TgMIF Promotes Hepatocyte Pyroptosis and Recruitment of Proinflammatory Macrophages During Severe Liver Injury in Acute Toxoplasmosis.

Liver injury is a common complication during infection of Toxoplasma gondii. However, the Toxoplasma effector proteins involved remain unknown. Herein, we identified that T. gondii macrophage migration inhibitory factor (TgMIF) is a critical pathogenic factor of liver injury in acute toxoplasmosis mouse model induced by a less virulent strain, which is widely prevalent in humans. We show that TgMIF is a novel activator of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in hepatocytes, resulting in subsequent pyroptosis. Furthermore, T. gondii promotes the TgMIF-dependent infiltration of Ly6Chi proinflammatory macrophages to release cytokines, leading to hepatocyte apoptosis. Although the intense inflammation induced by TgMIF inhibits the proliferation of intracellular parasites, it results in fatal liver damage. In contrast, parasites with TgMIF gene deletion significantly alleviate liver injury and prolong mice survival. The discovery of novel Toxoplasma virulence factor may expedite the development of human toxoplasmosis control strategies.

Dihydroartemisinin Attenuates Hypoxia-Induced Pulmonary Hypertension Through the ELAVL2/miR-503/PI3K/AKT Axis.

ABSTRACT: Dihydroartemisinin (DHA) is an active form of artemisinin extracted from the traditional Chinese medicine Artemisia annua , which is used to treat malaria. Previous studies have shown that DHA has a therapeutic effect on pulmonary hypertension (PH), but its specific mechanism has not been fully elucidated. In this study, a hypoxia-induced PH mouse model was established and DHA was administered as a therapeutic intervention. We measured hemodynamics and right ventricular hypertrophy and observed hematoxylin and eosin staining of lung tissue sections, proving the therapeutic effect of DHA on PH. Furthermore, cell counting kit-8 and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay kit were performed to examine cell proliferation of pulmonary artery smooth muscle cells cultured in hypoxia or in normoxia. Transwell migration chamber assay was performed to examine cell migration of the same cell model. Consistent with the therapeutic effect in vivo, DHA inhibited hypoxia-induced cell proliferation and migration. Through high-throughput sequencing of mouse lung tissue, we screened embryonic lethal abnormal vision-like 2 (ELAVL2) as a key RNA binding protein in PH. Mechanistically, DHA inhibited the proliferation and migration of pulmonary artery smooth muscle cells by promoting the expression of ELAVL2 and regulating the miR-503/PI3K/AKT pathway. The binding relationship between ELAVL2 and pre-miR-503 was verified by RNA binding protein immunoprecipitation assay. In conclusion, we first propose that DHA alleviates PH through the ELAVL2/miR-503/PI3K/AKT pathway, which may provide a basis for new therapeutic strategies of PH.

Telocinobufagin Has Antitumor Effects in Non-Small-Cell Lung Cancer by Inhibiting STAT3 Signaling.

Non-small-cell lung carcer (NSCLC), the main histological subtype of lung cancer, is responsible for significant morbidity and mortality worldwide. Telocinobufagin, an active compound of the Chinese traditional medicine ChanSu, has antitumor effects, but its mechanism of action remains unknown. Therefore, we investigated the effect of telocinobufagin on NSCLC growth and metastasis and its possible mechanism of action, in vitro and in vivo. Cell proliferation, migration, and apoptosis were measured by methyl thiazol tetrazolium assay, colony formation, 5-ethynyl-2'-deoxyuridine incorporation, Transwell migration, wound healing, and flow cytometry analysis. A mouse xenograft model was used to evaluate tumor formation in vivo. Telocinobufagin was found to suppress proliferation and metastasis and induce apoptosis in human NSCLC cells. Moreover, telocinobufagin was able to significantly inhibit STAT3 phosphorylation at tyrosine 705 (Y705) and its downstream targets. Additionally, telocinobufagin also impaired the IL-6-induced nuclear translocation of STAT3. Consistent with the in vitro experiments, telocinobufagin reduced the A549 xenograft tumor burden and the levels of P-STAT3Y705, MCL1, BCL2, and cleaved PARP1 in vivo. These results support telocinobufagin as a promising STAT3 signaling inhibitor candidate for the treatment of NSCLC patients.

Global proteome profiling of human livers upon ischemia/reperfusion treatment.

