Evaluation of Digital Mental Health Technologies in the United States: Systematic Literature Review and Framework Synthesis.

BACKGROUND: Digital mental health technologies (DMHTs) have the potential to enhance mental health care delivery. However, there is little information on how DMHTs are evaluated and what factors influence their use. OBJECTIVE: A systematic literature review was conducted to understand how DMHTs are valued in the United States from user, payer, and employer perspectives. METHODS: Articles published after 2017 were identified from MEDLINE, Embase, PsycINFO, Cochrane Library, the Health Technology Assessment Database, and digital and mental health congresses. Each article was evaluated by 2 independent reviewers to identify US studies reporting on factors considered in the evaluation of DMHTs targeting mental health, Alzheimer disease, epilepsy, autism spectrum disorder, or attention-deficit/hyperactivity disorder. Study quality was assessed using the Critical Appraisal Skills Program Qualitative and Cohort Studies Checklists. Studies were coded and indexed using the American Psychiatric Association's Mental Health App Evaluation Framework to extract and synthesize relevant information, and novel themes were added iteratively as identified. RESULTS: Of the 4353 articles screened, data from 26 unique studies from patient, caregiver, and health care provider perspectives were included. Engagement style was the most reported theme (23/26, 88%), with users valuing DMHT usability, particularly alignment with therapeutic goals through features including anxiety management tools. Key barriers to DMHT use included limited internet access, poor technical literacy, and privacy concerns. Novel findings included the discreetness of DMHTs to avoid stigma. CONCLUSIONS: Usability, cost, accessibility, technical considerations, and alignment with therapeutic goals are important to users, although DMHT valuation varies across individuals. DMHT apps should be developed and selected with specific user needs in mind.

The transcriptional landscape and clinico-biological characterization of human endogenous retroviruses in esophageal squamous cell carcinoma.

Human endogenous retroviruses (HERVs) are emerging as critical elements in host genomic regulation. Aberrant HERV transcription has been implicated in developmental and tissue-specific aging and pathological processes. In this study, we presented a comprehensive locus-specific characterization of the HERV expression landscape in esophageal squamous cell carcinoma (ESCC). We demonstrated the transcriptional diversity among patients and identified 12 clinically relevant HERVs in the SCH cohort, which were experimentally validated by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) in the CAMS cohort. ESCC patients were stratified into three HERV-based subtypes (HERVhigh, HERVmedian and HERVlow) with distinct clinical and biological characteristics. The HERVhigh subtype was associated with worse survival, increased CD4+ T cells infiltration and decreased metabolic activity, whereas the HERVlow subtype was characterized by abundant CD8+ T cells, increased metabolic activity, and better survival. The HERV-based tumor subtyping was further robustly validated by RNA sequencing and RT-qPCR in two additional external cohorts. Our findings demonstrate the clinical significance of HERVs for tumor subtyping and prognosis, provide insights into the functional role of HERVs and a valuable resource for developing novel biomarkers and therapeutic targets in ESCC.

Multimodal analysis of cfDNA methylomes for early detecting esophageal squamous cell carcinoma and precancerous lesions.

Detecting early-stage esophageal squamous cell carcinoma (ESCC) and precancerous lesions is critical for improving survival. Here, we conduct whole-genome bisulfite sequencing (WGBS) on 460 cfDNA samples from patients with non-metastatic ESCC or precancerous lesions and matched healthy controls. We develop an expanded multimodal analysis (EMMA) framework to simultaneously identify cfDNA methylation, copy number variants (CNVs), and fragmentation markers in cfDNA WGBS data. cfDNA methylation markers are the earliest and most sensitive, detectable in 70% of ESCCs and 50% of precancerous lesions, and associated with molecular subtypes and tumor microenvironments. CNVs and fragmentation features show high specificity but are linked to late-stage disease. EMMA significantly improves detection rates, increasing AUCs from 0.90 to 0.99, and detects 87% of ESCCs and 62% of precancerous lesions with >95% specificity in validation cohorts. Our findings demonstrate the potential of multimodal analysis of cfDNA methylome for early detection and monitoring of molecular characteristics in ESCC.

A growth factor-reduced culture system for colorectal cancer organoids.

