20 mice). The sepsis-induced AKI mouse model was reproduced by cecal ligation and puncture (CLP). The sham group only received laparotomy. SS-31 peptide (5 mg/kg) was intraperitoneally injected in SS-31 peptide group and positive control group 30 minutes after the operation, while an equivalent amount of normal saline was given in sham group and sepsis model group for 7 days. The blood samples were collected 24 hours after the operation from orbit, and the serum was collected to test the serum creatinine (SCr) and blood urea nitrogen (BUN). The mice were sacrificed 7 days after surgery. The kidney tissues were collected to observe the pathologic structure changes under the hematoxylin-eosin (HE) staining by light microscope. And the mitochondrial ultrastructure was checked under the transmission electron microscope. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL). The expression level of peroxisome proliferator-activated receptorγ coactivator-1α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), and cleaved caspase-3 protein were tested by Western blotting. RESULTS: Compared with sham group, the levels of SCr and BUN were significantly increased in sepsis model group [SCr (µmol/L): 93.12±11.80 vs. 32.94±3.37, BUN (mmol/L): 41.36±6.48 vs. 9.49±3.58, both P < 0.05]. The expression levels of AMPK, PGC-1α and cleaved caspase-3 protein increased (AMPK/ß-actin: 0.30±0.02 vs. 0.12±0.01, PGC-1α/ß-actin: 0.38±0.03 vs. 0.16±0.02, cleaved caspase-3/ß-actin: 0.20±0.01 vs. 0.11±0.02, all P < 0.05). HE staining showed that inflammatory cell was infiltrated, glomerular basement membrane was exposed and vacuole-like transparent casts were found in the lumen. Mitochondria were damaged under electron microscope with swelling, ridge disappearance and ruptured membranes, with increasing of apoptotic cells [cells: 24.00 (18.75, 31.00) vs. 2.00 (0.72, 3.25), P < 0.05]. Meanwhile, compared with sepsis model group, the levels of SCr, BUN and the expressions of AMPK, PGC-1α, cleaved caspase-3 protein were significantly decreased in the SS-31 peptide group [SCr (µmol/L): 71.33±10.14 vs. 93.12±11.80, BUN (mmol/L): 27.00±5.52 vs. 41.36±6.48, AMPK/ß-actin: 0.23±0.01 vs. 0.30±0.02, PGC-1α/ß-actin: 0.27±0.02 vs. 0.38±0.03, cleaved caspase-3/ß-actin: 0.13±0.01 vs. 0.20±0.01, all P < 0.05]. HE staining showed that cell swelling reduced, the mitochondrial structure was intact, the ridge swelling was also reduced, and the membrane structure was relatively intact, the number of apoptotic cells was significantly reduced [cells: 13.00 (9.00, 16.50) vs. 24.00 (18.75, 31.00), P < 0.05]. CONCLUSIONS: The protective effect of SS-31 peptide on organ dysfunction induced by sepsis-induced AKI is related to maintaining mitochondrial homeostasis and inhibiting cell apoptosis.

[Relationship between the timing of initiation of continuous renal replacement therapy and the prognosis of patients with sepsis-associated acute kidney injury].

OBJECTIVE: To investigate the relationship between the timing of initiation of continuous renal replacement therapy (CRRT) and the prognosis of patients with sepsis associated-acute kidney injury (SA-AKI). METHODS: The clinical data of SA-AKI patients undergoing CRRT in intensive care unit (ICU) of Ningbo First Hospital from January 2017 to November 2019 were retrospectively analyzed. According to the guidelines for Kidney Disease: Improving Global Outcomes (KDIGO), patients with AKI who started CRRT in stage 1 or 2 were included in the early treatment group, and those started CRRT in stage 3 were included in the late treatment group. The general clinical data, length of ICU stay, total length of hospital stay, 28-day and 90-day mortality, CRRT duration, 28-day and 90-day renal replacement therapy (RRT) disengagement rate, 28-day and 90-day RRT dependence rate in the survival patients were compared between the two groups. Kaplan-Meier survival analysis was performed to assess the 90-day cumulative survival rate of patients with SA-AKI between two groups. RESULTS: A total of 244 SA-AKI patients were enrolled in this study, including 71 patients in the early treatment group and 173 patients in the late treatment group. There were no significant differences in age, gender composition, acute physiology and chronic health evaluation II (APACHE II), proportion of surgical patients, infection site and anticoagulation program between the two groups. The CRRT duration in the early group was significantly shorter than that in the late group [hours: 26.0 (12.0, 49.0) vs. 41.0 (20.8, 87.0), P < 0.01], but there were no significant differences in the length of ICU stay [days: 9.0 (4.0, 15.0) vs. 10.0 (4.5, 18.0)], total length of hospital stay [days: 17.0 (10.0, 30.0) vs. 18.0 (10.0, 32.0)], 28-day mortality (45.1% vs. 48.0%), 90-day mortality (46.4% vs. 51.4%), 28-day RRT disengagement rate (49.3% vs. 45.1%) and 90-day RRT disengagement rate (52.1% vs. 47.4%) between the early treatment group and late treatment group (all P > 0.05). There were also no significant differences in 28-day RRT dependence rate [10.3% (4/39) vs. 13.3% (12/90)] and 90-day RRT dependence rate [2.6% (1/38) vs. 2.4% (2/84)] between early treatment group and late treatment group (both P > 0.05). Kaplan-Meier survival analysis suggested that there was no significant difference in the 90-day cumulative survival rate between two groups (Log-Rank test: χ2 = 0.791, P = 0.374). CONCLUSIONS: Early initiation of CRRT treatment in SA-AKI patients can reduce the duration of CRRT, but has no effect on length of ICU stay, total length of hospital stay, renal function recovery and mortality. At present, the optimal timing for initiation of CRRT in patients with SA-AKI remains unknown.