Hepatic ischemia/reperfusion (I/R) injury represents a major risk factor for liver transplantation and is related to graft dysfunction and acute/chronic rejection. However, a significant part of these processes remain poorly characterized. To reveal differences in the proteome during liver I/R injury, we collected human liver biopsy samples during hepatectomy before and after the Pringle maneuver and conducted a TMT-based proteomic analysis through quantitative high-throughput mass spectrometry. We used a fold-change threshold of 1.3 and a t-test p-value < 0.05 as the criteria to identify 5,257 total quantifiable proteins. The levels of 142 proteins were increased, while the levels of 103 proteins were decreased in response to hepatic I/R treatment. Bioinformatic analysis further revealed that these differentially expressed proteins are mainly involved in multiple biological functions and enzyme-regulated metabolic pathways. Most proteins whose expression was changed are related to the defense, immune and inflammatory responses as well as lipid and steroid metabolic processes. Based on this finding, we developed a panel for targeted proteomic analysis and used the parallel reaction monitoring (PRM) method, qPCR and western blotting experiments to validate alterations in the expression of some of the identified proteins. The upregulated proteins were found to be involved in immunity and inflammatory responses, and downregulated proteins were enriched in metabolic pathways. This study therefore may provide a research direction for the design of new therapeutic strategies for hepatic ischemia/reperfusion injury.

RTN1-C mediates cerebral ischemia/reperfusion injury via modulating autophagy.

It has been widely accepted that autophagic cell death exacerbates the progression of cerebral ischemia/reperfusion (I/R). Our previous study revealed that overexpression of reticulon protein 1-C (RTN1-C) is involved in cerebral I/R injury. However, the underlying mechanisms have not been studied intensively. This study was designed to evaluate the effect of RTN1-C on autophagy under cerebral I/R. Using an in vitro oxygen-glucose deprivation followed by reoxygenation and a transient middle cerebral artery occlusion model in rats, we found that the expression of RTN1-C protein was significantly upregulated. We also revealed that RTN1-C knockdown suppressed overactivated autophagy both in vivo and in vitro, as indicated by decreased expressions of autophagic proteins. The number of Beclin-1/propidium iodide-positive cells was significantly less in the LV-shRTN1-C group than in the LV-shNC group. In addition, rapamycin, an activator of autophagy, aggravated cerebral I/R injury. RTN1-C knockdown reduced brain infarct volume, improved neurological deficits, and attenuated cell vulnerability to cerebral I/R injury after rapamycin treatment. Taken together, our findings demonstrated that the modulation of autophagy from RTN1-C may play vital roles in cerebral I/R injury, providing a potential therapeutic treatment for ischemic brain injury.

Encephalitis is mediated by ROP18 of Toxoplasma gondii, a severe pathogen in AIDS patients.

The neurotropic parasite Toxoplasma gondii is a globally distributed parasitic protozoan among mammalian hosts, including humans. During the course of infection, the CNS is the most commonly damaged organ among invaded tissues. The polymorphic rhoptry protein 18 (ROP18) is a key serine (Ser)/threonine (Thr) kinase that phosphorylates host proteins to modulate acute virulence. However, the basis of neurotropism and the specific substrates through which ROP18 exerts neuropathogenesis remain unknown. Using mass spectrometry, we performed proteomic analysis of proteins that selectively bind to active ROP18 and identified RTN1-C, an endoplasmic reticulum (ER) protein that is preferentially expressed in the CNS. We demonstrated that ROP18 is associated with the N-terminal portion of RTN1-C and specifically phosphorylates RTN1-C at Ser7/134 and Thr4/8/118. ROP18 phosphorylation of RTN1-C triggers ER stress-mediated apoptosis in neural cells. Remarkably, ROP18 phosphorylation of RTN1-C enhances glucose-regulated protein 78 (GRP78) acetylation by attenuating the activity of histone deacetylase (HDAC), and this event is associated with an increase of neural apoptosis. These results clearly demonstrate that both RTN1-C and HDACs are involved in T. gondii ROP18-mediated pathogenesis of encephalitis during Toxoplasma infection.

Protozoan-Derived Cytokine-Transgenic Macrophages Reverse Hepatic Fibrosis.

