Ophiopogonin D alleviates acute lung injury by regulating inflammation via the STAT3/A20/ASK1 axis.

BACKGROUND: Acute lung injury (ALI) is characterized by acute pulmonary inflammatory infiltration. Alveolar epithelial cells (AECs) release numerous pro-inflammatory cytokines, which result in the pathological changes seen in ALI. Ophiopogonin D (OD), extracted from the roots of Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), reduces inflammation; however, the efficacy of OD in ALI has not been reported and the underlying molecular mechanisms remain unclear. PURPOSE: This study investigated the anti-inflammatory effects of OD, as well as the underlying mechanisms, in AECs and a mouse ALI model. METHODS: Lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α) were used to stimulate macrophages and A549 cells, and a mouse ALI model was established by intratracheal LPS administration. The anti-inflammatory effects and mechanisms of OD in the TNF-α-induced in vitro inflammation model was evaluated using real-time quantitative polymerase chain reaction qPCR), enzyme-linked immunosorbent assay (ELISA), western blotting, nuclear and cytoplasmic protein extraction, and immunofluorescence. The in vivo anti-inflammatory activity of OD was evaluated using hematoxylin and eosin staining, qPCR, ELISA, and western blotting. RESULTS: The bronchoalveolar lavage fluid and lung tissue of LPS-induced ALI mice exhibited increased TNF-α expression. TNF-α induced a significantly greater pro-inflammatory effect in AECs than LPS. OD reduced inflammation and mitogen-activated protein kinase (MAPK) and transcription factor p65 phosphorylation in vivo and in vitro and promoted signal transducer and activator of transcription 3 (STAT3) phosphorylation and A20 expression, thereby inducing apoptosis signal-regulating kinase 1 (ASK1) proteasomal degradation. CONCLUSION: OD exerts an anti-inflammatory effect by promoting STAT3-dependent A20 expression and ASK1 degradation. OD may therefore have therapeutic value in treating ALI and other TNF-α-related inflammatory diseases.

Activation of cannabinoid receptors 2 alleviates myocardial damage in cecal ligation and puncture-induced sepsis by inhibiting pyroptosis.

BACKGROUND: It has been reported that cannabinoid receptors 2 (CB2 receptors) play an important role in the pathophysiological process of sepsis, which may also be associated with the regulation of pyroptosis, an inflammatory programmed cell death. The present study aimed to investigate the protective effect of CB2 receptors on myocardial damage in a model of septic mice by inhibiting pyroptosis. METHODS: The C57BL/6 mice underwent cecal ligation and puncture (CLP) to induce sepsis. All mice were randomly divided into the sham, CLP, or CLP+HU308 group. Blood and heart tissue samples were collected 12 h after surgery. Hematoxylin and eosin staining was used for analyzing histopathological results. Creatine kinase isoenzymes (CK-MB) and IL-1ß were measured using ELISA, while lactate dehydrogenase (LDH) level was determined using photoelectric colorimetry. The expression levels of CB2 receptors and pyroptosis-associated proteins (NLRP3, caspase-1, and GSDMD) were measured using western blotting. The location and distribution of CB2 receptors and caspase-1 in myocardial tissues were assessed by immunofluorescence. TUNEL staining was used to quantify the number of dead cells in myocardial tissues. RESULTS: The CLP procedure increased CB2 receptor expression in mice. CB2 receptors were located in myocardial macrophages. Activating CB2 receptors decreased the levels of myocardial damage mediator LDH, CK-MB, and inflammatory cytokine IL-1ß. The results also showed that CLP increased the pyroptosis in myocardial tissues, while CB2 agonist HU308 inhibited pyroptosis by decreasing the level of NLRP3 and activating caspase-1 and GSDMD. CONCLUSIONS: CB2 receptor activation has a protective effect on the myocardium of mice with sepsis by inhibiting pyroptosis.

Asymmetric Epoxidation of Alkenes Catalyzed by a Cobalt Complex.