[Expression and significance of c-FLIP protein in sepsis mice with acute kidney injury].

OBJECTIVE: To observe the expression of cellular Fas-associated death domain-like interleukin-1ß converting enzyme inhibit protein (c-FLIP) in sepsis mice with acute kidney injury (SAKI) and explore its significance. METHODS: Thirty male ICR mice were divided into the normal control group (Normal group), sham operation group (Sham group) and SAKI group by random number table method, with 10 mice in each group. The SAKI model of mice was established by cecal ligation and puncture (CLP); the Sham group was not ligated and the cecum was not punctured, and other surgical procedures were the same as the SAKI group; the Normal group did not experience any treatment. The serum and renal tissues of mice in each group were harvested 24 hours after CLP model establishment. The levels of serum creatinine (SCr) and blood urea nitrogen (BUN) were detected by enzyme linked immunosorbent assay (ELISA). The renal tissues were stained with hematoxylin-eosin (HE), and the pathological changes of renal tissues were observed under light microscope and the severity of injury was determined. The expression of c-FLIP in renal tissues was detected by immunohistochemistry. The expression of c-FLIP, Bax and caspase-3 protein in renal tissue was detected by Western Blot. The correlation between c-FLIP expression and Bax, caspase-3 protein expressions in renal tissues were analyzed by Pearson test. RESULTS: In the Normal group and the Sham group, the renal tubular epithelial cells were regular and intact, and no interstitial inflammatory cell infiltration was observed; the renal injury score was both 1.30±0.48; immunohistochemistry showed a large amount of c-FLIP positive expression in renal tubular epithelial cells (IA: 120.20±3.87, 116.70±3.46); Western Blot showed high expression of c-FLIP in renal tissues (c-FLIP/GAPDH: 0.99±0.01, 0.98±0.02), and low expressions of Bax and caspase-3 (Bax/GAPDH: 0.16±0.04, 0.19±0.03, caspase-3/GAPDH: 0.24±0.04, 0.23±0.05). Compared with the Sham group, in the SAKI group, renal tubular epithelial cells were degenerated and necrosis, and a large number of interstitial inflammatory cells infiltrated, the renal injury score was significantly increased (4.60±0.52 vs. 1.30±0.48, P < 0.01); the levels of SCr and BUN were significantly increased [SCr (µmol/L): 193.90±13.54 vs. 24.50±3.78, BUN (mmol/L): 81.60±7.26 vs. 5.20±0.92, both P < 0.01]; the c-FLIP positive cells in renal tissues was significantly reduced (IA: 17.11±0.82 vs. 116.70±3.46, P < 0.01); the expression of c-FLIP protein in renal tissues was significantly decreased (c-FLIP/GAPDH: 0.29±0.03 vs. 0.98±0.02, P < 0.01), while the expressions of Bax and caspase-3 protein were significantly increased (Bax/GAPDH: 0.87±0.06 vs. 0.19±0.03, caspase-3/GAPDH: 0.88±0.07 vs. 0.23±0.05, both P < 0.01]. The correlation analysis showed that the c-FLIP protein was significantly negatively correlated with Bax (r = -0.468, P = 0.029) and caspase-3 protein expressions (r = -0.663, P = 0.004). CONCLUSIONS: The expression level of c-FLIP protein was significantly down-regulated in renal tissue of SAKI, and its down-regulation mechanism was associated with increased apoptosis of renal tubular epithelial cells, which could be an effective target for the treatment of SAKI.

Mitochondrial C4375T mutation might be a molecular risk factor in a maternal Chinese hypertensive family under haplotype C.

Here, we reported a Han Chinese essential hypertensive pedigree based on clinical hereditary and molecular data. To know the molecular basis on this family, mitochondrial genome of one proband from the family was identified through direct sequencing analysis. The age of onset year and affected degree of patients are different in this family. And matrilineal family members carrying C4375T mutation and belong to Eastern Asian halopgroup C. Phylogenetic analysis shows 4375C is highly conservative in 17 species. It is suggested that these mutations might participate in the development of hypertension in this family. And halopgroup C might play a modifying role on the phenotype in this Chinese hypertensive family.