Macrophage therapy for liver fibrosis is on the cusp of meaningful clinical utility. Due to the heterogeneities of macrophages, it is urgent to develop safer macrophages with a more stable and defined phenotype for the treatment of liver fibrosis. Herein, a new macrophage-based immunotherapy using macrophages stably expressing a pivotal cytokine from Toxoplasma gondii, a parasite that infects ≈ 2 billion people is developed. It is found that Toxoplasma gondii macrophage migration inhibitory factor-transgenic macrophage (Mφtgmif) shows stable fibrinolysis and strong chemotactic capacity. Mφtgmif effectively ameliorates liver fibrosis and deactivates aHSCs by recruiting Ly6Chi macrophages via paracrine CCL2 and polarizing them into the restorative Ly6Clo macrophage through the secretion of CX3CL1. Remarkably, Mφtgmif exhibits even higher chemotactic potential, lower grade of inflammation, and better therapeutic effects than LPS/IFN-γ-treated macrophages, making macrophage-based immune therapy more efficient and safer. Mechanistically, TgMIF promotes CCL2 expression by activating the ERK/HMGB1/NF-κB pathway, and this event is associated with recruiting endogenous macrophages into the fibrosis liver. The findings do not merely identify viable immunotherapy for liver fibrosis but also suggest a therapeutic strategy based on the evolutionarily designed immunomodulator to treat human diseases by modifying the immune microenvironment.

Integrative analyses of whole-transcriptome sequencing reveals CeRNA regulatory network in pulmonary hypertension treated with FGF21.

Noncoding RNAs have been shown to play essential roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Differentially expressed mRNAs, miRNAs, lncRNAs, and circRNAs regulated by hypoxia and FGF21 were selected through intersection analysis. Based on prediction databases and sequencing data, differentially co-expressed mRNAs, miRNAs, lncRNAs, and circRNAs were further screened, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. The ceRNA regulatory network of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. In addition, quantitative real-time PCR experiments were employed to verify the whole-transcriptome sequencing data. The results of luciferase reporter assays highlighted the relationship between miR-449c-5p and XR_878320.1, miR-449c-5p and Stab2, miR-449c-5p and circ_mtcp1, which suggesting that miR-449c-5p may be a key regulator of FGF21 in the treatment of PH. Taken together, this study provides potential biomarkers, pathways, and ceRNA regulatory networks in HPH treated with FGF21 and will provide an experimental basis for the clinical application of FGF21 in PH.

Parthenolide attenuates hypoxia-induced pulmonary hypertension through inhibiting STAT3 signaling.

BACKGROUND: Pulmonary hypertension (PH) is a chronic lung disease characterized by the progressive pulmonary vascular remodeling with increased pulmonary arterial pressure and right ventricular failure. Pulmonary vascular remodeling involves the proliferation, migration, and resistance to apoptosis of pulmonary artery smooth cells (PASMCs). Parthenolide (PTN) is a bioactive compound derived from a traditional medical plant feverfew (Tanacetum parthenium), and it has been studied for treatment of pulmonary fibrosis, lung cancer, and other related ailments. However, the function of PTN in the treatment of PH has not been studied. PURPOSE: This study aimed to evaluate the anti-proliferation and pro-apoptosis effects of PTN on PH and investigate its potential mechanisms. METHODS: An in vivo hypoxia-induced pulmonary hypertension (HPH) model was established by maintaining male rats in a hypoxia chamber (10% O2) for 3 weeks, and PTN was intraperitoneally administered at the dose of 10 or 30 mg/kg. We assessed the impact of PTN on mean pulmonary arterial pressure (mPAP), pulmonary vascular remodeling, and right ventricular hypertrophy. In vitro, we evaluated hypoxia-induced cellular proliferation, migration, and apoptosis of rat PASMCs. Proteins related to the STAT3 signaling axis were analyzed by western blotting and immunofluorescence assays. Recovery experiments were performed using the STAT3 activator, colivelin TFA. RESULTS: PTN significantly alleviated the symptoms of HPH rats by attenuating pulmonary arterial remodeling. It also prevented the proliferation and migration of PASMCs. PTN also induced the apoptosis of PASMCs. PTN could directly interact with STAT3 and markedly inhibited STAT3 phosphorylation and nuclear translocation. In vitro, and in vivo experiments demonstrated that overexpression of STAT3 partially suppressed the effect of PTN. CONCLUSION: Our study indicated that PTN alleviated hypoxia-induced pulmonary hypertension in rats by suppressing STAT3 activity.

