Clarifying the mechanism of apigenin against blood-brain barrier disruption in ischemic stroke using systems pharmacology.

Currently, recombinant tissue plasminogen activator (rtPA) is an effective therapy for ischemic stroke (IS). However, blood-brain barrier (BBB) disruption is a serious side effect of rtPA therapy and may lead to patients' death. The natural polyphenol apigenin has a good therapeutic effect on IS. Apigenin has potential BBB protection, but the mechanism by which it protects the BBB integrity is not clear. In this study, we used network pharmacology, bioinformatics, molecular docking and molecular dynamics simulation to reveal the mechanisms by which apigenin protects the BBB. Among the 146 targets of apigenin for the treatment of IS, 20 proteins were identified as core targets (e.g., MMP-9, TLR4, STAT3). Apigenin protects BBB integrity by inhibiting the activity of MMPs through anti-inflammation and anti-oxidative stress. These mechanisms included JAK/STAT, the toll-like receptor signaling pathway, and Nitrogen metabolism signaling pathways. The findings of this study contribute to a more comprehensive understanding of the mechanism of apigenin in the treatment of BBB disruption and provide ideas for the development of drugs to treat IS.

Pharmacological mechanism and therapeutic efficacy of Icariside II in the treatment of acute ischemic stroke: a systematic review and network pharmacological analysis.

BACKGROUND AND OBJECTIVE: Epimedii has long been used as a traditional medicine in Asia for the treatment of various common diseases, including Alzheimer's disease, cancer, erectile dysfunction, and stroke. Studies have reported the ameliorative effects of Icariside II (ICS II), a major metabolite of Epimedii, on acute ischemic stroke (AIS) in animal models. Based on network pharmacology, molecular docking, and molecular dynamics (MD) simulations, we conducted a systematic review to evaluate the effects and neuroprotective mechanisms of ICS II on AIS. METHODS: First, we have searched 6 databases using studies with ICS II treatment on AIS animal models to explore the efficacy of ICS II on AIS in preclinical studies. The literature retrieval time ended on March 8, 2022 (Systematic Review Registration ID: CRD42022306291). There were no restrictions on the language of the search strategy. Systematic review follows the Patient, Intervention, Comparison and Outcome (PICO) methodology and framework. SYCLE's RoB tool was used to evaluate the the risk of bias. In network pharmacology, AIS-related genes were identified and the target-pathway network was constructed. Then, these targets were used in the enrichments of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and gene ontology (GO). Molecular docking and MD simulation were finally employed between ICS II and the potential target genes. RESULTS: Twelve publications were included describing outcomes of 1993 animals. The literature details, animal strains, induction models, doses administered, duration of administration, and outcome measures were extracted from the 12 included studies. ICS II has a good protective effect against AIS. Most of the studies in this systematic review had the appropriate methodological quality, but some did not clearly state the controlling for bias of potential study. Network pharmacology identified 246 targets with SRC, CTNNB1, HSP90AA1, MAPK1, and RELA as the core target proteins. Besides, 215 potential pathways of ICS II were identified, such as PI3K-Akt, MAPK, and cGMP-PKG signaling pathway. GO enrichment analysis showed that ICS II was significantly enriched in subsequent regulation such as MAPK cascade. Molecular docking and MD simulations showed that ICS II can closely bind with important targets. CONCLUSIONS: ICS II is a promising drug in the treatment of AIS. However, this systematic review reveals key knowledge gaps (i.e., the protective role of ICS II in women) that ICS II must address before it can be used for the treatment of human AIS. Our study shows that ICS II plays a protective role in AIS through multi-target and multi-pathway characteristics, providing ideas for the development of drugs for the treatment of AIS.

Simultaneous detection of circulating tumor DNAs using a SERS-based lateral flow assay biosensor for point-of-care diagnostics of head and neck cancer.