Dexmedetomidine Based Sedation for Post-surgery Critically Ill Adults: A Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Using dexmedetomidine (Dex) as a sedative agent may benefit the clinical outcomes of post-surgery patients. We reviewed randomized controlled trials (RCTs) to assess whether use of a Dex could improve the outcomes in post-surgery critically ill adults. METHODS: We searched Medline, Embase, PubMed, and the Cochrane databases for RCTs comparing Dex with propofol or a placebo in post-operative patients, all included RCTs should be published in English before Jul 2016. Citations meeting inclusion criteria were full screened, and trial available data were abstracted independently and the Cochrane risk of bias tool was used for quality assessment. RESULTS: Sixteen RCTs involving 2568 patients were subject to this meta-analysis. The use of a Dex sedative regimen was associated with a reduce delirium prevalence [odd ratio (OR):0.33, 95% confidence intervals (CI): 0.24-0.45, I2= 5%, P<0.001], a shorter the length of ICU stay [mean difference (MD): -0.60, 95%CI: -0.69 to -0.50, I2=40%, P<0.001] and the length of hospital stay [MD: -0.68, 95%CI: -1.21 to -0.16, I2=0%, P=0.01]. However, using of Dex could not shorter the duration of mechanical ventilation [MD: -10.18. 95%CI: -31.08-10.72, I2=99%, P=0.34], but could shorter the time to extubation in post-surgery patients [MD: -47.46, 95%CI: -84.63-10.67, I2=98%, P=0.01]. CONCLUSION: The use of a Dex sedative regimen was associated with a reduce delirium prevalence, a shorter the length of ICU and hospital stay, and a shorter time to extubation in post-surgery critical ill patients.

Thrombocytopenia as a predictor of severe acute kidney injury in patients with heat stroke.

BACKGROUND: Abnormalities of blood system often occur several days before acute kidney injury (AKI) in patients with heat stroke (HS). We aimed to investigate the prevalence and prognostic value of the early hematological markers in patients with AKI induced by HS. METHODS: In a retrospective cohort study, we analyzed the case records of 176 patients with HS and evaluated the hematological markers for early prediction and risk classification in the patients with AKI. RESULTS: Of 176, 103 (58%) HS cases developed AKI, and men comprised more than half (75%) of the sample population. The nadir platelet count significantly correlated with the levels of peak serum creatinine (r = -0.608, p < 0.01) and blood urea nitrogen (r = -0.546, p < 0.01), and the length of hospital stay (r = -0.393, p < 0.01). The areas under the receiver operating characteristic curves (AU-ROC) indicated the prognostic accuracy of hematological markers, AU-ROC was significantly higher with the nadir platelet count than that with the admission platelet count (AU-ROC of the nadir platelet: 0.73; 95% CI: 0.67-0.82; vs. AU-ROC of the admission platelet: 0.67; 95% CI: 0.59-0.75; p < 0.01). Multiple logistic regression results indicated that the nadir platelet count (adjusted ORs: 37.92; 95% CI: 2.18-87.21; p < 0.01) was independent predictor of AKI in HS. CONCLUSION: The high mortality observed in HS complicated with AKI, and among the various hematological parameters assessed, thrombocytopenia is associated with AKI induced by HS independently.

Losartan prevents sepsis-induced acute lung injury and decreases activation of nuclear factor kappaB and mitogen-activated protein kinases.

Lack of specific and efficient therapy leads to the high mortality rate of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Losartan is a potent pharmaceutical drug for ALI/ARDS. However, the protective effects and mechanisms of losartan remain incompletely known. This study evaluates the effects of losartan on ALI/ARDS and further investigates the possible mechanisms of these protective effects. Mice received i.p. injections of the AT1 inhibitor losartan (15 mg/kg), or control vehicle, half hour after cecal ligation and puncture (CLP). Plasma TNF-alpha, IL-1beta, and IL-6 cytokines were assayed 6 h after CLP. Blood gas, wet/dry lung weight ratio, lung tissue histology for occurrence of ALI/ARDS, and survival were examined. Lastly, nuclear factor kappaB (NF-kappaB) activations, IkappaB-alpha degradations, phosphorylations of p38 MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase expressions were evaluated in lung tissue. Losartan treatment significantly attenuated TNF-alpha, IL-6, and IL-1beta 6 h after CLP. Furthermore, losartan prevented blood gas and histopathologic appearance of ALI/ARDS after sepsis and significantly improved survival. Finally, losartan given after sepsis led to inhibition of lung tissue NF-kappaB activation (P < 0.01 vs. CLP group), attenuated degradation of IkappaB-alpha, and inhibited phosphorylation of p38MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase, pathways critical for cytokine release. These data reveal that losartan exerts a protective effect on ALI/ARDS, and this protective effect may be dependent, at least in part, on NF-kappaB and MAPK mechanisms.