Signaling Pathway Inhibitor Research Articles

IL-27 induces autophagy through regulation of the DNMT1/lncRNA MEG3/ERK/p38 axis to reduce pulmonary fibrosis

PurposePrevious studies have shown that interleukin-27 (IL-27) can reduce bleomycin (BLM)-induced pulmonary fibrosis (PF). However, the underlying mechanism by which IL-27 attenuates PF is not fully clear.MethodsIn this research, we used BLM to construct a PF mouse model, and MRC-5 cells stimulated by transforming growth factor-β1 (TGF-β1) were used to construct a PF model in vitro. The lung tissue status was observed by Masson and hematoxylin and eosin (HE) staining. To detect gene expression, RT‒qPCR was used. The protein levels were detected by western blotting and immunofluorescence staining. EdU and ELISA were used to detect cell proliferation viability and hydroxyproline (HYP) content, respectively.ResultsAberrant IL-27 expression was observed in BLM-induced mouse lung tissues, and the use of IL-27 attenuated mouse lung tissue fibrosis. TGF-β1 induced autophagy inhibition in MRC-5 cells, and IL-27 alleviated MRC-5 cell fibrosis by activating autophagy. The mechanism is inhibition of DNA methyltransferase 1 (DNMT1)-mediated lncRNA MEG3 methylation and ERK/p38 signaling pathway activation. Overexpression of DNMT1, knockdown of lncRNA MEG3, autophagy inhibitor or ERK/p38 signaling pathway inhibitors reversed the positive effect of IL-27 in a lung fibrosis model in vitro.ConclusionIn conclusion, our study shows that IL-27 upregulates MEG3 expression through inhibition of DNMT1-mediated lncRNA MEG3 promoter methylation, which in turn inhibits ERK/p38 signaling pathway-induced autophagy and attenuates BLM-induced PF, providing a contribution to the elucidation of the potential mechanisms by which IL-27 attenuates PF.

Small Molecules as Anti‐inflammatory Agents: Molecular Mechanisms and Heterocycles as Inhibitors of Signaling Pathways

AbstractCancer and inflammation interlink with many pathways. Millions of people died because of these two disorders. Major pathways that cause both inflammation and cancer are STAT3, NF‐κB, COX, and histone acetylation. However, these are the target for medicinally potent heterocycles such as pyrimidines, indole, and pyrazole. Many NSAIDs reported in the literature belong to both natural and synthetic molecules. This review encompasses molecular mechanisms for inflammation, NSAIDs derived from natural sources and synthetic methods, and recent reports on heterocycles as potent anti‐inflammatory agents and their significance in cancer treatment. Literature for the study, accomplished from 2019 to 2022.

Sodium selenite inhibits proliferation of lung cancer cells by inhibiting NF-κB nuclear translocation and down-regulating PDK1 expression which is a key enzyme in energy metabolism expression

As a trace element that maintains homeostasis in human body, selenium has significant anti-tumor activity. However, its exact molecular mechanism remains to be elucidated. Sodium selenite (SSe) is the most widely-distributed inorganic selenium in nature. In this study, we selected SSe as the research object to explore its anti-tumor mechanism in lung cancer. In vitro experiment showed that SSe could inhibit the activation of NF-κB signaling pathway, knowing that NF-κB is an important intracellular nuclear transcription factor that regulates the expression of pyruvate dehydrogenase kinase 1 (PDK1), a key energy metabolism switch affecting the survival status of the whole cell.At the same time, Bay11–7082(NF-κB signaling pathway inhibitors) and SSe resulted in phosphorylation of p65 and IκBα, decreased expression of PDK1 and Bcl-2,and increased expression of Bax in lung cancer cells. Our further study demonstrated that the reduction of PDK1 activity inhibited lactate secretion, reduced mitochondrial membrane potential, caused the release of Cytochrome C (Cyto C), activated mitochondrial respiration, and promoted the apoptosis of lung cancer cells. The in vivo experiment revealed that SSe inhibited the activation of NF-κB signaling pathway, decreased the expression of PDK1, and induced lung cancer cell proliferation and apoptosis. All these findings indicated that SSe promoted lung cancer cell apoptosis by inhibiting the activation of NF-κB signaling pathway, down-regulating PDK1 and activating mitochondrial apoptosis pathway.

Palmitic acid combined with γ-interferon inhibits gastric cancer progression by modulating tumor-associated macrophages' polarization via the TLR4 pathway.