Circulating tumor DNA (ctDNA) has recently emerged as an ideal target for biomarker analytes. Thus, the development of rapid and ultrasensitive ctDNA detection methods is essential. In this study, a high-throughput surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) strip is proposed. The aim of this method is to achieve accurate quantification of TP53 and PIK3CA E545K, two types of ctDNAs associated with head and neck squamous cell carcinoma (HNSCC), particularly for point-of-care testing (POCT). Raman reporters and hairpin DNAs are used to functionalize the Pd-Au core-shell nanorods (Pd-AuNRs), which serve as the SERS probes. During the detection process, the existence of targets could open the hairpins on the surface of Pd-AuNRs and trigger the first step of catalytic hairpin assembly (CHA) amplification. The next stage of CHA amplification is initiated by the hairpins prefixed on the test lines, generating numerous "hot spots" to enhance the SERS signal significantly. By the combination of high-performing SERS probes and a target-specific signal amplification strategy, TP53 and PIK3CA E545K are directly quantified in the range of 100 aM-1 nM, with the respective limits of detection (LOD) calculated as 33.1 aM and 20.0 aM in the PBS buffer and 37.8 aM and 23.1 aM in human serum, which are significantly lower than for traditional colorimetric LFA methods. The entire detection process is completed within 45 min, and the multichannel design realizes the parallel detection of multiple groups of samples. Moreover, the analytical performance is validated, including reproducibility, uniformity, and specificity. Finally, the SERS-LFA biosensor is employed to analyze the expression levels of TP53 and PIK3CA E545K in the serum of patients with HNSCC. The results are verified as consistent with those of qRT-PCR. Thus, the SERS-LFA biosensor can be considered as a noninvasive liquid biopsy assay for clinical cancer diagnosis.

Puerarin alleviates the ototoxicity of gentamicin by inhibiting the mitochondria­dependent apoptosis pathway.

Gentamicin (GM) is a commonly used antibiotic, and ototoxicity is one of its side effects. Puerarin (PU) is an isoflavone in kudzu roots that exerts a number of pharmacological effects, including antioxidative and free radical scavenging effects. The present study investigated whether PU could protect against GM­induced ototoxicity in C57BL/6J mice and House Ear Institute­Organ of Corti 1 (HEI­OC1) cells. C57BL/6J mice and HEI­OC1 cells were used to establish models of GM­induced ototoxicity in this study. Auditory brainstem responses were measured to assess hearing thresholds, and microscopy was used to observe the morphology of cochlear hair cells after fluorescent staining. Cell viability was examined with Cell Counting Kit­8 assays. To evaluate cell apoptosis and reactive oxygen species (ROS) production, TUNEL assays, reverse transcription­quantitative PCR, DCFH­DA staining, JC­1 staining and western blotting were performed. PU protected against GM­induced hearing damage in C57BL/6J mice. PU ameliorated the morphological changes of mouse cochlear hair cells and reduced the apoptosis rate of HEI­OC1 cells after GM­mediated damage. GM­induced ototoxicity may be closely related to the upregulation of p53 expression and the activation of endogenous mitochondrial apoptosis pathways, and PU could protect cochlear hair cells from GM­mediated damage by reducing the production of ROS and inhibiting the mitochondria­dependent apoptosis pathway.

SERS Based Lateral Flow Assay for Rapid and Ultrasensitive Quantification of Dual Laryngeal Squamous Cell Carcinoma-Related miRNA Biomarkers in Human Serum Using Pd-Au Core-Shell Nanorods and Catalytic Hairpin Assembly.