Analyzing the molecular mechanism of xuefuzhuyu decoction in the treatment of pulmonary hypertension with network pharmacology and bioinformatics and verifying molecular docking.

BACKGROUND: XueFuZhuYu (XFZY), a typical Chinese herbal formula, has remarkable clinical effects for treating Pulmonary Hypertension (PH) with unclear mechanisms. Our research involved the utilization of network pharmacology to explore the traditional Chinese herbal monomers and their related targets within XFZY for PH treatment. Furthermore, molecular docking verification was performed. METHODS: The XFZY's primary active compounds, along with their corresponding targets, were both obtained from the TCMSP, ChEMBL, and UniProt databases. The target proteins relevant to PH were sifted through OMIM, GeneCards and TTD databases. The common "XFZY-PH" targets were evaluated with Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses with the assistance of R software. The Protein-Protein Interaction (PPI) network and compound-target-pathway network were constructed and a systematic analysis of network parameters was performed by the powerful software Cytoscape. Molecular docking was employed for assessing and verifying the interactions between the core targets and the top Chinese herbal monomer. RESULTS: The screening included 297 targets of active compounds in XFZY and 8400 PH-related targets. DO analysis of the above common 268 targets indicated that the treatment of the diseases by XFZY is mediated by genes related to Chronic Obstructive Pulmonary Disease (COPD), Obstructive Lung Disease (OLD), ischemia, and myocardial infarction. The findings from molecular docking indicated that the binding energies of 57 ligand-receptor pairs in PH and 20 ligand-receptor pairs in COPD-PH were lower than -7kJ•mol-1. CONCLUSIONS: This study indicates that XFZY is a promising option within traditional Chinese medicine compound preparation for combating PH, particularly in cases associated with COPD. Our demonstration of the specific molecular mechanism of XFZY anti-PH and its effective active ingredients provides a theoretical basis for better clinical application of the compound.

Dual delivery of an NF-κB inhibitor and IL-10 through supramolecular hydrogels polarizes macrophages and promotes cardiac repair after myocardial infarction.

The use of anti-inflammatory strategies has the potential to be a definitive treatment for ventricular remodeling post myocardial infarction (MI). The regulation of macrophage phenotypes by anti-inflammatory agents contributes to the alleviation of myocardial fibrosis. However, their poor retention rates severely affect treatment efficacy. Here, we propose a supramolecular compound, NapFFY, to co-assemble with IL-10 and SN50 as a novel anti-inflammatory SN50/IL-10/NapFFY hydrogel with cardioprotective properties. Results from the in vitro and in vivo experiments in murine cell line and rats, respectively, demonstrated that the SN50/IL-10/NapFFY hydrogel exhibits an ideal and sustained release of IL-10 and SN50. Intramyocardial injection of the SN50/IL-10/NapFFY hydrogel in a rat model of MI significantly inhibited the expression of proinflammatory cytokines. It promoted the polarization of M2 macrophages, which reduced cardiomyocyte apoptosis and improved vascularization at the border zones. Specifically, the SN50/IL-10/NapFFY hydrogel significantly improved heart function and ameliorated ventricular remodeling 28 days post MI. We envision that the SN50/IL-10/NapFFY hydrogel could serve as a new anti-inflammatory agent for the clinical treatment of MI in future studies. STATEMENT OF SIGNIFICANCE: Anti-inflammation is an ideal strategy for the treatment of ventricular remodeling post myocardial infarction (MI). SN50 and IL-10 have been shown to have diverse roles in antiinflammatory process, respectively. However, direct intravenous administration or intramyocardial injection of SN50 or IL-10 is not a viable option given its poor half-life in vivo. This study aimed to evaluate the synergistic cardioprotective effects of a supramolecular hydrogel loaded with an NF-κB inhibitor (SN50) and IL-10. Animal experiments showed that the SN50/IL-10/NapFFY hydrogels ameliorated the inflammatory microenvironment, and improved cardiac function to the infarct area in a rat model of MI. We anticipate that SN50/IL10NapFFY hydrogel could be used clinically to treat MI in the near future.

Piceatannol alleviates liver ischaemia/reperfusion injury by inhibiting TLR4/NF-κB/NLRP3 in hepatic macrophages.