Tumor-associated macrophages (TAMs) constitute the main infiltrating immune cells in the solid tumor microenvironment. Amounting studies have analyzed the antitumor effect on immune response induced by Toll-like receptor (TLR) agonists, such as lipopolysaccharide (LPS), γ-interferon (γ-IFN), and palmitic Acid (PA). However, their combined treatment for gastric cancer (GC) has not been illuminated. We investigated the relevance of macrophage polarization and the effect of PA and γ-IFN in GC in vitro and in vivo. M1 and M2 macrophage-associated markers were measured by real-time quantitative PCR and flow cytometry, and the activation level of the TLR4 signaling pathways was evaluated by western blot analysis. The effect of PA and γ-IFN on the proliferation, migration, and invasion of GC cells (GCCs) was evaluated by Cell-Counting Kit-8, transwell assays, and wound-healing assays. In vivo animal models were used to verify the effect of PA and γ-IFN on tumor progression, and the M1 and M2 macrophage markers, CD8 + T lymphocytes, regulatory T cells (Treg) cells, and the myeloid-derived suppressor cells (MDSCs) in tumor tissues were analyzed by flow cytometry and immunohistochemical (IHC). The results showed that this combination strategy enhanced M1-like macrophages and diminished M2-like macrophages through the TLR4 signaling pathway in vitro. In addition, the combination strategy impairs the proliferative and migratory activity of GCC in vitro and in vivo. While, the antitumor effect was abolished using the TAK-424 (a specific TLR-4 signaling pathway inhibitor) in vitro. The combined treatment of PA and γ-IFN inhibited GC progression by modulating macrophages polarization via the TLR4 pathway.

JNK/c-Jun pathway activation is essential for HBx-induced IL-35 elevation to promote persistent HBV infection.

Immunoregulation plays pivotal roles during chronic hepatitis B virus (HBV) infection. Studies have shown that Interleukin (IL)-35 is an important molecule associated with inadequate immune response against HBV. However, the mechanisms involved in the up-regulation of IL-35 expression during persistent HBV infection remain unknown. In this study, we constructed a plasmid expressing the HBV X protein (pCMV-HBx) to evaluate the relationship between HBx and IL-35. Activation of the JNK/c-Jun pathway was analyzed and chromatin immunoprecipitation followed by sequencing and luciferase reporter assays were performed to determine whether c-Jun could regulate IL-35 transcription. HBx can significantly activate IL-35 promoter in both LO2 and HepG2 cells compared to the control plasmid (pCMV-Tag2) using the dual-luciferase assay. Whereas other viral proteins, such as S, preS1, the core protein, had no significant effect on IL-35 expression. Similarly, WB and qRT-PCR also showed that HBx can significantly promote IL-35 expression at protein and mRNA levels in the aforementioned cells. The relevant pathway mechanism showed that the expression of JNK and c-Jun genes was significantly higher in transfected cells carrying pCMV-HBx than in the pCMV-Tag2-transfected and -untransfected cells. WB analysis revealed that phosphorylated JNK and c-Jun were overexpressed after HBx action. Conversely, the addition of the JNK/c-Jun signaling pathway inhibitor could significantly suppress HBx-induced IL-35 expression in a dose-dependent manner. A novel molecular mechanism of HBV-induced IL-35 expression was revealed, which involves JNK/c-Jun signaling in up-regulating IL-35 expression via HBx, resulting in transactivation of the IL-35 subunit EBI3 and p35 promoter.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of communication, learning, and behavioral problems. The syndromic form of ASD is caused by monogenic mutations, in the case when it is not possible to find genetic or other known mechanisms, the term “idiopathic autism” is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody-induced autism and childhood autoimmune neuropsychiatric disorders such as Sydenham’s chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the possibility of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

Inhibitors of the Ubiquitin-Mediated Signaling Pathway Exhibit Broad-Spectrum Antiviral Activities against New World Alphaviruses.

New World alphaviruses including Venezuelan Equine Encephalitis Virus (VEEV) and Eastern Equine Encephalitis Virus (EEEV) are mosquito-transmitted viruses that cause disease in humans and equines. There are currently no FDA-approved therapeutics or vaccines to treat or prevent exposure-associated encephalitic disease. The ubiquitin proteasome system (UPS)-associated signaling events are known to play an important role in the establishment of a productive infection for several acutely infectious viruses. The critical engagement of the UPS-associated signaling mechanisms by many viruses as host-pathogen interaction hubs led us to hypothesize that small molecule inhibitors that interfere with these signaling pathways will exert broad-spectrum inhibitory activity against alphaviruses. We queried eight inhibitors of the UPS signaling pathway for antiviral outcomes against VEEV. Three of the tested inhibitors, namely NSC697923 (NSC), bardoxolone methyl (BARM) and omaveloxolone (OMA) demonstrated broad-spectrum antiviral activity against VEEV and EEEV. Dose dependency and time of addition studies suggest that BARM and OMA exhibit intracellular and post-entry viral inhibition. Cumulatively, our studies indicate that inhibitors of the UPS-associated signaling pathways exert broad-spectrum antiviral outcomes in the context of VEEV and EEEV infection, supporting their translational application as therapeutic candidates to treat alphavirus infections.