Asymmetric epoxidation of alkenes catalyzed by nonheme chiral Mn-O and Fe-O catalysts has been well established, but chiral Co-O catalysts for the purpose remain virtually undeveloped due to the oxo wall. Herein is first reported a chiral cobalt complex to realize the enantioselective epoxidation of cyclic and acyclic trisubstituted alkenes by using PhIO as the oxidant in acetone, wherein the tetra-oxygen-based chiral N,N'-dioxide with sterically hindered amide subunits plays a crucial role in supporting the formation of the Co-O intermediate and enantioselective electrophilic oxygen transfer. Mechanistic studies, including HRMS measurements, UV-vis absorption spectroscopy, magnetic susceptibility, as well as DFT calculations, were carried out, confirming the formation of Co-O species as a quartet Co(III)-oxyl tautomer. The mechanism and the origin of enantioselectivity were also elucidated based on control experiments, nonlinear effects, kinetic studies, and DFT calculations.

Efficacy and cost-effectiveness analysis of pretreatment percutaneous endoscopic gastrostomy in unresectable locally advanced esophageal cancer patients treated with concurrent chemoradiotherapy (GASTO 1059).

BACKGROUND: We launched a single-arm phase II study to determine the efficacy and cost-effectiveness of percutaneous endoscopic gastrostomy (PEG) before concurrent chemoradiotherapy (CCRT) in patients with esophageal squamous cell carcinoma (ESCC). METHODS: Eligible patients received pretreatment PEG and enteral nutrition during CCRT. The primary outcome was the change of weight during CCRT. The secondary outcome included nutrition status, loco-regional objective response rate (ORR), loco-regional progression-free survival (LRFS), overall survival (OS), and toxicities. A 3-state Markov model was applied for cost-effectiveness analysis. Eligible patients were matched and compared with those who had nasogastric tube feeding (NTF) or oral nutritional supplements (ONS). RESULTS: Sixty-three eligible patients received pretreatment PEG-based CCRT. The mean change of weight during CCRT was -1.4% (standard deviation, 4.4%), and after CCRT, 28.6% of patients gained weight and 98.4% had normal albumin levels. The loco-regional ORR and 1-year LRFS were 98.4% and 88.3%. The incidence of grade ≥3 esophagitis was 14.3%. After matching, another 63 patients were included in the NTF group and 63 in the ONS group. More patients gained weight after CCRT in the PEG group (p = 0.001). The PEG group showed higher loco-regional ORR (p = 0.036) and longer 1-year LRFS (p = 0.030). In cost analysis, the PEG group showed an incremental cost-effectiveness ratio of $3457.65 per quality-adjusted life-years (QALY) compared with the ONS group with a probability of cost-effectiveness of 77.7% at the $10,000 per QALY willingness-to-pay threshold. CONCLUSION: Pretreatment PEG is associated with better nutritional status and treatment outcome in ESCC patients treated with CCRT compared with ONS and NTF. Pretreatment of PEG can be cost-effective because of its significant clinical benefits.

SERBP1 affects the apoptotic level by regulating the expression and alternative splicing of cellular and metabolic process genes in HeLa cells.

Background: RNA-binding proteins (RBPs) have important roles in orchestrating posttranscriptional regulation and modulating many tumorigenesis events. SERBP1 has been recognized as an important regulator in multiple cancers, while it remains unclear whether SERBP1-regulated gene expression at the transcriptome-wide level is significantly correlated with tumorigenesis. Methods: We overexpressed SERBP1 in HeLa cells and explored whether SERBP1 overexpression (SERBP1-OE) affects the proliferation and apoptosis of HeLa cells. We analyzed the transcriptome-wide gene expression changes and alternative splicing changes mediated by SERBP1-OE using the transcriptome sequencing method (RNA-seq). RT-qPCR was conducted to assay SERBP1-regulated alternative splicing. Results: SERBP1-OE induced the apoptosis of HeLa cells. The downregulated genes were strongly enriched in the cell proliferation and apoptosis pathways according to the GO analysis, including FOS, FOSB, PAK6 and RAB26. The genes undergoing at least one SERBP1-regulated alternative splicing event were enriched in transcriptional regulation, suggesting a mechanism of the regulation of gene expression, and in pyruvate and fatty acid metabolic processes critical for tumorigenesis events. The SERBP1-regulated alternative splicing of ME3, LPIN3, CROT, PDP1, SLC27A1 and ALKBH7 was validated by RT-qPCR analysis. Conclusions: We for the first time demonstrated the cellular function and molecular targets of SERBP1 in HeLa cells at transcriptional and post-transcriptional levels. The SERBP1-regulated gene expression and alternative splicing networks revealed by this study provide important information for exploring the functional roles and regulatory mechanisms of SERBP1 in cancer development and progression.