BACKGROUND: Macrophages present strong immunomodulatory ability and are considered to be core immune cells in the process of hepatic ischaemia‒reperfusion (I/R). The NLRP3 inflammasome is a kind of intracellular multimolecular complex that actively participates in innate immune responses and proinflammatory signalling pathways. Piceatannol (PIC) is a derivative of the natural phenolic compound resveratrol and has antioxidant and anti-inflammatory effects. The purpose of this study was to examine whether pretreatment with PIC can alleviate hepatic I/R injury by targeting NLRP3 inflammasome-induced macrophage pyroptosis. METHODS: PIC-pretreated primary hepatic macrophages were subjected to hypoxia/reoxygenation, and liver ischaemia/reperfusion was performed in mice. RESULTS: PIC pretreatment ameliorated histopathological changes, oxidative stress and inflammation while enhancing antioxidant and anti-inflammasome markers through downregulation of Toll-like receptor 4 (TLR4), p-IκBα (S32), p-NF-κBp65 (S536), NLRP3, caspase-1 (p20), IL-1ß, IL-18 and GSDMD-N expression during liver ischaemia‒reperfusion. Moreover, PIC inhibited the translocation of NF-κB p65 after stimulation with hypoxia/reoxygenation in primary hepatic macrophages. CONCLUSIONS: The results indicated that PIC protected the liver against hepatic I/R injury, which was mediated by targeting TLR4-NF-κB-NLRP3-mediated hepatic macrophage pyroptosis.

Preparation and Preliminary Application of Epitope Peptide-Based Antibody against Toxoplasma gondii GRA3.

Toxoplasma gondii dense granule protein GRA3 has been shown to promote Toxoplasma gondii transmission and proliferation by interacting with the host cell endoplasmic reticulum (ER) through calcium-regulated cyclophilin ligands (CAMLG). Although many studies have focused on the interaction between the host cell endoplasmic reticulum and GRA3, no polyclonal antibodies (PcAbs) against GRA3 have been reported to date. According to the antigenicity prediction and exposure site analysis, three antigen peptide sequences were selected to prepare polyclonal antibodies targeting GRA3. Peptide scans revealed that the major antigenic epitope sequences were 125ELYDRTDRPGLK136, 202FFRRRPKDGGAG213, and 68NEAGESYSSATSG80, respectively. The GRA3 PcAb specifically recognized the GRA3 of T. gondii type Ⅱ ME49. The development of PcAbs against GRA3 is expected to elucidate the molecular mechanisms by which GRA3 regulates host cell function and contribute to the development of diagnostic and therapeutic strategies for toxoplasmosis.

Baicalin alleviates bleomycin-induced early pulmonary fibrosis in mice via the mitoKATP signaling pathway.

Bleomycin (BLM), a frequently employed chemotherapeutic agent, exhibits restricted clinical utility owing to its pulmonary toxicity. Meanwhile, baicalin (BA)-an active ingredient extracted from the roots of Scutellaria baicalensis Georgi -has been shown to alleviate BLM-induced pulmonary fibrosis (PF). Hence, the objective of this study was to examine the protective effects of BA in the context of BLM-induced early PF in mice and elucidate the underlying mechanism(s). We established an in vivo BLM (3.5 mg/kg)-induced PF murine model and in vitro BLM (35 µM)-damaged MLE-12 cell model. On Day 14 of treatment, the levels of fibrosis and apoptosis were evaluated in mouse lungs via hydroxyproline analysis, western blotting (COL1A1, TGF-ß, Bax, Bcl-2, cleaved caspase-3), and Masson, immunohistochemical (α-SMA, AIF, Cyto C), and TUNEL staining. Additionally, in vitro, apoptosis was assessed in MLE-12 cells exposed to BLM for 24 h using the Annexin V/PI assay and western blotting (Bax, Bcl-2, cleaved caspase-3, AIF, Cyto C). To elucidate the role of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the protective effect of BA, we utilised diazoxide (DZX)-a mitoKATP agonist-and 5-hydroxydecanoate sodium (5-HD)-a mitoKATP inhibitor. Results revealed the involvement of mitoKATP in the protective effect of BA in BLM-induced PF. More specifically, mitoKATP activation can attenuate BLM-induced PF progression and mitigate alveolar epithelial type II cell death by reducing mitochondrial ROS, maintaining the mitochondrial membrane potential, and impeding the mitochondrial apoptotic pathway. Collectively, the findings offer pharmacological support to use BA for the treatment or prevention of BLM-induced PF and suggest that mitoKATP might serve as an effective therapeutic target for this condition.