Although organoids derived from tumor tissues have been widely used in cancer research, it is a great challenge for cultured organoids to retain the characteristics of the original tumor tissues due to their heterogeneity. In this study, we explore organoid culture recipes to capture tumor features of colorectal cancers. We find that the activation of Wnt and EGF signaling and inhibition of BMP signaling are non-essential for the survival of most colorectal cancer organoids (CRCOs). We design a growth factor-reduced culture medium containing FGF10, A83-01 (TGF-ß type I receptor inhibitor), SB202190 (p38 MAPK inhibitor), gastrin, and nicotinamide. Using this medium, we can maintain tumor features in long-term CRCO cultivation, as evidenced by histopathology, genetic stability, tumorigenicity, and response of clinical treatments. Our findings offer a reliable and economical strategy for CRCO culture, facilitating the utilization of organoids in colorectal cancer research and treatment.

The transcriptional landscape and diagnostic potential of long non-coding RNAs in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with no clinically relevant biomarkers for early detection. Here, we comprehensively characterized the transcriptional landscape of long non-coding RNAs (lncRNAs) in paired tumor and normal tissue specimens from 93 ESCC patients, and identified six key malignancy-specific lncRNAs that were integrated into a Multi-LncRNA Malignancy Risk Probability model (MLMRPscore). The MLMRPscore performed robustly in distinguishing ESCC from normal controls in multiple in-house and external multicenter validation cohorts, including early-stage I/II cancer. In addition, five candidate lncRNAs were confirmed to have non-invasive diagnostic potential in our institute plasma cohort, showing superior or comparable diagnostic accuracy to current clinical serological markers. Overall, this study highlights the profound and robust dysregulation of lncRNAs in ESCC and demonstrates the potential of lncRNAs as non-invasive biomarkers for the early detection of ESCC.

Characteristics and key differences between patient populations receiving imaging modalities for coronary artery disease diagnosis in the US.

BACKGROUND: There are limited data on the impact of imaging modality selection for the assessment of coronary artery disease (CAD) risk on downstream resource utilisation. This study sought to identify differences between patient populations in the US undergoing stress echocardiography, single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI), positron emission tomography (PET) MPI, and coronary computed tomography angiography (cCTA) for the assessment of CAD risk, and associated physician referral patterns. METHODS: Claims and electronic health records data for 2.5 million US patients who received stress echocardiography, cCTA, SPECT MPI or PET MPI between January 2016 and March 2018, from the Decision Resources Group Real-World Evidence US Data Repository, were analysed. Patients were stratified into suspected and existing CAD cohorts, and further stratified by pre-test risk and presence and recency of interventions or acute cardiac events (within 1-2 years pre-index test). Linear and logistic regression were used to compare numeric and categorical variables. RESULTS: Physicians were more likely to refer patients to standalone SPECT MPI (77%) and stress echocardiography (18%) than PET MPI (3%) and cCTA (2%). Overall, 43% of physicians referred more than 90% of their patients to standalone SPECT MPI. Just 3%, 1% and 1% of physicians referred more than 90% of their patients to stress echocardiography, PET MPI or cCTA. At the aggregated imaging level, patients who underwent stress echocardiography or cCTA had similar comorbidity profiles. Comorbidity profiles were also similar for patients who underwent SPECT MPI and PET MPI. CONCLUSION: Most patients underwent SPECT MPI at the index date, with very few undergoing PET MPI or cCTA. Patients who underwent cCTA at the index date were more likely to undergo additional imaging tests compared with those who underwent other imaging modalities. Further evidence is needed to understand factors influencing imaging test selection across patient populations.

VEGF to CITED2 ratio predicts the collateral circulation of acute ischemic stroke.

Objective: The research objective was to evaluate the predicting role of the vascular endothelial growth factor to CBP/P300-interacting transactivator with Glu/Asp-rich C-terminal domain 2 Ratio (VEGF/CITED2) from peripheral blood mononuclear cells (PBMCs) in the collateral circulation of acute ischemic stroke (AIS). Methods: In an observational study of patients with AIS, the western blot was applied to test the protein expression of VEGF and CITED2. Then, we calculated the VEGF/CITED2 and collected other clinical data. Binary logistic regression analysis between collateral circulation and clinical data was performed. Finally, receiver operating characteristic (ROC) curve analysis was used to explore the predictive value of VEGF/CITED2. Results: A total of 67 patients with AIS were included in the study. Binary logistic regression analysis indicated the VEGF/CITED2 (OR 165.79, 95%CI 7.25-3,791.54, P = 0.001) was an independent protective factor. The ROC analyses showed an area under the ROC curve of the VEGF/CITED2 was 0.861 (95%CI 0.761-0.961). The optimal cutoff value of 1.013 for VEGF/CITED2 had a sensitivity of 89.1% and a specificity of 85.7%. Conclusion: In patients with AIS, the VEGF/CITED2 was related to the establishment of collateral circulation. The VEGF/CITED2 is a potentially valuable biomarker for predicting collateral circulation. Clinical trial registration: ClinicalTrials.gov, identifier: NCT05345366.