Non-invasive early diagnosis is of great significant in disease pathologic development and subsequent medical treatments, and microRNA (miRNA) detection has attracted critical attention in early cancer screening and diagnosis. However, it was still a challenge to report an accurate and sensitive method for the detection of miRNA during cancer development, especially in the presence of its analogs that produce intense background noise. Herein, we developed a surface-enhanced Raman scattering (SERS)-based lateral flow assay (LFA) biosensor, assisted with catalytic hairpin assembly (CHA) amplification strategy, for the dynamic monitoring of miR-106b and miR-196b, associated with laryngeal squamous cell carcinoma (LSCC). In the presence of target miRNAs, two hairpin DNAs could self-assemble into double-stranded DNA, exposing the biotin molecules modified on the surface of palladium (Pd)-gold (Au) core-shell nanorods (Pd-AuNRs). Then, the biotin molecules could be captured by the streptavidin (SA), which was fixed on the test lines (T1 line and T2 line) beforehand. The core-shell spatial structures and aggregation Pd-AuNRs generated abundant active "hot spots" on the T line, significantly amplifying the SERS signals. Using this strategy, the limits of detections were low to aM level, and the selectivity, reproducibility, and uniformity of the proposed SERS-LFA biosensor were satisfactory. Finally, this rapid analysis strategy was successfully applied to quantitatively detect the target miRNAs in clinical serum obtained from healthy subjects and patients with LSCC at different stages. The results were consistent with the quantitative real-time PCR (qRT-PCR). Thus, the CHA-assisted SERS-LFA biosensor would become a promising alternative tool for miRNAs detection, which showed a tremendous clinical application prospect in diagnosing LSCC.

Construction of prognostic risk prediction model based on high-throughput sequencing expression profile data in childhood acute myeloid leukemia.

This study aimed to identify critical prognostic molecular markers in Childhood acute myeloid leukemia (AML) and construct nomogram-based model for prognostic prediction. The RNA-sequencing profiles and corresponding clinical information were downloaded from TCGA database. Differential expressed genes (DEG) were screened using limma package, subsequently following by GO and KEGG pathway analysis. Univariate and multivariate cox regression analysis were performed to screen critical DEGs. Nomogram-based prediction model were constructed to identify clinical factors with independent prognostic values, and the accuracy of this model was validated. A total of 214 DEGs were identified from relapse AML samples compared with non-relapse samples. These DEGs were mainly involved in twenty GO terms and three signaling pathways, such as chromatin assembly or disassembly, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway. Among these genes, Univariate and multivariate cox regression analysis results showed that relapse and risk score were significantly correlated with survival outcomes. Finally, the accuracy ability of nomogram-based prediction model was validated. These six DEGs (ABCA5, CYP7A1, HERC5, etc.) play major roles in AMLs progression. Our nomogram-based prognostic predictive model might be an effective method to estimate survival probability of AML patients with different risk status.

Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play an important role in the regulation of gene expression related to inflammatory responses. Human adipose stem cells are characterized by pluripotent differentiation potential and isolated from adipose tissues. These cells regulate inflammation mainly by interacting with immune cells and affecting the secretion of immune factors; details of this interaction are currently unknown. In the current study, we successfully established an acute inflammation model and a chronic inflammation model involving adipose stem cells. We used high-throughput miRNA microarray analysis to identify miRNAs that were significantly (p < 0.05) differentially expressed during both acute and chronic inflammation. Lipopolysaccharide (LPS) significantly (p < 0.05) reduced the expression of miR-223-3P and miR-2909, while promoting the production of pro-inflammatory cytokines, interleukin (IL) 6, IL-1ß, and tumor necrosis factor (TNF)-α via the Toll-like receptor (TLR) 4/TLR2/nuclear factor (NF)-κB/signal transducer and activator of transcription (STAT) 3 signaling pathway in human adipose stem cells. Further, miR-223-3P expression was significantly (p < 0.05) reduced in human adipose stem cells during activation by IL-6 stimulation. The inducible down-regulation of miR-223-3P resulted in the activation of STAT3, which was directly targeted by miR-223-3P. STAT3 directly targeted TLR4 and TLR2, promoting the production of the pro-inflammatory cytokine, IL-6, and formed a positive feedback loop to regulate IL-6 levels. Similarly, TNF-α significantly (p < 0.05) increased the expression of miR-223-3p, with LPS and TLR4/TLR2/NF-κB/STAT3 forming a negative feedback loop to regulate TNF-α levels. In addition, miR-2909, which depends on NF-κB, targeted Krueppel-like factor (KLF) 4 to regulate the levels of pro-inflammatory cytokines, IL-6, IL-1ß, and TNF-α. We conclude that miR-223-3p and miR-2909 form a complex regulatory network with pro-inflammatory factors and signaling pathways in adipose stem cells stimulated by LPS. These findings will inform the development of therapies against autoimmune and inflammatory diseases.