Roles of Embryonic Lethal Abnormal Vision-Like RNA Binding Proteins in Cancer and Beyond.

Embryonic lethal abnormal vision-like (ELAVL) proteins are RNA binding proteins that were originally discovered as indispensable regulators of the development and functioning of the nervous system. Subsequent studies have shown that ELAVL proteins not only exist in the nervous system, but also have regulatory effects in other tissues. ELAVL proteins have attracted attention as potential therapeutic targets because they stabilize multiple mRNAs by binding within the 3'-untranslated region and thus promote the development of tumors, including hepatocellular carcinoma, pancreatic cancer, ovarian cancer, breast cancer, colorectal carcinoma and lung cancer. Previous studies have focused on these important relationships with downstream mRNAs, but emerging studies suggest that ELAVL proteins also interact with non-coding RNAs. In this review, we will summarize the relationship of the ELAVL protein family with mRNA and non-coding RNA and the roles of ELAVL protein family members in a variety of physiological and pathological processes.

Toxoplasma gondii CDPK3 Controls the Intracellular Proliferation of Parasites in Macrophages.

Interferon-γ (IFN-γ)-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. The growth of the less virulent type II strain of Toxoplasma gondii (such as ME49) was strongly inhibited by IFN-γ-activated murine macrophages. However, the mechanism of resistance is poorly understood. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Previous studies showed the cassette of autophagy-related proteins including Atg7, Atg3, and Atg12-Atg5-Atg16L1 complex, plays crucial roles in the proper targeting of IFN-γ effectors onto the parasitophorous vacuole (PV) membrane of Toxoplasma gondii and subsequent control of parasites. TgCDPK3 is a calcium dependent protein kinase, located on the parasite periphery, plays a crucial role in parasite egress. Herein, we show that the less virulent strain CDPK3 (ME49, type II) can enhance autophagy activation and interacts with host autophagy proteins Atg3 and Atg5. Infection with CDPK3-deficient ME49 strain resulted in decreased localization of IRGs and GBPs around PV membrane. In vitro proliferation and plaque assays showed that CDPK3-deficient ME49 strain replicated significantly more quickly than wild-type parasites. These data suggested that TgCDPK3 interacts with the host Atg3 and Atg5 to promote the localization of IRGs and GBPs around PV membrane and inhibits the intracellular proliferation of parasites, which is beneficial to the less virulent strain of Toxoplasma gondii long-term latency in host cells.

Dihydroartemisinin Attenuates Hypoxic Pulmonary Hypertension via the Downregulation of miR-335 Targeting Vangl2.

Dihydroartemisinin (DHA) is a traditional antimalarial drug. DHA plays a crucial role in preventing pulmonary hypertension (PH); however, its regulatory function on microRNAs (miRNAs) in PH remains unclear. This study aimed to investigate whether DHA exerts its protective functions by regulating miR-335 in PH. Hypoxia-induced PH models were induced both in vitro and in vivo. Mice were treated with various concentrations of DHA, and pulmonary arterial smooth muscle cells (PASMCs) were treated with DHA, miR-335 inhibitor, miR-335 mimic, or Van Gogh-like 2 (Vangl2) plasmid. The expression of miR-335 and Vangl2, pulmonary arterial remodeling index; right ventricular hypertrophy index; and proliferation and migration indexes were measured. DHA improved pulmonary vascular remodeling and alleviated PH in vivo. miRNA sequencing and real-time PCR results further show that the increase in hypoxia-induced miR-335 was avoided by DHA administration, and miR-335 increased the hypoxia-induced PASMC proliferation and migration. MiRNA databases and dual-luciferase reporter assay show that miR-335 directly targets Vangl2, and Vangl2 decreased the hypoxia-induced PASMC proliferation and migration. The miR-335 inhibitor failed to inhibit hypoxia-induced proliferation and migration upregulation in Vangl2 knockdown PASMCs, and the effect of DHA can be blocked by miR-335 upregulation. In hypoxic PH, MiR-335 is increased, whereas Vangl2 is decreased. MiR-335 can significantly promote the hypoxia-induced proliferation and migration of PASMCs by targeting the Vangl2 gene. DHA effectively reverses the hypoxia-induced upregulation of miR-335 expression, avoiding the miR-335-mediated downregulation of Vangl2 and thereby promoting the expression of Vangl2 to prevent PH.