The Deubiquitinase USP39 Promotes Esophageal Squamous Cell Carcinoma Malignancy as a Splicing Factor.

Esophageal squamous cell carcinoma (ESCC) is an aggressive epithelial malignancy and the underlying molecular mechanisms remain elusive. Here, we identify that the ubiquitin-specific protease 39 (USP39) drives cell growth and chemoresistance by functional screening in ESCC, and that high expression of USP39 correlates with shorter overall survival and progression-free survival. Mechanistically, we provide evidence for the role of USP39 in alternative splicing regulation. USP39 interacts with several spliceosome components. Integrated analysis of RNA-seq and RIP-seq reveals that USP39 regulates the alternative splicing events. Taken together, our results indicate that USP39 functions as an oncogenic splicing factor and acts as a potential therapeutic target for ESCC.

CSPGs promote the migration of meningeal fibroblasts via p38 mitogen-activated protein kinase signaling pathway under OGD conditions.

AIMS: Usually glial scar that occurs after central nervous system injury has significantly affected the local neural microenvironment. Meningeal fibroblasts play an essential role in the formation of the glial scar. However, how and why meningeal fibroblasts migrate to lesion sites is still unclear. MAIN METHODS: Astrocytes were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) injury. And then, we measured the glial fibrillary acidic protein(GFAP) and chondroitin sulfate proteoglycans (CSPGs) expression of reactive astrocytes by western blot and quantitative polymerase chain reaction (qPCR) after they were co-cultured with meningeal fibroblasts. Following, we clarified the possibility that CSPGs induce the migration of meningeal fibroblasts to glial scar by transwell migration assay and the activation of the p38 MAPK signaling pathway during the migration by western blot. KEY FINDINGS: We found that co-cultured meningeal fibroblasts could alleviate the significantly increased expression of GFAP and CSPGs in the activation of reactive astrocytes induced by OGD/R. Additionally, CSPGs secreted by reactive astrocytes could induce the migration of meningeal fibroblasts and the expression of phospho-p38 in meningeal fibroblasts when meningeal fibroblasts were co-cultured with supernatant of reactive astrocytes. What's more, we could observe a noticeable increase in CSPGs that chondroitinase ABC could reverse their functions. Moreover, phospho-p38 could cause the expression of phospho-cofilin and the migration of CSPGs-induced meningeal fibroblasts. SIGNIFICANCE: Our study provides reliable evidence for explaining scar formation mechanisms and further studying to improve regeneration after an injury to the central nervous system.

Paraspeckle Promotes Hepatocellular Carcinoma Immune Escape by Sequestering IFNGR1 mRNA.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the most common type of hepatic malignancies, with poor prognosis and low survival rate. Paraspeckles, which are unique subnuclear structures, are recently found to be involved in the development of various tumors, including HCC, and are related to induction in chemoresistance of HCC. This study aimed to investigate the possibility of paraspeckle in HCC cells participating in immune escape and its underlying mechanism in vitro and in vivo. METHODS: Expression of NEAT1_2, the framework of paraspeckle, in HCC cells and tissues was detected by qRT-PCR and RNA-FISH. mRNAs interacted with NEAT1_2 were pull-downed and sequenced in C-terminal S1-aptamer-tagged NEAT1_2 endogenously expressed HCC cells constructed using CRISPR-CAS9 knock-in technology. The effects of paraspeckle on HCC sensitivity to T-cell-mediated cytolysis were detected by T-cell mediated tumor cell killing assay. The roles of NEAT1_2 or NONO on IFNGR1 expression and IFN-γ signaling by applying gene function loss analysis in HCC cells were detected by qRT-PCR, RNA immunoprecipitation, Western blotting, and ELISA. The role of paraspeckle during adoptive T-cell transfer therapy for HCC in vivo was performed with a subcutaneous xenograft mouse. RESULTS: Paraspeckle in HCC cells is negatively related to T-cell-mediated cytolysis. Destruction of paraspeckle in HCC cells by knockdown of NEAT1_2 or NONO significantly improved the sensibility of resistant HCC cells to T-cell killing effects. Furthermore, IFNGR1 mRNA, which is sequestered by NEAT1_2 and NONO, is abundant in paraspeckle of T-cell killing-resistant HCC cells. Incapable IFN-γ-IFNGR1 signaling accounts for paraspeckle mediated-adoptive T-cell therapy resistance. Moreover, NEAT1_2 expression negatively correlates with IFNGR1 expression in clinical HCC tissues. CONCLUSIONS: Paraspeckle in HCC cells helps tumor cells escape from immunosurveillance through sequestering IFNGR1 mRNA to inhibiting IFN-γ-IFNGR1 signaling, thereby avoiding T-cell killing effects. Collectively, our results hint that NEAT1_2 highly expressed HCC patient is more resistant to T-cell therapy in clinic, and NEAT1_2 may be potential target for HCC immunotherapy.