Protective Effects of Cannabinoid Type 2 Receptor Activation Against Microglia Overactivation and Neuronal Pyroptosis in Sepsis-Associated Encephalopathy.

Sepsis-associated encephalopathy (SAE) has close association with long-term cognitive deficits, resulting in increased mortality. The mechanism of SAE is complicated, including excessive microglial activation and neuroinflammation. Cannabinoid type 2 receptor (CB2R) has been proved to be effective in neuronal protection and survival promotion. Microglia play a role in CB2R mediated neuronal protection when neurons are exposed to noxious stimuli. Pyroptosis is a type of programmed proinflammatory cell death. However, the underlying mechanisms involved in this process still remain to be explored. Here, the SAE model was derived from cecal ligation and puncture (CLP). Tests used to evaluate behavior phenotypes included the open-field test (OFT), novel object recognition test (NORT), and Morris water maze (MWM). Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assays (ELISA), Western blotting, and immunofluorescence staining were performed to detect cell injury, cytokine, CB2R and pyroptosis-associated protein expression. Conclusion from these results, we conclude that CLP could induce microglia hyperactivation and neuronal pyroptosis, aggravating brain tissue destruction and cognitive dysfunction. The CB2R-specific agonist HU308 could have protective effects against SAE by inhibiting microglia activity and neuronal pyroptosis. This study provides a new therapeutic option for the treatment of SAE.

Medical Thoracoscopic Thermal Ablation Therapy for Metastatic Pleural Tumors with Malignant Effusion: An Exploratory Retrospective Study.

PURPOSE: Malignant pleural effusion (MPE) is an intractable condition. The current mainstream therapies for MPE, ie, indwelling pleural catheter and pleurodesis, have some drawbacks. In this retrospective study, we explored the efficacy and safety of medical thoracoscopic thermal ablation (argon plasma coagulation, APC) therapy for metastatic pleural tumors with MPE. PATIENTS AND METHODS: A total of 176 patients were enrolled and divided into catheter pleural drainage (CPD) group (n = 77), non-ablation group (n = 46), and thermal ablation group (n = 53). Propensity score matching (PSM) was used for between-group comparisons to minimize bias. The primary endpoints were pleural effusion objective response rate (ORR) and time to progression (TTP); secondary endpoints included overall survival (OS), chest-tube duration, and safety. RESULTS: Thermal ablation group and non-ablation group showed significantly higher ORR and shorter chest-tube duration versus the CPD group (ORR: thermal ablation, 88.2% vs 66.7%, P = 0.004; non-ablation, 88.4% vs 64.4%, P = 0.042; chest-tube duration: thermal ablation, 4.90 vs 7.24 days, P < 0.001; non-ablation, 5.73 vs 7.33 days, P = 0.010). Thermal ablation group exhibited longer TTP than the CPD group (median, 13.7 vs 7.3 months, P = 0.001) and the non-ablation group (median, 13.6 vs 10.3 months, P = 0.037). OS in the thermal ablation group was numerically longer than that in the CPD group with marginally significant difference (P = 0.055). There was no significant difference in the frequency of adverse events or changes in vital signs between thermal ablation and non-ablation groups. CONCLUSION: Medical thoracoscopic thermal ablation (APC technique) therapy was effective and safe in the treatment of metastatic pleural tumors with MPE for improving ORR and TTP.

