Regulation of the AGEs-induced inflammatory response in human periodontal ligament cells via the AMPK/NF-κB/ NLRP3 signaling pathway.

The heightened prevalence and accelerated progression of periodontitis in individuals with diabetes is primarily attributed to inflammatory responses in human periodontal ligament cells (HPDLCs). This study is aimed at delineating the regulatory mechanism of nucleotide-binding oligomerization domain-like receptors (NLRs) in mediating inflammation incited by muramyl dipeptide (MDP) in HPDLCs, under the influence of advanced glycation end products (AGEs), metabolic by-products associated with diabetes. We performed RNA-seq in HPDLCs induced by AGEs treatment and delineated activation markers for the receptor of AGEs (RAGE). It showed that advanced glycation end products modulate inflammatory responses in HPDLCs by activating NLRP1 and NLRP3 inflammasomes, which are further regulated through the NF-κB signaling pathway. Furthermore, AGEs synergize with NOD2, NLRP1, and NLRP3 inflammasomes to augment MDP-induced inflammation significantly.

Low expression of GRM4 is associated with poor prognosis and tumor immune infiltration in glioma.

INTRODUCTION: The metabotropic glutamate receptor 4 (mGlu4, GRM4) exhibits significant expression within the central nervous system (CNS) and has been implicated to be correlated with a poor prognosis. OBJECTIVE: This study was aimed to elucidate the relationship between the expression profile of GRM4 and the prognosis of glioma patients. METHODS: RNA-sequencing datasets from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and China Glioma Genome Atlas (CGGA) repositories were used to evaluate the potential relationship. The value of clinical prognostic about GRM4 was assessed using clinical survival data from CGGA and TCGA. The GEPIA database was used to select genes like GRM4. PPI network was constructed by the database of (STRING), GO and KEGG analyses were performed. TargetScan, TarBase, miRDB, and starBase were used to explore miRNAs that could regulate GRM4 expression. EWAS Data Hub, MethSurv, and MEXPRESS were used for the analysis and relationship between DNA methylation and GRM4 expression and prognosis in glioma. TIMER2.0 and CAMOIP databases were used to assess the association between immune cell infiltration and GRM4. Human GBM cell lines were used to validate the function of GRM4. RESULTS: Our study shows that GRM4 is under expressed among gliomas and accompanied by poorer OS. Multivariate analysis showed that low mRNA expression of GRM4 was an independent factor of prognostic for shorter OS in all glioma patients. MiR-1262 affects the malignant phenotype of gliomas through GRM4. Methylation of DNA plays an important role in the instruction of GRM4 expression, the methylation level of GRM4 in glioma tissue is higher in comparison to normal tissue, and the higher methylation level was accompanied with the worse prognosis. Further analysis showed that GRM4 mRNA expression in GBM linked negatively with common lymphoid progenitor, Macrophage M1, Macrophage, and T cell CD4+ Th2, but not with the tumor purity. Overexpression of GRM4 prevents the migration of human GBM cell lines in vitro. CONCLUSION: GRM4 may have a substantial impact on the infiltration of immune cells and serve as a valuable prognostic biomarker in gliomas.

Safety and Effectiveness of High-dose Liposomal Amphotericin B: A Systematic Review and Meta-analysis.

Background: Although the level of medical care has been improved in recent years, the probability of patients contracting pathogens has increased greatly, with a rising incidence of invasive fungal infections. Deep-seated fungi have become common pathogens of nosocomial infections. Objective: This study aims to systematically assess the effectiveness, mortality, survival rate, and adverse reactions (ARs) of high-dose (HD) liposomal amphotericin B (L-AMB) for human diseases. Methods: Ten articles (1661 patients) of randomized controlled trials (RCTs; whether randomized, single-blind, or double-blind) from January 1, 1960, to December 31, 2020, of HD-L-AMB treatment of diseases were retrieved from the PubMed, Embase, Scopus, Web of Science, and Cochrane Central Register of Controlled Trials databases. The primary outcome measure was the overall therapeutic effect, and the secondary outcome measures were mortality, ≥10-week survival, and ARs. Data were meta-analyzed using RevMan 5.3. Results: Ten RCTs involving 1661 patients were included. HD-L-AMB did not show a significant therapeutic advantage in anti-infection treatment. In addition, HD-L-AMB treatment of invasive Aspergillus infection led to high mortality and low survival (≥10 weeks, OR = 0.57, 95%CI 0.34-0.94, P = .03). According to subgroup analysis, the incidence of ARs and the incidence of renal dysfunction associated with invasive fungal infection treatment were higher with HD-L-AMB than with regular-dose L-AMB. Conclusion: HD-L-AMB had no obvious advantage for the treatment of diseases and was accompanied by increased mortality, reduced long-term survival, and increased ARs (including renal insufficiency). Therefore, the use of HD-L-AMB to control infections is recommended with caution only when the preferred treatment is contraindicated.