RNA helicase DDX5 suppresses IFN-I antiviral innate immune response by interacting with PP2A-Cß to deactivate IRF3.

DEAD-box (DDX) helicases are critical for recognizing viral nucleic acids to regulate antiviral innate immunity. Although DDX5 has been reported to participate in various virus infection, whether DDX5 regulates innate immune responses and its underlying mechanisms are still unknown. Here, we report that DDX5 is a negative regulator of type I IFN (IFN-I) production in antiviral responses. DDX5 knockdown significantly promoted DNA or RNA virus infection-induced IFN-I production and IFN-stimulated genes (ISGs) expression, while ectopic expression of DDX5 inhibited IFN-I production and promoted viral replication. Furthermore, we found that DDX5 specifically interacted with serine/threonine-protein phosphatase 2 A catalytic subunit beta (PP2A-Cß) and viral infection enhanced the interaction between DDX5 and PP2A-Cß. Besides, PP2A-Cß interacted with IFN regulatory factor 3 (IRF3), and PP2A-Cß knockdown promoted viral infection-induced IRF3 phosphorylation and IFN-I production. In addition, DDX5 knockdown rendered the mice more resistant to viral infection and enhanced antiviral innate immunity in vivo. Thus, DDX5 suppresses IFN-I antiviral innate immune response by interacting with PP2A-Cß to deactivate IRF3. Together, these findings identify a negative role of DDX5 on regulating IFN-I signaling in innate immune responses.

microRNA-421-3p prevents inflammatory response in cerebral ischemia/reperfusion injury through targeting m6A Reader YTHDF1 to inhibit p65 mRNA translation.

OBJECTIVE: Ischemic stroke is one of the leading causes of death globally, and inflammation is considered as a vital contributor to the pathophysiology of ischemic stroke. Recently, microRNA-421-3p-derived macrophages is found to promote motor function recovery in spinal cord injury. Here, we explored whether microRNA-421-3p is involved in inflammation responses during cerebral ischemia/reperfusion (I/R) injury and its molecular mechanism. METHODS: An in vivo experimental animal model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and in vitro model of microglial subjected to oxygen-glucose deprivation and reoxygenation (OGD/R) were used. The effects of microRNA-421-3p on cerebral I/R injury and its underlying mechanism were detected by quantitative real-time PCR, western blotting, immunofluorescence staining, RNA immunoprecipitation, flow cytometry, luciferase reporter assay, and bioinformatics analysis. RESULTS: We find that microRNA-421-3p is significantly decreased in cerebral I/R injury in vitro and in vivo. Furthermore, overexpression of microRNA-421-3p evidently suppresses pro-inflammatory factor expressions and inhibits NF-κB p65 protein expression and nuclear translocation in BV2 microglia cells treated with OGD/R. However, microRNA-421-3p neither promotes p65 mRNA expression, nor affects p65 mRNA or protein stability. Moreover, we find the m6A 'reader' protein YTH domain family protein 1 (YTHDF1) is the specific target of microRNA-421-3p, and YTHDF1 specifically binds to the m6a site of p65 mRNA to promote its translation. CONCLUSION: microRNA-421-3p prevents inflammatory response in cerebral ischemia/reperfusion injury through targeting YTHDF1 to inhibit p65 mRNA translation. These findings provide novel insights into understanding the molecular pathogenesis of cerebral I/R injury.