The expression and function of PD-L1 in CD133(+) human liver cancer stem-like cells

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.

Research advances on the mechanism of Wnt/β-catenin signaling pathway in body surface wound healing

Wound healing is a slow and complex biological process, including inflammatory reaction, cell proliferation, cell differentiation, cell migration, angiogenesis, extracellular matrix deposition, tissue remodeling, and so on. Wnt signaling pathway can be divided into classical pathway and non-classical pathway. Wnt classical pathway, also known as Wnt/β-catenin signaling pathway, plays an important role in cell differentiation, cell migration, and maintenance of tissue homeostasis. Many inflammatory factors and growth factors are involved in the upstream regulation of this pathway. The activation of Wnt/β-catenin signaling pathway plays an important role in the occurrence, development, regeneration, repair and related treatment of skin wounds. This article review the relationship between Wnt/β-catenin signaling pathway and wound healing, meanwhile summarizes its effects on important processes of wound healing, such as inflammation, cell proliferation, angiogenesis, hair follicle regeneration, and skin fibrosis, as well as the role of inhibitors of Wnt signaling pathway in wound healing.

Bile duct ligation increased dopamine levels in the cerebral cortex of rats partly due to induction of tyrosine hydroxylase.

Liver failure is associated with psychiatric alterations, partly resulting from the increased brain dopamine levels. We investigated the relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and the mechanism by which liver failure increased dopamine levels in SH-SY5Y cells. Behavioural tests were carried out on day 13 and 27 following BDL, along with measurements of dopamine and metabolites, expressions of enzymes and transporters related to dopamine metabolism, and its transport into the cortex and the hippocampus. SH-SY5Y cells were used to investigate whether NH4 Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase or not. Tyrosine hydroxylase (TH) expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. Open-field test results demonstrated BDL rats showed anxiety-like behaviour, accompanied by increased dopamine levels and expression of TH protein in the cortex. Membrane bound long form (MB)-COMT, slightly but significantly decreased. SH-SY5Y cells indicated that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY 11-7082 and silencing NF-κB p65 reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in increases of TH protein expressions and dopamine levels were measured using hyperbilirubinemia rats. Anxiety-like behaviour, was associated with increased dopamine levels and TH protein expressions in hyperbilirubinemia rats. BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signalling pathway.

CEBPA-Regulated Expression of SOCS1 Suppresses Milk Protein Synthesis through mTOR and JAK2-STAT5 Signaling Pathways in Buffalo Mammary Epithelial Cells

Milk protein content is a key quality indicator of milk, and therefore elucidating its synthesis mechanism has been the focus of research in recent years. Suppressor of cytokine signaling 1 (SOCS1) is an important inhibitor of cytokine signaling pathways that can inhibit milk protein synthesis in mice. However, it remains elusive whether SOCS1 plays roles in the milk protein synthesis in the buffalo mammary gland. In this study, we found that the mRNA and protein expression levels of SOCS1 in buffalo mammary tissue during the dry-off period was significantly lower than those during lactation. Overexpression and knockdown experiments of SOCS1 showed that it influenced the expression and phosphorylation of multiple key factors in the mTOR and JAK2-STAT5 signaling pathways in buffalo mammary epithelial cells (BuMECs). Consistently, intracellular milk protein content was significantly decreased in cells with SOCS1 overexpression, while it increased significantly in the cells with SOCS1 knockdown. The CCAAT/enhancer binding protein α (CEBPA) could enhance the mRNA and protein expression of SOCS1 and its promoter activity in BuMECs, but this effect was eliminated when CEBPA and NF-κB binding sites were deleted. Therefore, CEBPA was determined to promote SOCS1 transcription via the CEBPA and NF-κB binding sites located in the SOCS1 promoter. Our data indicate that buffalo SOCS1 plays a significant role in affecting milk protein synthesis through the mTOR and JAK2-STAT5 signaling pathways, and its expression is directly regulated by CEBPA. These results improve our understanding of the regulation mechanism of buffalo milk protein synthesis.