Prognostic Characteristics of Patients With Colorectal Cancer Who Have Benign Mesenteric Lymph Node Enlargement: A Multi-institutional Cohort Study.

BACKGROUND: The characteristics of patients with colorectal cancer who have benign mesenteric lymph node enlargement are not well documented. OBJECTIVE: The aim of this study is to assess the clinical and prognostic significance of benign mesenteric lymph node enlargement in patients with colorectal cancer. DESIGN: This is a prospective cohort study. SETTING: This study was conducted at multitertiary institutions. PATIENTS: We included 601 patients with stage 0, I, and II colorectal cancer in Tianjin, Shandong, and Zhejiang from January 2010 to April 2014. Patients underwent curative surgery and were separated into 2 groups by the presence of benign mesenteric lymph node enlargement: the enlargement group (n = 275) and the control group (n = 326). MAIN OUTCOME MEASURES: Univariate log rank and multivariate Cox regression analyses were constructed to identify risk factors for recurrence and mortality. RESULTS: The risk of recurrence in the enlargement group after curative resection was significantly lower than in the control group, with the 1-, 3-, and 5-year disease-free survival rates being 97.1%, 91.6%, and 86.9% in the enlargement group and 95.7%, 86.2%, and 78.2% in the control group (p = 0.004). The postoperative 1-, 3-, and 5-year overall survival rates were 99.6%, 94.9%, and 90.5% in the enlargement group and 99.4%, 91.4%, and 82.1% in the control group (p = 0.001). Patients in the enlargement group had a higher percentage of patients at a younger age, family tumor history, right-sided tumors, and larger tumor size compared with the control group. For patients in the enlargement group, no significant correlation was observed between the number of enlarged lymph nodes and disease-free survival or overall survival (p = 0.113 and 0.386). Adjusted Cox regression model showed that benign mesenteric lymph node enlargement was an independent prognostic risk factor for both disease-free survival (HR, 0.587; 95% CI, 0.399-0.861; p = 0.007) and overall survival (HR, 0.506; 95% CI, 0.328-0.779; p = 0.002). LIMITATIONS: No immunological results could be compared with clinicopathological findings. CONCLUSIONS: The study indicates that benign mesenteric lymph node enlargement can be a useful positive factor in predicting recurrence and long-term survival concerning patients with colorectal cancer. See Video Abstract at http://links.lww.com/DCR/B785. CARACTERSTICAS PRONSTICAS DE LOS PACIENTES PORTADORES DE CNCER COLORRECTAL CON AGRANDAMIENTO BENIGNO DE LOS GANGLIOS LINFTICOS MESENTRICOS UN ESTUDIO DE COHORTE MULTIINSTITUCIONAL: ANTECEDENTES:Las características de los pacientes portadores de cáncer colorrectal con agrandamiento benigno de los ganglios linfáticos mesentéricos no se encuentran bien documentados.OBJETIVO:El objetivo de este estudio es evaluar la importancia clínica y pronóstica del agrandamiento benigno de los ganglios linfáticos mesentéricos en pacientes con cáncer colorrectal.DISEÑO:Este es un estudio de cohorte de tipo prospectivo.AJUSTE:Este estudio se llevó a cabo en instituciones de educación superior.PACIENTES:Incluimos a 601 pacientes con cáncer colorrectal en estadio 0, I, II en Tianjin, Shandong y Zhejiang desde enero de 2010 hasta abril de 2014. Los pacientes fueron sometidos a cirugía curativa y fueron separaron en dos grupos tomando en cuenta la presencia del agrandamiento benigno de los ganglios linfáticos mesentéricos: grupo con agrandamiento (n = 275) y grupo control (n = 326).PRINCIPALES MEDIDAS DE RESULTADO:Se construyeron análisis de rango logarítmico de una variante y de regresión de Cox con variante múltiple para identificar los factores de riesgo de recurrencia y mortalidad.RESULTADOS:El riesgo de recurrencia en el grupo con agrandamiento tras la resección curativa fue significativamente menor que en el grupo de control, con tasas de periodo libre de enfermedad a los 1, 3 y 5 años de 97,1, 91,6, y 86,9% en el grupo de agrandamiento y con tasas de 95,7, 86,2, y 78,2% en el grupo control respectivamente (p = 0,004). Las tasas postoperatorias de supervivencia general a los 1, 3 y 5 años fueron 99,6, 94,9, y 90,5% en el grupo de agrandamiento y de 99,4, 91,4, y 82,1% en el grupo de control, respectivamente (p = 0,001). Los pacientes del grupo con agrandamiento tenían un porcentaje más elevado de menor edad, antecedente familiar tumoral, tumores del lado derecho y de mayor tamaño tumoral con respecto al grupo de control. Para los pacientes con agrandamiento, no se observó una correlación significativa entre el número de ganglios linfáticos agrandados y el periodo libre de enfermedad o la supervivencia general (p = 0,113 y 0,386). El modelo de regresión de Cox ajustado mostró que el agrandamiento benigno de los ganglios linfáticos mesentéricos era un factor de riesgo pronóstico independiente tanto para la supervivencia libre de enfermedad (cociente de riesgo 0,587; IC del 95%: 0,399-0,861; p = 0,007) como para la supervivencia global (cociente de riesgo 0,506; IC del 95%: 0,328- 0,779; p = 0,002).LIMITACIONES:No fue posible comparar los resultados inmunológicos con los hallazgos clínico-patológicos.CONCLUSIONES:El estudio indica que el agrandamiento benigno de los ganglios linfáticos mesentéricos puede ser un factor positivo útil para predecir la recurrencia y la supervivencia a largo plazo en pacientes con cáncer colorrectal. Consulte Video Resumen en http://links.lww.com/DCR/B785. (Traducción-Dr. Osvaldo Gauto).

