OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

OBJECTIVE: To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD). METHODS: Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins. RESULTS: In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa. CONCLUSION Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.
Humans ; Mice ; Animals ; Caco-2 Cells ; beta Catenin/metabolism* ; Culture Media, Conditioned/pharmacology* ; Tight Junctions/metabolism* ; Intestinal Mucosa ; Inflammatory Bowel Diseases ; Tight Junction Proteins/metabolism* ; Inflammation/metabolism* ; Fibroblasts/metabolism* ; Mice, Inbred C57BL ; Glycoproteins/metabolism* ; Wnt Proteins/pharmacology* ; Frizzled Receptors/metabolism*

OX40 is a costimulatory receptor that is expressed primarily on activated CD4⁺, CD8⁺, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.
Mice ; Animals ; Receptors, Tumor Necrosis Factor/physiology* ; Receptors, OX40 ; Membrane Glycoproteins ; Ligands ; Antibodies, Monoclonal/pharmacology* ; Antineoplastic Agents/pharmacology*

Animals ; Aquaporins/metabolism* ; Disease Models, Animal ; Glycoproteins/pharmacology* ; Humans ; Lung/metabolism* ; Protective Agents/pharmacology* ; Respiratory Distress Syndrome/physiopathology* ; Sus scrofa ; Trypsin Inhibitors/pharmacology* ; Up-Regulation

OBJECTIVE: To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2 (ACE2) receptor and viral spike protein by virtual screening. METHODS: The three-dimensional (3D)-coordinate file of the receptor-binding domain (RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration (FDA)-approved drugs from DrugBank and natural compounds from Traditional Chinese Medicine Systems Pharmacology (TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine (CM) herb sources were listed in this paper. RESULTS: It was found that digitoxin, a cardiac glycoside in DrugBank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen (), a CM formula reportedly exerting activity against severe acute respiratory syndrome (SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. CONCLUSIONS A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-CoV-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.
China ; Computer Simulation ; Coronavirus Infections ; diagnosis ; drug therapy ; Drug Repositioning ; methods ; Drugs, Chinese Herbal ; pharmacology ; Glycoproteins ; drug effects ; metabolism ; Humans ; Imaging, Three-Dimensional ; Mass Screening ; methods ; Molecular Docking Simulation ; methods ; Pandemics ; Peptidyl-Dipeptidase A ; drug effects ; Pneumonia, Viral ; diagnosis ; drug therapy ; Protein Binding ; United States ; United States Food and Drug Administration

Depression is a debilitating psychiatric disorder with a huge socioeconomic burden, and its treatment relies on antidepressants including selective serotonin reuptake inhibitors (SSRIs). Recently, the melatonergic system that is closely associated with the serotonergic system has been implicated in the pathophysiology and treatment of depression. However, it remains unknown whether combined treatment with SSRI and melatonin has synergistic antidepressant effects. In this study, we applied a sub-chronic restraint stress paradigm, and evaluated the potential antidepressant effects of combined fluoxetine and melatonin in adult male mice. Sub-chronic restraint stress (6 h/day for 10 days) induced depression-like behavior as shown by deteriorated fur state, increased latency to groom in the splash test, and increased immobility time in the forced-swim test. Repeated administration of either fluoxetine or melatonin at 10 mg/kg during stress exposure failed to prevent depression-like phenotypes. However, combined treatment with fluoxetine and melatonin at the selected dose attenuated stress-induced behavioral abnormalities. Moreover, we found that the antidepressant effects of combined treatment were associated with the normalization of brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling in the hippocampus, but not in the prefrontal cortex. Our findings suggest that combined fluoxetine and melatonin treatment exerts synergistic antidepressant effects possibly by restoring hippocampal BDNF-TrkB signaling.
Animals ; Antidepressive Agents ; pharmacology ; Behavior, Animal ; drug effects ; Brain-Derived Neurotrophic Factor ; drug effects ; metabolism ; Depression ; Drug Synergism ; Drug Therapy, Combination ; Fluoxetine ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Male ; Melatonin ; pharmacology ; Membrane Glycoproteins ; drug effects ; metabolism ; Mice, Inbred C57BL ; Protein-Tyrosine Kinases ; drug effects ; metabolism ; Restraint, Physical ; Signal Transduction ; drug effects