Absorption and Transport Mechanism of Red Meat-Derived N-glycolylneuraminic Acid and Its Damage to Intestinal Barrier Function through the NF-κB Signaling Pathway.

N-glycolylneuraminic acid (Neu5Gc) is a specific factor in red meat that induces intestinal disease. Our aim was to investigate the effect of Neu5Gc on the intestinal barrier as well as its mechanism of endocytosis and exocytosis. Ten specific inhibitors were used to explore the mechanism of Neu5Gc endocytosis and exocytosis by Caco-2 cells. Amiloride hydrochloride and cytochalasin D had the strongest inhibitory effect on the endocytosis of Neu5Gc. Sodium azide, dynasore, chlorpromazine hydrochloride, and nystatin also inhibited Neu5Gc endocytosis. Dynasore exhibited a stronger inhibitory effect than that of chlorpromazine hydrochloride or nystatin alone. Exocytosis inhibitors, including nocodazole, brefeldin A, monensin, and bafilomycin A, inhibited the transmembrane transport of Neu5Gc. Monensin promoted the exocytosis of Neu5Gc from Caco-2 cells. In another experiment, we observed no significant inhibitory effects of monensin and brefeldin A. Dietary concentrations of Neu5Gc induced prominent damage to intestinal tight junction proteins zonula occludens-1 (ZO-1), occludin, and claudin-1 and promoted the phosphorylation of IκB-α and P65 to activate the canonical Nuclear Factor kappa-B (NF-κB) pathway. Neu5Gc increased the RNA levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α and inhibited those of anti-inflammatory factors TGF-β and IL-10. BAY, an NF-κB signaling pathway inhibitor, attenuated these changes. Reductions in the levels of ZO-1, occludin, and claudin-1 were recovered in response to BAY. Our data reveal the endocytosis and exocytosis mechanism of Neu5Gc and prove that Neu5Gc can activate the canonical NF-κB signaling pathway, regulate the transcription of inflammatory factors, thereby damaging intestinal barrier function.

Upregulation of long intergenic non-coding RNA LINC00326 inhibits non-small cell lung carcinoma progression by blocking Wnt/β-catenin pathway through modulating the miR-657/dickkopf WNT signaling pathway inhibitor 2 axis

BackgroundLong intergenic non-coding RNA 326 (LINC00326) modulates hepatocarcinogenic lipid metabolism. However, the ability of LINC00326 to modulate the highly aggressive non-small cell lung carcinoma (NSCLC) is unknown. Here, LINC00326 in NSCLC was investigated, together with its effects on tumor malignancy and the underlying mechanisms of action.MethodsLINC00326 levels in tumor tissues and cell lines were measured by Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and RNA fluorescence in situ hybridization (FISH). Proliferation and apoptosis were assessed in cell lines by Cell Counting Kit-8 (CCK-8), EdU staining assays and flow cytometry, respectively, and tumor growth was measured in mouse models. Possible microRNA targets of LINC00326 were predicted by bioinformatics and verified by RNA pull-down and immunoprecipitation and luciferase reporter assays. Western blotting was used to evaluate the expression of Wnt/β-catenin-associated proteins.Results LINC00326 was downregulated in tumor tissues and cell lines. Knockdown of LINC00326 stimulated NSCLC cell proliferation and suppressed apoptosis in vitro, as well as enhancing xenograft tumor growth. LINC00326 sponged miR-657, and dickkopf WNT signaling pathway inhibitor 2 (DKK2) was found to be directly targeted by miR-657, with LINC00326 positively regulating its expression through sponging miR-657. The actions of LINC00326 knockdown on proliferation and apoptosis were reversed by stimulation of the miR-657/DKK2 axis. Furthermore, overexpression of miR-657 mitigated DKK2 inhibition on Wnt/β-catenin signaling.ConclusionsLINC00326/miR-657/DKK2 axis signaling blocked tumor-associated functions in NSCLC cells through the targeting Wnt/β-catenin pathway. This suggests that this pathway could be a target for NSCLC treatment.