Role of ferroptosis-related genes in coronary atherosclerosis and identification of key genes: integration of bioinformatics analysis and experimental validation.

BACKGROUND: Coronary atherosclerosis (CA) is the most common type of atherosclerosis. However, the inherent pathogenesis and mechanisms of CA are unclear, and the relationship with ferroptosis-related genes (FRGs) has not been reported. The purpose of this study was to use bioinformatics techniques to evaluate potential therapeutic targets for CA.Please provide the given name for author "Dingshun".Please provide the given name for author "Dingshun". METHODS: First, the GSE132651 dataset was acquired from the Gene Expression Omnibus database. Gene Ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and Protein-Protein interaction network were successively conducted. Next, overlapping genes between hub genes and CA genes were found. FRGs were found when comparing the CA group with the normal group. The correlation between overlapping genes and FRGs was further analyzed. At last, we performed Elisa to validate the expression of these genes in human blood specimens. Mice aortic tissues were used for western blot to detect the expression of proteins. RESULTS: Based on the GSE132651 dataset, 102 differentially expressed genes were identified. Five overlapping genes between hub genes and CA genes were found (CCNA2, RRM2, PBK, PCNA, CDK1). TFRC and GPX4 were found to be FRGs. TFRC was positively correlated with CCNA2, PBK, PCNA, CDK1, RRM2, with CDK1 being the strongest correlation. GPX4 was negatively correlated with these genes, among which CCNA2 was the strongest correlation. The ELISA results showed that CCNA2, CDK1, and TFRC expression were markedly increased in serum of the CA samples compared with controls, while GPX4 expression was markedly decreased in the CA samples. The western blot results show that GPX4 expression was lower in the model group, TFRC, CDK1, and CCNA2 protein expression were high in the model group. CONCLUSIONS: Ferroptosis-related genes GPX4 and TFRC were closely correlated with the identified overlapping genes CCNA2 and CDK1, which may serve as targeted therapies for the treatment of CA.

Discovery of Novel Quinazoline Derivatives as Potent Antitumor Agents.