PURPOSE: Early application of protease inhibitors through the intestinal lumen could increase survival following experimental shock by blocking the pancreatic digestive enzymes. Hence, it was hypothesized that two-route injection (intraintestinal + intravenous) of ulinastatin (UTI), a broad-spectrum protease inhibitor, could better alleviate intestinal injury than single-route injection (either intravenous or intraintestinal). METHODS: A sepsis model induced by lipopolysaccharide on rats was established. The rats were randomly divided into five groups: sham, sepsis, UTI intravenous injection (Uiv), UTI intraintestinal injection (Uii), and UTI intraintestinal + intravenous injection (Uii + Uiv) groups. The mucosal barrier function, enzyme-blocking effect, levels of systemic inflammatory cytokines, and 5-day survival rate were compared among groups. The small intestinal villus height (VH), crypt depth (CD), and two components of mucosal barrier (E-cadherin and mucin-2) were measured to evaluate the mucosal barrier function. The levels of trypsin and neutrophil elastase (NE) in the intestine, serum, and vital organs were measured to determine the enzyme-blocking effect. RESULTS: Compared with the single-route injection group (Uiv or Uii), the two-route injection (Uii + Uiv) group displayed: (1) significantly higher levels of VH, VH/CD, E-cadherin, and mucin-2; (2) decreased trypsin and NE levels in intestine, plasma, and vital organs; (3) reduced systemic inflammatory cytokine levels; and (4) improved survival of septic rats. CONCLUSION Two-route UTI injection was superior to single-route injection in terms of alleviating intestinal injury, which might be explained by extensive blockade of proteases through different ways.
Animals ; Cadherins ; metabolism ; Cytokines ; metabolism ; Disease Models, Animal ; Glycoproteins ; administration & dosage ; pharmacology ; Inflammation Mediators ; metabolism ; Injections, Intralesional ; Injections, Intravenous ; Intestinal Diseases ; drug therapy ; etiology ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; Leukocyte Elastase ; metabolism ; Male ; Mucin-2 ; metabolism ; Rats, Wistar ; Sepsis ; complications ; Trypsin ; metabolism ; Trypsin Inhibitors ; administration & dosage ; pharmacology

To explore the effect of ulinastatin on perioperative glycocalyx and lung function in patients undergoing mitral valve replacement surgery.
 Methods: Fourty patients, undergoing mitral valve replacement, were randomly allocated into a control group and an ulinastatin group, which were administrated 50 mL normal saline or 2×104 U/kg ulinastatin at the beginning of cardiopulmonary bypass (CPB), respectively. The radical artery blood was collected at 4 time points: After induction of anesthesia (T0), at 10 min after the start of CPB (T1), 1 h after the end of CPB (T2), and 8 h after operation. The concentration of syndecan-1 and TNF-α in blood was measured. Moreover, the blood gas analysis was preformed and the oxygen index (OI) and difference in alveolar arterial oxygen partial pressure (PA-aO2) were calculated at T0, T2, and T3.
 Results: There were no significant difference between the 2 groups in OI, PA-aO2, and the concentration of syndecan-1 and TNF-α at T0 (P>0.05). The concentration of syndecan-1 and TNF-α was significantly increased at T1 and T2 in the 2 groups, and reached peak at T2. Compared with the control group, the concentration of syndecan-1 and TNF-α was decreased in the ulinastatin group at T1, T2, and T3 (P<0.05). Compared with T0, OI was lower and PA-aO2 was higher at T2 and T3 in both groups, but the 2 indexes were improved in the ulinastatin group compared with those in the control group (P<0.05).
 Conclusion: Ulinastatin can improve the post-operative pulmonary ventilation function in patients with mitral valve replacement. The mechanism may be associated with the inhibition of TNF-α release and the reduction of glycocalyx shedding induced by ulinastatin.
Cardiopulmonary Bypass ; Glycocalyx ; drug effects ; Glycoproteins ; pharmacology ; Heart Valve Prosthesis Implantation ; Humans ; Lung ; drug effects ; Mitral Valve ; surgery ; Oxygen ; blood ; Syndecan-1 ; blood ; Time Factors ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVEThis work aims to examine the effects of paeonol treatment on the ability of bone marrow-derived macrophage (BMM) to excrete inflammatory factors and to differentiate into osteoclasts upon induction with Porphyromonas gingivalis (P. gingivalis). This work also aims to investigate the underlying mechanisms of these abilities.