Abnormal mTOR Signaling Pathway Activity in Autism Spectrum Disorders: Prospects of Mechanism-Based Therapy

Autism spectrum disorder (ASD) is a developmental disorder characterized by the early onset of problems with communication, learning, and behavior. The syndromic form of ASD is caused by monogenic mutations. When it is not possible to find genetic or other known mechanisms, the term "idiopathic autism" is used. A significant part of both syndromic and idiopathic autism is associated with translational deregulation dependent on the mechanistic target of rapamycin (mTOR). In this review, we present both bioinformatic and experimental data that link the mTOR signaling pathway to maternal autoantibody related autism and childhood autoimmune neuropsychiatric disorders such as Sydenham's chorea and pediatric autoimmune neuropsychiatric disorder associated with streptococcal infections (PANDAS). The need for ASD subtyping and the prospects of mechanism-based therapy with inhibitors of the mTOR signaling pathway are also discussed.

MicroRNA21-5p alleviates hyperoxia-induced acute lung injury in rats through activating phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway by regulating type II alveolar epithelial cell apoptosis

To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type II alveolar epithelial cell (AEC II). Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 μL titer (1×1012 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AEC II cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AEC II cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AEC II cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AEC II cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (μg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1β (μg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AEC II cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg ≈ 0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2-ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AEC II cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. miR-21-5p attenuates HALI by inhibiting AEC II cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.

Dickkopf-1 inhibits the secretion of MUC5AC induced by Mycoplasma pneumoniae P1-C in mouse lung epithelial cells

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M. pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M. pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M. pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

In silico approach to identify novel allosteric intracellular antagonist for blocking the interleukin-8/CXCR2 receptor signaling pathway

The role of interleukin-8 (IL-8) and its receptor CXCR2 in inflammatory responses and tumor development and progression has been well documented. Our study aims to discover novel compounds as CXCR2 antagonists to block the IL-8 signaling pathway using an in silico drug design. Herein, a structure-based pharmacophore model was developed based on the crystal structure of inactive CXCR2 in a complex with an allosteric inhibitor. This model was validated and refined, followed by virtual screening with the ZINC15 database. Subsequent molecular docking allows for predicting the best pose of a ligand inside a receptor binding site. We found that the 35 top-ranked hits exhibited docking scores from −30.81 to −25.28 kJ/mol and better interaction potential comparing the reference inhibitor. Analysis of ADME and toxicity properties revealed the efficacy and safety of the selected seven compounds. To validate the stability of the protein-ligand complex structure MD simulations approach has also been performed and confirmed via the critical parameters. The MD results explained that the CXCR2 receptor bound with two best-proposed molecules, including ZINC77105530 and ZINC93176465, was quite stable states as observed from low RMSD, RMSF, Rg, SASA values, and high occupancy of the interaction types. Finally, our data identified that these compounds play as potential inhibitors of IL-8 signaling pathways with the MM/GBSA binding free energies of −41.77 ± 6.45 kcal/mol and −38.84 ± 6.16 kcal/mol, respectively. Communicated by Ramaswamy H. Sarma

AR and PI3K/AKT in Prostate Cancer: A Tale of Two Interconnected Pathways.

Prostate cancer (PCa) is the most common cancer in men. The androgen receptor (AR) has a pivotal role in the pathogenesis and progression of PCa. Many therapies targeting AR signaling have been developed over the years. AR signaling inhibitors (ARSIs), including androgen synthesis inhibitors and AR antagonists, have proven to be effective in castration-sensitive PCa (CSPC) and improve survival, but men with castration-resistant PCa (CRPC) continue to have a poor prognosis. Despite a good initial response, drug resistance develops in almost all patients with metastatic CRPC, and ARSIs are no longer effective. Several mechanisms confer resistance to ARSI and include AR mutations but also hyperactivation of other pathways, such as PI3K/AKT/mTOR. This pathway controls key cellular processes, including proliferation and tumor progression, and it is the most frequently deregulated pathway in human cancers. A significant interaction between AR and the PI3K/AKT/mTOR signaling pathway has been shown in PCa. This review centers on the current scene of different AR and PI3K signaling pathway inhibitors, either as monotherapy or in combination treatments in PCa, and the treatment outcomes involved in both preclinical and clinical trials. A PubMed-based literature search was conducted up to November 2022. The most relevant and recent articles were selected to provide essential information and current evidence on the crosstalk between AR and the PI3K signaling pathways. The ClinicalTrials.gov registry was used to report information about clinical studies and their results using the Advanced research tool, filtering for disease and target.