In this work, we designed and synthesized a novel series of quinazoline derivatives 6-19 and then evaluated their broad-spectrum antitumor activity against MGC-803, MCF-7, PC-9, A549, and H1975, respectively. Most of them demonstrated low micromolar cytotoxicity towards five tested cell lines. In particular, compound 18 exhibited nanomolar level inhibitory activity against MGC-803 cells with an IC50 value of 0.85 µM, indicating approximately a 32-fold selectivity against GES-1 (IC50 = 26.75 µM). Further preclinical evaluation showed that compound 18 remarkably inhibited the migration of MGC-803 cells, induced cell cycle arrest at G2/M, and induced MGC-803 apoptosis, resulting in decreasing the expression of both Bcl-2 and Mcl-1, and up-regulating the expression of both Bax and cleaved PARP. No death or obvious pathological damage was observed in mice by acute toxicity assay. The in vivo antitumor evaluation suggested that compound 18 significantly decreased the average tumor volume and tumor weight without any effect on body weight, which is better than 5-Fu. Therefore, compound 18 can be used as a lead compound for the further development of antitumor drugs in the future.

Exosome-delivered miR-221/222 exacerbates tumor liver metastasis by targeting SPINT1 in colorectal cancer.

MicroRNAs (miRNAs) are involved in the progression of many cancers through largely unelucidated mechanisms. The results of our present study identified a gene cluster, miR-221/222, that is constitutively upregulated in serum exosome samples of patients with colorectal carcinoma (CRC) with liver metastasis (LM); this upregulation predicts a poor overall survival rate. Using an in vitro cell coculture model, we demonstrated that CRC exosomes harboring miR-221/222 activate liver hepatocyte growth factor (HGF) by suppressing SPINT1 expression. Importantly, miR-221/222 plays a key role in forming a favorable premetastatic niche (PMN) that leads to the aggressive nature of CRC, which was further shown through in vivo studies. Overall, our results show that exosomal miR-221/222 promotes CRC progression and may serve as a novel prognostic marker and therapeutic target for CRC with LM.

Simultaneous measurement of tadehaginoside and its principal metabolite in rats by HPLC-MS/MS and its application in pharmacokinetics and tissue distribution study.

CONTEXT: Tadehaginoside, an active ingredient isolated from Tadehagi triquetrum (Linn.) Ohashi (Leguminosae), exhibited various biological activities. However, the pharmacokinetics and tissue distribution which affect tadehaginoside's therapeutic actions and application remain elusive. OBJECTIVE: To clarify the metabolism of tadehaginoside in vivo. MATERIALS AND METHODS: The pharmacokinetics and tissue distribution of tadehaginoside and its metabolite p-hydroxycinnamic acid (HYD) were investigated using LC-MS/MS. Pharmacokinetic parameters were determined in 10 Sprague-Dawley rats divided into two groups, the intravenous group (5 mg/kg) and the oral group (25 mg/kg). For the tissue-distribution study, 20 rats were intravenously given tadehaginoside (5 mg/kg) before the experiment (n = 4). Biological samples were collected before drug administration (control group) and after drug administration. RESULTS: The linearity, accuracy, precision, stability, recovery and matrix effect of the method were well-validated and the results satisfied the requirements of biological sample measurement. Treatment with tadehaginoside via intragastric and intravenous administration, the calculated Cmax in rats was 6.01 ± 2.14 ng/mL and 109.77 ± 4.29 ng/mL, and Tmax was 0.025 ± 0.08 h and 0.08 h, respectively. The results indicated that the quick absorption of tadehaginoside was observed following intravenous administration, and tadehaginoside in plasma of rats with intragastric administration showed relatively low concentration may be due to the formation of its metabolite. Tissue-distribution study indicated that kidney and spleen were the major distribution organs for tadehaginoside in rats and there was no long-term accumulation in most tissues. DISCUSSION AND CONCLUSION: These results could provide clues for exploring the bioactivity of tadehaginoside based on its pharmacokinetic characteristics.

MicroRNA-9 overexpression suppresses vulnerable atherosclerotic plaque and enhances vascular remodeling through negative regulation of the p38MAPK pathway via OLR1 in acute coronary syndrome.