Comparison of the eighth version of the American Joint Committee on Cancer manual to the seventh version for colorectal cancer: A retrospective review of our data.

AIM: To analyze the survival trends in colorectal cancer (CRC) based on the different classifications recommended by the seventh and eighth editions of the American Joint Committee on Cancer staging system (AJCC-7th and AJCC-8th). METHODS: The database from our institution was queried to identify patients with pathologically confirmed stage 0-IV CRC diagnosed between 2006 and 2012. Data from 2080 cases were collected and 1090 cases were evaluated through standardized inclusion and exclusion criteria. CRC was staged by AJCC-7th and then restaged by AJCC-8th. Five-year disease-free survival (DFS) and overall survival (OS) were compared. SPSS 21.0 software was used for all data. DFS and OS were compared and analyzed by Kaplan-Meier and Log-rank test. RESULTS: Linear regression and automatic linear regression showed lymph node positive functional equations by tumor-node-metastasis staging from AJCC-7th and tumor-node-metastasis staging from AJCC-8th. Neurological invasion, venous infiltration, lymphatic infiltration, and tumor deposition put forward stricter requirements for pathological examination in AJCC-8th compared to AJCC-7th. After re-analyzing our cohort with AJCC-8th, the percentage of stage IVB cases decreased from 2.8% to 0.8%. As a result 2% of the cases were classified under the new IVC staging. DFS and OS was significantly shorter (P = 0.012) in stage IVC patients compared to stage IVB patients. CONCLUSION: The addition of stage IVC in AJCC-8th has shown that peritoneal metastasis has a worse prognosis than distant organ metastasis in our institution's CRC cohort. Additional datasets should be analyzed to confirm these findings.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic MIR17HG and tumor-suppressive MIR100HG clusters. We demonstrate functional significance of these mature senescence-associated-microRNAs in the process of replicative senescence of human adipose-derived stem cells ex-vivo and define a set of senescence-associated-microRNA gene targets that are able to elicit, modulate and, most importantly, balance intimate connections between oncogenic and senescent events.

Transcriptional profiling of interleukin-2-primed human adipose derived mesenchymal stem cells revealed dramatic changes in stem cells response imposed by replicative senescence.

Inflammation is a double-edged sword with both detrimental and beneficial consequences. Understanding of the mechanisms of crosstalk between the inflammatory milieu and human adult mesenchymal stem cells is an important basis for clinical efforts. Here, we investigate changes in the transcriptional response of human adipose-derived stem cells to physiologically relevant levels of IL-2 (IL-2 priming) upon replicative senescence. Our data suggest that replicative senescence might dramatically impede human mesenchymal stem cell (MSC) function via global transcriptional deregulation in response to IL-2. We uncovered a novel senescence-associated transcriptional signature in human adipose-derived MSCs hADSCs after exposure to pro-inflammatory environment: significant enhancement of the expression of the genes encoding potent growth factors and cytokines with anti-inflammatory and migration-promoting properties, as well as genes encoding angiogenic and anti-apoptotic promoting factors, all of which could participate in the establishment of a unique microenvironment. We observed transcriptional up-regulation of critical components of the nitric oxide synthase pathway (iNOS) in hADSCs upon replicative senescence suggesting, that senescent stem cells can acquire metastasis-promoting properties via stem cell-mediated immunosuppression. Our study highlights the importance of age as a factor when designing cell-based or pharmacological therapies for older patients and predicts measurable biomarkers characteristic of an environment that is conducive to cancer cells invasiveness and metastasis.

Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/-4.03%, which were comparable to Biotrans (8.94%+/-3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.