METHODSBMM culture was treated with different paeonol concentrations at for 1 h and then stimulated with P. gingivalis for 24 h before programmed death-ligand 1 (PD-L1) was quantified with flow cytometry. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The BMM culture was treated with the receptor activator for nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), and then with paeonol for 1 h prior to induction with P. gingivalis. Then, osteoclast formation was assessed using tartrate resistant acid phosphatase (TRAP) staining. The osteoclast-related proteins TRAP and receptor activator of nuclear factor-κB (RANK) were quantified by Western blotting.

RESULTSPaeonol was nontoxic to BMM within a range of 10-50 μmol·L⁻¹. Flow cytometry showed that paeonol inhibited PD-L1 expression in P. gingivalis-induced BMM in a dose-dependent manner. ELISA indicated that paeonol dose-dependently inhibited the excretion of TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM (P<0.01). TRAP staining revealed that paenol treatment inhibited the differentiation of P. gingivalis-induced BMM into osteoclasts. Western blot results suggested that paeonol decreased the expression of TRAP and RANK in BMM.

CONCLUSIONSPaeonol dose-dependently inhibited the excretion of the inflammatory factors TNF-α, IL-1β, and IL-6 by P. gingivalis-induced BMM in a dose-dependent manner. Moreover, paenol treatment prevented the differentiation of P. gingivalis-induced BMM differentiation into osteoclasts.
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Acetophenones ; pharmacology ; Acid Phosphatase ; Animals ; Carrier Proteins ; Cell Differentiation ; Interleukin-1beta ; Interleukin-6 ; Isoenzymes ; Macrophage Colony-Stimulating Factor ; Macrophages ; Membrane Glycoproteins ; Mice ; Osteoclasts ; Porphyromonas gingivalis ; RANK Ligand ; Receptor Activator of Nuclear Factor-kappa B ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the effect of ulinastatin (UTI) for early drug intervention on the serum levels of tumor necrosis factor-α (TNF-α), P-selectin, and thrombin-antithrombin complex (TAT) in young rats with sepsis.

METHODSA total of 120 male rats aged 4 weeks were randomly divided into normal control group, sham-operation group, sepsis group, low-dose UTI group (50 000 U/kg), and high-dose UTI group (200 000 U/kg), with 24 rats in each group. Modified cecal ligation and puncture was performed to establish a rat model of sepsis, and the rats in the low- and high-dose UTI groups were given caudal vein injection of UTI after model establishment. ELISA was used to measure the serum levels of TNF-α, P-selectin, and TAT at 6, 12, and 24 hours after model establishment.

RESULTSThe sepsis group had significant increases in the serum levels of TNF-α, P-selectin, and TAT at 6 hours, and the serum levels of TNF-α and TAT continued to increase by 24 hours (P<0.05); P-selectin reached the peak at 12 hours and decreased slightly at 24 hours (P<0.05). The UTI groups had similar change patterns in the levels of P-selectin and TAT as the sepsis group. The UTI groups had significant increases in the level of TNF-α at 6 hours, but gradually decreased over time. The changes in serum levels of TNF-α, P-selectin, and TAT in the UTI groups were significantly smaller than in the sepsis group (P<0.05). The high-dose UTI group had significantly smaller changes in serum levels of TNF-α, P-selectin, and TAT than the low-dose UTI group (P<0.05).

CONCLUSIONSEarly intervention with UTI can significantly improve coagulation function and inhibit the production of TNF-α, P-selectin, and TAT in young rats with sepsis. High-dose UTI has a significantly greater effect than low-dose UTI.

Animals ; Antithrombin III ; Glycoproteins ; pharmacology ; therapeutic use ; Male ; P-Selectin ; blood ; Peptide Hydrolases ; blood ; Rats ; Rats, Sprague-Dawley ; Sepsis ; blood ; drug therapy ; Tumor Necrosis Factor-alpha ; blood