Activation of CB2 receptor inhibits pyroptosis and subsequently ameliorates cecal ligation and puncture-induced sepsis.

BACKGROUND: Cannabinoid receptor 2 (CB2), whose activities are upregulated during sepsis, may be related to the regulation of inflammatory programmed cell death called pyroptosis. The aim of this study is to investigate the role of CB2 activation in attenuation of inflammation through inhibiting pyroptosis in cecal ligation puncture (CLP)-induced sepsis andlipopolysaccharide (LPS) + ATP-stimulated macrophages. METHODS: C57BL/6 mice were subjected to CLP procedure and treated with CB2 agonist HU308 and CB2 antagonist AM630. Lung tissues were collected for analyses of lung W/D ratio, inflammatory factors levels, and pyroptosis-related protein expression. Murine bone-marrow-derived macrophages (BMDM) were treated with LPS and ATP to construct a septic model in vitro in the presence of HU308 and AM630 for assessment of cell injury, cytokine levels and pyroptosis-related protein expression accordingly. To verify the relationship between CB2 receptors and pyroptosis in the process of inflammatory response, BMDM were transduced with CB2 receptors knockdown lentiviral vectors in the presence of HU308 and AM630 for assessment of pyroptosis-related protein expression. RESULTS: CB2 activation ameliorated the release of inflammatory mediators. The results showed that CLP-induced pyroptosis was elevated, and CB2 agonist HU308 treatment inhibited the pyroptosis activity through a decrease of the protein levels of NLRP3 as well as caspase-1 and GSDMD activation. Similar results were obtained in BMDM after LPS and ATP treatment. Treatment with CB2 knockdown lentiviral particles prevented the HU308-induced decreases in cell pyroptosis, demonstrating that endogenous CB2 receptors are required for the cannabinoid-induced cell protection. CONCLUSIONS: CB2 receptors activation plays a protective role in sepsis through inhibition of pyroptosis. The effect of CB2 receptors against pyroptosis depends on the existence of endogenous CB2 receptors.

Asymmetric Catalytic Epoxidation of Terminal Enones for the Synthesis of Triazole Antifungal Agents.

An enantioselective epoxidation of α-substituted vinyl ketones was realized to construct the key epoxide intermediates for the synthesis of various triazole antifungal agents. The reaction proceeded efficiently in high yields with good enantioselectivities by employing a chiral N,N'-dioxide/ScIII complex as the chiral catalyst and 35% aq. H2O2 as the oxidant. It enabled the facile transformation for optically active isavuconazole, efinaconazole, and other potential antifungal agents.

Enantioselective dicarbofunctionalization of (E)-alkenyloxindoles with pyridinium salts by chiral Lewis acid/photo relay catalysis.

A highly efficient enantioselective dicarbofunctionalization reaction of (E)-alkenyloxindoles with pyridinium salts was realized. The process includes the chiral N,N'-dioxide-Sc(iii) complex-catalyzed regio-, diastereo-, and enantioselective [3+2] cycloaddition reaction and the following photo-promoted aza-Norrish II type rearrangement. A series of 2-pyridyl substituted oxindole derivatives were obtained in good yields with moderate to good diastereo- and enantioselectivities.

Diversified Transformations of Tetrahydroindolizines to Construct Chiral 3-Arylindolizines and Dicarbofunctionalized 1,5-Diketones.

Enantioselective diverse synthesis of a small-molecule collection with structural and functional similarities or differences in an efficient manner is an appealing but formidable challenge. Asymmetric preparation and branching transformations of tetrahydroindolizines in succession present a useful approach to the construction of N-heterocycle-containing scaffolds with functional group, and stereochemical diversity. Herein, we report a breakthrough toward this end via an initial diastereo- and enantioselective [3 + 2] cycloaddition between pyridinium ylides and enones, following diversified sequential transformations. Chiral N,N'-dioxide-earth metal complexes enable the generation of optically active tetrahydroindolizines in situ, across the strong background reaction for racemate-formation. In connection with deliberate sequential transformations, involving convenient rearomatic oxidation, and light-active aza-Norrish II rearrangement, the tetrahydroindolizine intermediates were converted into the final library including 3-arylindolizine derivatives and dicarbofunctionalized 1,5-dicarbonyl compounds. More importantly, the stereochemistry of four-stereogenic centered tetrahydroindolizine intermediates could be efficiently transferred into axial chirality in 3-arylindolizines and vicinal pyridyl and aryl substituted 1,5-diketones. In addition, densely functionalized cyclopropanes and bridged cyclic compounds were also discovered depending on the nature of the pyridinium ylides. Mechanism studies were involved to explain the stereochemistry during the reaction processes.