Upregulation of miR-219a-5p Decreases Cerebral Ischemia/Reperfusion Injury In Vitro by Targeting Pde4d.

BACKGROUND: Ischemic stroke is the leading cause of disability and death globally. Micro-RNAs (miRNAs) have been reported to play important roles in the development and pathogenesis of the nervous system. However, the exact function and mechanism of miRNAs have not been fully elucidated about brain damage caused by cerebral ischemia/reperfusion (I/R). METHODS: In this study, we explored the neuroprotective effects of miR-219a-5p on brain using an in vitro ischemia model (mouse neuroblastoma N2a cells treated with oxyglucose deprivation and reperfusion), and in vivo cerebral I/R model in mice. Western blot assay and Reverse Transcription-Polymerase Chain Reaction were used to check the expression of molecules involved. Flow cytometry and cholecystokinin were used to examine cell apoptosis, respectively. RESULTS: Our research shows that miR-219a-5p gradually decreases in cerebral I/R models in vivo and in vitro. In vitro I/R, we find that miR-219a-5p mimics provided evidently protection for cerebral I/R damage, as shown by increased cell viability and decreased the release of LDH and cell apoptosis. Mechanically, our findings indicate that miR-219a-5p binds to cAMP specific 3', 5'-cyclic phosphodiesterase 4D (PDE4D) mRNA in the 3'-UTR region, which subsequently leads to a decrease in Pde4d expression in I/R N2a cells. CONCLUSIONS: Our results provide new ideas for the study of the mechanism of cerebral ischemia/reperfusion injury, and lay the foundation for further research on the treatment of brain I/R injury. Upregulation of miR-219a-5p decreases cerebral ischemia/reperfusion injury by targeting Pde4d in vitro.

Isosteviol Sodium Protects against Ischemic Stroke by Modulating Microglia/Macrophage Polarization via Disruption of GAS5/miR-146a-5p sponge.

Recent studies have shown that transforming microglia phenotype from pro-inflammation of M1 phenotype to anti-inflammation and tissue-repairing M2 phenotype may be an effective therapeutic strategy for preventing ischemic stroke brain injury. Isosteviol Sodium (STV-Na) has shown promise as a neuroprotective agent in cerebral ischemia model, although its effect on microglial polarization and chronic recovery after stroke is not clear. Here, we demonstrated that STV-Na treatment significantly reduced cerebral ischemic damage at both acute and chronic time points. STV-Na has a profound regulatory effect on microglia response after stroke. It can promote M2 polarization and inhibit microglia-mediated inflammation (M1) response following stroke in vivo and in vitro. Furthermore, we also found that Growth Arrest-Specific 5 (GAS5) altered OGD/R-induced microglial activation by increasing Notch1 expression via miR-146a-5p, the mRNA level of GAS5 and the protein level of Notch1 in vivo and in vitro, were discovered that both downgraded with STV-Na. Taken together, the present study demonstrated that STV-Na exerted neuroprotective effects by modulating microglia/macrophage polarization in ischemic stroke via the GAS5/miR-146a-5p sponge. These findings provide new evidence that targeting STV-Na could be a treatment for the prevention of stroke-related brain damage.

Comparison between in-hospital stroke and community-onset stroke treated with endovascular thrombectomy.

OBJECTIVE: In-hospital stroke (IHS) is an uncommon but serious medical emergency. Early recanalization through endovascular thrombectomy (EVT) may offer a vital therapeutic choice. This study compared the clinical features and outcomes between IHS and community-onset stroke (COS). METHODS: From a single-center registry of 2813 patients with ischemic stroke, those who had received EVT for acute ischemic stroke were included and classified into the IHS and COS groups based on their stroke onset scenario. We compared the outcomes including successful recanalization, symptomatic intracranial hemorrhage, functional independence (modified Rankin Scale score, 0-2) at 90 days, and mortality between the two groups. RESULTS: A total of 24 patients with IHS (mean age, 70 years; 54% men) and 105 patients with COS (mean age, 73 years; 47% men) were included. The most frequently reported reasons for admission in patients with IHS were cardiovascular and oncological diseases. The initial National Institutes of Health Stroke Scale (NIHSS) scores and main occluded vessels were similar between the two groups. Patients with IHS received a higher number of active malignancy diagnoses, were more likely to withhold antithrombotic agents, and exhibited higher prestroke functional dependency. The median onset-to-puncture time was 192 min in IHS and 217 min in COS (P = 0.15). The percentages of successful recanalization (79% vs 71%), symptomatic hemorrhage (0% vs 9%), functional independence (42% vs 40%), and mortality (17% vs 12%) were comparable between the two groups. After adjustment for covariates, initial NIHSS scores and successful recanalization were the most important predictors for functional independence at 90 days. CONCLUSIONS: Despite having disadvantages at baseline, patients with IHS could still benefit from timely EVT to achieve favorable outcomes. A well-designed acute stroke protocol tailored for IHS should be developed.