Toxoplasma gondii dense granule protein 3 promotes endoplasmic reticulum stress-induced apoptosis by activating the PERK pathway.

BACKGROUND: Toxoplasma gondii is a neurotropic single-celled parasite that can infect mammals, including humans. Central nervous system infection with T. gondii infection can lead to Toxoplasma encephalitis. Toxoplasma infection can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, which ultimately can lead to apoptosis of host cells. The dense granule protein GRA3 has been identified as one of the secretory proteins that contribute to the virulence of T. gondii; however, the mechanism remains enigmatic. METHODS: The expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT-PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24-36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated. RESULTS: GRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased. CONCLUSION: GRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.

Acetylshikonin exerts anti-tumor effects on non-small cell lung cancer through dual inhibition of STAT3 and EGFR.

BACKGROUND: Lung cancer is one of the most common types of malignant tumor. It has one of the highest morbidity and mortality rates worldwide, and approximately 85% of cases are non-small cell lung cancer (NSCLC). Clinically, several EGFR inhibitors have been used to treat NSCLC, but resistance can develop. Studies have shown that cross talk between signal transducer and activator of transcription 3 (STAT3) and epidermal growth factor receptor (EGFR) can mediate drug resistance. Acetylshikonin has obvious antitumor effects, but the mechanism of action is still unclear. PURPOSE: To analyze the antitumor activity of acetylshikonin in lung cancer and clarify its molecular mechanism. METHODS: Methyl thiazolyl tetrazolium (MTT), colony formation and 5-ethynyl-2'-deoxyuridine (EDU) assays were performed to examine the effects of acetylshikonin in inhibiting the proliferation of NSCLC cells (PC-9, H1975 and A549). Scratch wound and transwell assays were used to evaluate the migration and invasion of NSCLC cells. Flow cytometry was employed to determine whether acetylshikonin could induce apoptosis. Proteome sequencing was used to identify the targets of acetylshikonin. Immunofluorescence staining and western blotting were utilized to verify the inhibition of STAT3 and EGFR phosphorylation. A xenotransplantation model was established to evaluate the efficacy of acetylshikonin in nude mice. RESULTS: Our data demonstrated that acetylshikonin significantly decreased the survival rate of human NSCLC cells, increased the apoptotic rate and inhibited cell migration dose-dependently. Immunofluorescence staining and western blotting analyses revealed that acetylshikonin inhibited EGFR and STAT3 pathways. Acetylshikonin also inhibited tumor growth in a xenograft model better than inhibitors of EGFR and STAT3. CONCLUSION: Acetylshikonin has anti-cancer effects on NSCLC cells by inhibiting EGFR and STAT3, indicating that acetylshikonin may be a new antitumor drug to treat NSCLC.

Live-attenuated ME49Δcdpk3 strain of Toxoplasma gondii protects against acute and chronic toxoplasmosis.

Toxoplasmosis, a common parasitic disease, is caused by Toxoplasma gondii, which infects approximately 30% of the world's population. This obligate intracellular protozoan causes significant economic losses and poses serious public health challenges worldwide. However, the development of an effective toxoplasmosis vaccine in humans remains a challenge to date. In this study, we observed that the knockout of calcium-dependent protein kinase 3 (CDPK3) in the type II ME49 strain greatly attenuated virulence in mice and significantly reduced cyst formation. Hence, we evaluated the protective immunity of ME49Δcdpk3 as a live attenuated vaccine against toxoplasmosis. Our results showed that ME49Δcdpk3 vaccination triggered a strong immune response marked by significantly elevated proinflammatory cytokine levels, such as IFN-γ, IL-12, and TNF-α, and increased the percentage of CD4+ and CD8+ T-lymphocytes. The high level of Toxoplasma-specific IgG was maintained, with mixed IgG1/IgG2a levels. Mice vaccinated with ME49Δcdpk3 were efficiently protected against the tachyzoites of a variety of wild-type strains, including type I RH, type II ME49, Chinese 1 WH3 and Chinese 1 WH6, as well as the cysts of wild-type strains ME49 and WH6. These data demonstrated that ME49Δcdpk3 inoculation induced effective cellular and humoral immune responses against acute and chronic Toxoplasma infections with various strains and was a potential candidate to develop a vaccine against toxoplasmosis.