A chiral cobalt(ii) complex catalyzed enantioselective aza-Piancatelli rearrangement/Diels-Alder cascade reaction.

A chiral N,N'-dioxide/cobalt(ii) complex catalyzed highly diastereoselective and enantioselective tandem aza-Piancatelli rearrangement/intramolecular Diels-Alder reaction has been disclosed. Various valuable hexahydro-2a,5-epoxycyclopenta[cd]isoindoles bearing six contiguous stereocenters have been obtained in good yields with excellent diastereo- and enantio-selectivities from a wide range of both readily available 2-furylcarbinols and N-(furan-2-ylmethyl)anilines.

Pyruvate Protects Against Intestinal Injury by Inhibiting the JAK/STAT Signaling Pathway in Rats With Hemorrhagic Shock.

BACKGROUND: This study aimed to investigate the role of Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway in protection by peritoneal resuscitation (PR) using pyruvate-peritoneal dialysis solution (PY-PDS) against intestinal injury from hemorrhagic shock (HS) in rats. MATERIALS AND METHODS: Sixty-four rats were assigned to eight groups: group SHAM; group intravenous resuscitation (VR); groups NS, LA, and PY in which the rats were subjected to HS and PR with normal saline (NS), lactate-peritoneal dialysis solution (LA-PDS), and PY-PDS, respectively, combined with VR; and groups DMSO, RPM, and AG490 in which the rats were subjected to HS and VR with pretreatment of dimethyl sulfoxide (DMSO), rapamycin (RPM), and tyrphostin B42 (AG490). RESULTS: At 2 h after HS and resuscitation, the levels of diamine oxidase, 15-F2t-isoprostane, thromboxane B2, and endothelin-1, in the blood and the intestinal mucosal apoptotic index and caspase-3 were lower in groups PY, RPM, and AG490 than in groups VR, NS, LA, and DMSO. Group PY showed lower levels of malondialdehyde and myeloperoxidase and a higher level of superoxide dismutase than groups VR, NS, and LA. Phosphorylated JAK2 and phosphorylated STAT3 levels were lower in groups PY, RPM, AG490, and LA than in groups VR, NS, and DMSO. CONCLUSIONS: The protection mechanism of PR with PY-PDS combined with VR was related to the inhibition of the JAK/STAT signaling pathway during HS and resuscitation. The process might include suppression of oxidative stress, reduction of neutrophil infiltration, regulation of microcirculation, and inhibition of apoptosis.

The analysis of nitrogen load and simulation uncertainty using SWAT in a catchment with paddy field in China.

Excessive load of nitrogen from anthropogenic sources is a threat to sustaining a healthy aquatic ecosystem. The difficulty in identifying the critical source areas (CSAs) of nitrogen load and apportioning the in-stream nitrogen to individual sources spatially and seasonally has made the Soil and Water Assessment Tool (SWAT) useful for analyzing nitrogen load at the catchment scale. However, the uncertainty of the nitrogen load simulated by SWAT has rarely been analyzed. The two simulations with the highest or the lowest PBIAS of total nitrogen (TN) load were proposed in this study to represent the range of the prediction uncertainty and therefore were used to generate the uncertainty of CSAs and nitrogen source apportionment. The model was set up for the Yuan River Catchment, which is under threat of extensive nitrogen load. Results indicated the highest nitrogen load was from downstream paddy fields with a denser population and 85% of the load was from fertilizer and feedlots. The relatively high prediction uncertainty was observed on both CSAs and source apportionment, which emphasizes the limitation of calibration only based on certain processes and the necessity to consider parameter uncertainty in the application of nitrogen load simulation.