Real-world experience of calcipotriene and betamethasone dipropionate foam 0.005%/0.064% in the treatment of adults with psoriasis in the United States.

Background: Clinical trials have established calcipotriene 0.005%/betamethasone dipropionate 0.064% (Cal/BD) foam as a well-tolerated and efficacious topical therapy for psoriasis. Methods: A chart review of 24 US healthcare providers gathered real-world information (clinical characteristics and outcomes, safety, and resource utilization) for patients (≥18 years old) prescribed Cal/BD foam between January 1 and October 31, 2016, along with healthcare provider characteristics and perceptions of Cal/BD foam. Results: Data were reported from 105 patients and 177 active psoriatic lesions. Cal/BD foam was applied once-daily; the prescription was 4 weeks for 69/177 (39%) lesions (median 4, range 1-26 weeks). Knees (n = 41; 23%) and elbows (n = 37; 20%) were the most frequently treated areas. Among 114 lesions, severity improved from "mild"/"moderate"/"severe" to "clear"/"almost clear" in 71%, and 54% had a clinically significant improvement (two-step/greater improvement) in lesion severity from baseline. Of 128 lesions with baseline itch, 90% were not itchy at the best treatment response. Most healthcare providers reported prescribing Cal/BD foam because of its overall efficacy (n = 20/23; 87%). Adverse events were reported in 1/105 patients (1%). Conclusion: Data from real clinical practice demonstrate that Cal/BD foam improves psoriasis disease severity and associated itch in patients and further extend results of clinical trials.

The journey for US psoriasis patients prescribed a topical: a retrospective database evaluation of patient progression to oral and/or biologic treatment.

Background: A specific sequence for psoriasis (PsO) therapy has not been defined. Objectives: This retrospective, observational cohort study characterized pathways of PsO treatment over 3 years for newly diagnosed patients initially treated with a topical medication. Methods: Adult PsO patients from the Explorys database (March 1 2011 to June 30 2015) were grouped according to medication-use patterns: 1) discontinued therapy; 2) topical therapy only; 3) switched/added an oral agent; and 4) switched/added a biologic agent. Results: Of 6875 patients, 907 (13.2%) discontinued treatment; 2544 (37.0%) used topical only, and 3319 (45.7%) and 819 (11.9%) switched/added-on an oral and/or biologic agent, respectively. Patients progressed to biologic treatment faster than to oral agents (median 254 vs. 378 d; p < .0001). Using an oral agent before a biologic significantly delayed biologic initiation compared to progressing to biologic directly from topical (median 456 vs. 90 d; p < .0001). Limitations: Disease severity and the reason for treatment transitions were not assessed. Conclusions: Patients initiating topical PsO treatment progressed to biologics faster than to oral agent using an oral agent prior to a biologic significantly delayed biologic initiation. Maintaining patients on an effective topical treatment may minimize the need for a switch to oral and biologics.

Isosteviol Sodium Protects Neural Cells Against Hypoxia-Induced Apoptosis Through Inhibiting MAPK and NF-κB Pathways.

BACKGROUND: Stevioside, isolated from the herb Stevia rebaudiana, has been widely used as a food sweetener all over the world. Isosteviol Sodium (STV-Na), an injectable formulation of isosteviol sodium salt, has been proved to possess much greater solubility and bioavailability and exhibit protective effects against cerebral ischemia injury in vivo by inhibiting neuron apoptosis. However, the underlying mechanisms of the neuroprotective effects STV-Na are still not completely known. In the present study, we investigated the effects of STV-Na on neuronal cell death caused by hypoxia in vitro and its underlying mechanisms. METHODS: We used cobalt chloride (CoCl2) to expose mouse neuroblastoma N2a cells to hypoxic conditions in vitro. RESULTS: Our results showed that pretreatment with STV-Na (20 µM) significantly attenuated the decrease of cell viability, lactate dehydrogenase release and cell apoptosis under conditions of CoCl2-induced hypoxia. Meanwhile, STV-Na pretreatment significantly attenuated the upregulation of intracellular Ca2+ concentration and reactive oxygen species production, and inhibited mitochondrial depolarization in N2a cells under conditions of CoCl2-induced hypoxia. Furthermore, STV-Na pretreatment significantly downregulated expressions of nitric oxide synthase, interleukin-1ß, tumor necrosis factor-α, interleukin-6, nuclear factor kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) signalings in N2a cells under conditions of CoCl2-induced hypoxia. CONCLUSIONS: Taken together, STV-Na protects neural cells against hypoxia-induced apoptosis through inhibiting MAPK and NF-κB pathways.