Abstract PR015: Dormant mammary tumors persist long-term despite adaptive immunity by establishing a Treg-dominated niche via DKK3

Abstract A significant portion of breast cancer survivors eventually experience locoregional or metastatic recurrence. It is clear that the adaptive immune system controls proliferative tumor outgrowth, but how dormant tumor cells escape to eventually form new lesions remains unclear. Previous studies showed that proliferative D2A1 or D2.OR tumors are restricted by adaptive immunity in Balb/c versus SCID mice whereas dormant D2.1 tumors are largely unaffected. Further, D2.1 tumors are highly infiltrated with T cells, particularly CD4+ cells and Tregs. Thus, the aim of the present study was to determine the extent to which dormant D2.1 tumors evade antigen-specific immunity and how these tumors shape the T cell niche. Experiments were performed using Balb/c syngeneic D2 mammary tumor cells – D2A1 cells grow rapidly upon orthotopic injection, D2.OR cells proliferate but to a lesser degree than D2A1, and D2.1 cells remain dormant long term. Cells were also modified to express enhanced green fluorescent protein (eGFP) for use with Just eGFP Death Inducing (JeDI) T cells containing a T cell receptor (TCR) targeted to eGFP200-208. Transfer of JeDI T cells caused complete rejection of D2A1-eGFP tumors in the mammary fat pad (MFP) while no difference was observed in D2.1-eGFP tumors. In contrast, treatment with anti-CTLA4 alone, anti-PD-1 alone, or combined anti-CTLA4/PD-1 reduced D2.1 tumor progression compared to isotype control. Whole JeDI splenocytes were also stimulated in vitro with eGFP200-208 peptide in the presence of D2A1 or D2.1 conditioned medium (CM). Surprisingly, while CD8 cells proliferated in both D2A1 and D2.1 CM, CD4+ T cells also proliferated in D2.1 CM and the Treg fraction of CD4+ cells was significantly elevated in D2.1 CM compared to D2A1 CM or control medium. RNA-sequencing confirmed substantial immune activity in D2.1 tumors compared to D2A1. D2.1 tumors also displayed a hybrid epithelial/mesenchymal (E/M) phenotype and expressed genes associated with both mammary epithelium (Krt8, Krt14, Cdh1) and mesenchymal cells (Itga5, Snai2, Notch1, Zeb1). Moreover, Gene Set Enrichment Analysis (GSEA) showed an enrichment of mammary stem and luminal progenitor cell signatures in D2.1 tumors. Of the top upregulated genes, Dickkopf WNT Signaling Pathway Inhibitor 3 (DKK3) was chosen for further investigation. Interestingly, increased CD8 proliferation and IFNγ production, but decreased CD4 and Treg abundance, was observed in DKK3 shRNA knockdown (k/d) D2.1 CM compared to shScramble control. In vivo, overexpressing DKK3 in D2A1 cells significantly increased MFP tumor growth in Balb/c but not SCID mice, altered T cell activation locally and systemically, and decreased eGFP-specific antibodies. Similar results were observed in DKK3-overexpressing D2.OR tumors. Conversely, DKK3 k/d in D2.1 cells resulted in ~60% complete tumor rejection. Together, these data indicate that CD4 cells, particularly Tregs, are crucial in establishing a protective niche for long-term tumor dormancy with tumor-derived DKK3 emerging as a mediator of the Treg niche. Citation Format: Timothy N. Trotter, Delila Serra, Carina Dagotto, Tao Wang, Xiao Yang, Junping Wei, Gangjun Lei, H. Kim Lyerly, Zachary C. Hartman. Dormant mammary tumors persist long-term despite adaptive immunity by establishing a Treg-dominated niche via DKK3 [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR015.

COVID-19: A Comprehensive Review on Cardiovascular Alterations, Immunity, and Therapeutics in Older Adults.

Here, we present a review focusing on three relevant issues related to COVID-19 and its impact in older adults (60 years and older). SARS-CoV-2 infection starts in the respiratory system, but the development of systemic diseases accompanied by severe clinical manifestations has also been reported, with cardiovascular and immune system dysfunction being the major ones. Additionally, the presence of comorbidities and aging represent major risk factors for the severity and poor prognosis of the disease. Since aging-associated decline has been largely related to immune and cardiovascular alterations, we sought to investigate the consequences and the underlying mechanisms of these pathologies to understand the severity of the illness in this population. Understanding the effects of COVID-19 on both systems should translate into comprehensive and improved medical care for elderly COVID-19 patients, preventing cardiovascular as well as immunological alterations in this population. Approved therapies that contribute to the improvement of symptoms and a reduction in mortality, as well as new therapies in development, constitute an approach to managing these disorders. Among them, we describe antivirals, cytokine antagonists, cytokine signaling pathway inhibitors, and vaccines.