Host-guest interaction enabled chiroptical photo-switching and enhanced circularly polarized luminescence.

A cyanostilbene conjugated gelator was incorporated into the cavities of cyclodextrins via the host-guest interaction and the resulting supra-gelator from γ-CyD enabled chiroptical photo-switching, while the other gels showed enhanced circularly polarized luminescences.

Rearrangement of the transmembrane domain interfaces associated with the activation of a GPCR hetero-oligomer.

G protein-coupled receptors (GPCRs) can integrate extracellular signals via allosteric interactions within dimers and higher-order oligomers. However, the structural bases of these interactions remain unclear. Here, we use the GABAB receptor heterodimer as a model as it forms large complexes in the brain. It is subjected to genetic mutations mainly affecting transmembrane 6 (TM6) and involved in human diseases. By cross-linking, we identify the transmembrane interfaces involved in GABAB1-GABAB2, as well as GABAB1-GABAB1 interactions. Our data are consistent with an oligomer made of a row of GABAB1. We bring evidence that agonist activation induces a concerted rearrangement of the various interfaces. While the GB1-GB2 interface is proposed to involve TM5 in the inactive state, cross-linking of TM6s lead to constitutive activity. These data bring insight for our understanding of the allosteric interaction between GPCRs within oligomers.

Chronic CaMKII inhibition reverses cardiac function and cardiac reserve in HF mice.

AIMS: The present study was to explore the impact of KN93 - a specific inhibitor of CaMKII - on cardiac function and cardiac reserve in HF mice. MAIN METHODS: We have generated pressure-overload HF mice using modified transverse aortic constriction (TAC) method. For acute inhibition (AI) experiment, HF mice were randomly divided into HF group, HF + KN93 AI group and HF + KN92 AI group, using sham mice as control. Mice in HF + KN93 AI group and HF + KN92 AI group were injected with CaMKII inhibitor KN93 or its inactive analogue KN92 on post-TAC day 15, while mice in HF group and Sham group were treated with saline. For chronic inhibition (CI) experiment, mice were injected daily with KN93, KN92 or saline for one week. At baseline and after isoproterenol (Iso) injection, in vivo cardiac function was assessed by echocardiography and left ventricular pressure-volume catheter. KEY FINDINGS: Acute inhibition of CaMKII leads to decreased -dP/dtmin, increased EF, FS, longitudinal strain, longitudinal strain rate, ESPVR, dP/dtmax-EDV, PRSW, Tau and EDPVR, and unaltered reactivity to Iso in HF mice. Chronic inhibition results in increased EF, FS, longitudinal strain, longitudinal strain rate, ESPVR, dP/dtmax-EDV and PRSW, without alteration in -dP/dtmin, Tau and EDPVR. In addition, chronic inhibition reverses the effect of Iso on HF mice. SIGNIFICANCE: Although acute CaMKII inhibition can repair systolic function in HF mice, it also exacerbates the diastolic function, whereas chronic inhibition improves both systolic function and cardiac reserve to ß-adrenergic stimulation without impairing diastolic function.

Cannabinoid receptor 2: a potential novel therapeutic target for sepsis?