Neuroprotective effects of isosteviol sodium through increasing CYLD by the downregulation of miRNA-181b.

NF-κB signaling pathway plays a critical role in cerebral ischemic stroke. MicroRNA-181b (miR-181b) induces the expression of NF-κB signaling pathways indirectly, and isosteviol sodium (STVNa) protects against ischemic injury via the inhibition of NF-κB-mediated inflammatory and apoptotic responses. However, the function of miR-181b and the actual relationship between STVNa and miR-181b in the ischemia-induced activation of NF-κB signaling pathways remains unclear. In this study, we found that miR-181b expression was significantly decreased in N2A neuroblastoma cells after CoCl2-induced hypoxic injury in vitro. We further found, via western blot analysis and quantitative polymerase chain reaction assay, that altering miR-181b expression could induce changes in one of its target proteins, cylindromatosis (CYLD). Specifically, upregulation and downregulation of miR-181b (through transfection of either pre- or small interfering miR-181b, respectively) could negatively regulate CYLD protein levels as well as N2A cell survival rate and apoptosis following CoCl2-induced injury. Furthermore, STVNa treatment following ischemic injury significantly downregulated the expression of miR-181b to alter apoptotic proteins downstream of the NF-κB signaling pathway through increasing CYLD protein levels in vivo and in vitro. STVNa also had a protective effect on CoCl2-injured N2A cells, increasing cell survival rate, inhibiting apoptosis, reducing the damage of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Together, these results suggest that STVNa may downregulate miRNA-181b to protect mouse brain with ischemia stroke and against hypoxic injury in N2A cells by repressing NF-κB signaling pathways through the activation of CYLD, providing a novel therapy for ischemic stroke.

Isosteviol sodium injection improves outcomes by modulating TLRs/NF-κB-dependent inflammatory responses following experimental traumatic brain injury in rats.

Previous studies have shown that isosteviol sodium (STVNa) protects against permanent cerebral ischemia injury by inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammatory responses. Overwhelming evidence shows that toll-like receptors (TLRs) are the upstream regulators of NF-κB. On the basis of the similarity of the pathology caused by traumatic brain injury (TBI) and stroke, we speculated that STVNa may have a therapeutic effect against TBI through regulation of the TLRs/NF-κB signaling-mediated inflammatory response. Thus, we studied the potential therapeutic effects of STVNa and the underlying mechanisms. Male rats, subjected to controlled cortical impact (CCI) injury, were injected intraperitoneally with STVNa (5, 10, 20, 40, and 80 mg/kg, daily for 3 or 7 days) after trauma. Neurobehavioral scores, relative numbers of cortical lesions, and histology were examined. We also measured the mRNA and protein expression levels of TLRs/NF-κB signaling pathway-related genes including TLR2, TLR4, and NF-κB by quantitative real-time-PCR and western blotting, respectively, and concentrations of tumor necrosis factor-α and interleukin-1ß by an enzyme-linked immunosorbent assay. The results indicated that STVNa (20 mg/kg) showed significant neuroprotective effects 3 and 7 days after TBI, including the reduction of cortical lesions, improvement of the neurological severity score, significantly increased number of restored neurons, decreased number of astrocytes, and lower concentrations of tumor necrosis factor-α and interleukin-1ß. Results from quantitative real-time-PCR and western blotting also show that the mRNA and protein expression levels of TLR2, TLR4, and NF-κB were significantly lower in STVNa-treated rats compared with the vehicle-treated rats. The administration of STVNa attenuates the TLR/NF-κB signaling pathway-mediated inflammatory responses in the injured rat brain, and this may be the mechanism by which STVNa improves the outcome following TBI.