OBJECTIVE: Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. It is the most common cause of death among critically ill patients in non-coronary intensive care units and the incidence continues to rise. Although advanced management was applied, the prognosis of sepsis patients remains poor. As a G-protein coupled receptor, cannabinoid receptor 2 (CB2R) was implicated in a wide variety of diseases. In this study, we aimed to investigate the role of CB2R in sepsis. METHODS: Literature search was performed using "sepsis" and "Cannabinoid Receptor" as search terms in PubMed, EMbase, and the Cochrane Library. RESULTS AND DISCUSSION: Briefly, 97 records were identified by the search strategy, of which 76 were duplicate or irrelevant publications. With the anti-inflammatory and immunomodulatory effects, CB2R is a novel and promising therapeutic target in the management of sepsis. Indeed, specific CB2R agonists have been reported to attenuate leukocyte recruitment, oxidative burst, systemic inflammatory mediator release, bacteremia, and lung tissue damage, while improving survival in different sepsis models. In addition, autophagy has also been implicated in the protective role of CB2R activation in sepsis. However, almost all of the current outcomes result from animal studies or in vitro cultured cells. Due to the lack of clinical evidence and the ambiguous mechanisms underlying, the clinical application of CB2R stimulation in sepsis is limited. Further studies are needed to delineate the therapeutic effect and the related-pathways of CB2R agonists in sepsis.

M3 receptor is involved in the effect of penehyclidine hydrochloride reduced endothelial injury in LPS-stimulated human pulmonary microvascular endothelial cell.

LPS has been recently shown to induce muscarinic acetylcholine 3 receptor (M3 receptor) expression and penehyclidine hydrochloride (PHC) is an anticholinergic drug which could block the expression of M3 receptor. PHC has been demonstrated to perform protective effect on cell injury. This study is to investigate whether the effect of PHC on microvascular endothelial injury is related to its inhibition of M3 receptor or not. HPMVECs were treated with specific M3 receptor shRNA or PBS, and randomly divided into LPS group (A group), LPS+PHC group (B group), LPS + M3 shRNA group (C group) and LPS + PHC + M3 shRNA group (D group). Cells were collected at 60 min after LPS treatment to measure levels of LDH, endothelial permeability, TNF-α and IL-6 levels, NF-κB p65 activation, I-κB protein expression, p38MAPK, and ERK1/2 activations as well as M3 mRNA expression. PHC could decrease LDH levels, cell permeability, TNF-α and IL-6 levels, p38 MAPK, ERK1/2, NF-κB p65 activations and M3 mRNA expressions compared with LPS group. When M3 receptor was silence, the changes of these indices were much more obvious. These findings suggest that M3 receptor plays an important role in LPS-induced pulmonary microvascular endothelial injury, which is regulated through NF-κB p65 and MAPK activation. And knockout of M3 receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. Regulative effects of PHC on pulmonary microvascular permeability and NF-κB p65 as well as MAPK activations are including but not limited to inhibition of M3 receptor.

Celastrol inhibits IL-1ß-induced inflammation in orbital fibroblasts through the suppression of NF-κB activity.

Graves' disease is an autoimmune disease of the thyroid gland, which is characterized by hyperthyroidism, diffuse goiter and Graves' ophthalmopathy (GO). Although several therapeutic strategies for the treatment of GO have been developed, the effectiveness and the safety profile of these therapies remain to be fully elucidated. Therefore, examination of novel GO therapies remains an urgent requirement. Celastrol, a triterpenoid isolated from traditional Chinese medicine, is a promising drug for the treatment of various inflammatory and autoimmune diseases. CCK­8 and apoptosis assays were performed to investigate cytotoxicity of celastrol and effect on apoptosis on orbital fibroblasts. Reverse transcription­polymerase chain reaction, western blotting and ELISAs were performed to examine the effect of celastrol on interleukin (IL)­1ß­induced inflammation in orbital fibroblasts from patients with GO. The results demonstrated that celastrol significantly attenuated the expression of IL­6, IL­8, cyclooxygenase (COX)­2 and intercellular adhesion molecule­1 (ICAM­1), and inhibited IL­1ß­induced increases in the expression of IL­6, IL­8, ICAM­1 and COX­2. The levels of prostaglandin E2 in orbital fibroblasts induced by IL­1ß were also suppressed by celastrol. Further investigation revealed that celastrol suppressed the IL­1ß­induced inflammatory responses in orbital fibroblasts through inhibiting the activation of nuclear factor (NF)­κB. Taken together, these results suggested that celastrol attenuated the IL­1ß­induced pro­inflammatory pathway in orbital fibroblasts from patients with GO, which was associated with the suppression of NF-κB activation.