Acute coronary syndrome (ACS) is characterized by atherosclerotic plaque rupture with a high incidence of recurrent ischemic events. Several microRNAs are found to be aberrantly expressed in atherosclerotic plaques. This study aims to investigate the effects of microRNA-9 (miR-9) on vulnerable atherosclerotic plaque and vascular remodeling in ACS and underlying mechanisms. Microarray-based gene expression profiling was used to identify differentially expressed genes related to ACS and regulatory miRNAs. Oxidized low-density lipoprotein (lectin-like) receptor 1 (OLR1) was identified to be aberrantly activated in ACS and regulated by miR-9. OLR1 was verified as a target gene of miR-9 by bioinformatics prediction and dual luciferase reporter gene assay. The atherosclerotic models were induced in ApoE-/- mice, in which the agomir or antagomir of miR-9, or small interfering RNA (siRNA) against OLR1 were separately introduced. Serum lipid levels and expression of vascular remodeling and inflammatory response-related factors were determined, respectively. On the basis of the obtained results, in the atherosclerosis mice treated with the agomir of miR-9 and siRNA against OLR1, the p38-mitogen-activated protein kinase (p38MAPK) pathway was inhibited; levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor were reduced, but the high-density lipoprotein cholesterol level was increased, along with decreased vulnerable atherosclerotic plaque area and enhanced vascular remodeling. Taken together, these findings suggested an inhibitory role miR-9 acts in the formation of vulnerable atherosclerotic plaques in ACS mice, along with a promoted vascular remodeling, via a negative feedback regulation of OLR1-mediated p38MAPK pathway.

FCRL3 promotes IL-10 expression in B cells through the SHP-1 and p38 MAPK signaling pathways.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system that is caused by the interaction of genetic and environmental factors. Current studies have shown that Fc-receptor like-3 (FCRL3) is closely related to MS, but the specific role of FCRL3 in MS has not yet been clarified. This study further found that FCRL3 and interleukin 10 (IL-10) expression was downregulated in MS patients, but the expression of these proteins was higher in the remission phase than that in the acute phase. The C allele of rs7528684 was associated with MS, and the CC genotype could lead to the upregulation of FCRL3 expression and the increase in IL-10 secretion. Further in vitro experiments with B cells found that lipopolysaccharide (LPS) promoted FCRL3 expression in a dose- and time-dependent manner, thereby promoting IL-10 secretion. LPS regulated Src homology region 2 domain-containing phosphatase-1 (SHP-1) expression and p38 mitogen-activated protein kinase (MAPK) pathway activation through FCRL3, and FCRL3 upregulated the SHP-1 expression and p38 phosphorylation levels. When SHP-1 small interfering RNA or a p38 pathway inhibitor was added, the effect of FCRL3 on IL-10 secretion was significantly inhibited. In addition, FCRL3 inhibited the secretion of inflammatory factors (tumor necrosis factor-α, IL-1ß, IL-6, and IL-8); after inhibiting the expression of IL-10, the abovementioned effects of FCRL3 were blocked. These results suggest that FCRL3 can activate the SHP-1 and p38 MAPK pathways and then promote the secretion of IL-10 in B cells, thus inhibiting the secretion of inflammatory factors. Therefore, FCRL3 may play an immunoprotective role in MS, and it will be an effective target for the diagnosis and treatment of MS.

Holothuria Leucospilota Polysaccharides Ameliorate Hyperlipidemia in High-Fat Diet-Induced Rats via Short-Chain Fatty Acids Production and Lipid Metabolism Regulation.

Holothuria leucospilota polysaccharides (HLP) are expected to become potential resources for the treatment of hyperlipidemia because of their various bioactivities. In the study, the treatment of HLP on improving hyperlipidemia in rats was explored. Oral administration of HLP at 100 or 200 mg/kg body weight effectively alleviated serum lipid levels and liver histological abnormalities in high-fat-diet rats. HLP regulated abnormal mRNA, lipogenesis-related hormones and inflammatory cytokines (tumor necrosis factor-α, interleukin-6 and interleukin-12) levels. HLP improved the ability of gut microbiota to produce short-chain fatty acids (SCFAs). SCFAs have been found to ameliorate liver lesions. Therefore, HLP alleviated hyperlipidemia by improving the levels of SCFAs to regulate lipid metabolism. These results indicated that HLP could be used as beneficial polysaccharides to alleviate hyperlipidemia.

Elucidation of the FKBP25-60S Ribosomal Protein L7a Stress Response Signaling During Ischemic Injury.

BACKGROUND/AIMS: Peptidyl-prolyl cis-trans isomerase FKBP25 is a member of the FK506-binding proteins family which has peptidyl-prolyl cis/trans isomerase domain. The biological function and pathophysiologic role of FKBP25 remain elusive. METHODS: The spatio-temporal changes in expression of endothelial FKBP25 upon oxygen-glucose deprivation (OGD) treatment were examined by Western blot and immunofluorescence. The immunoprecipitation and fluorescence resonance energy transfer (FRET) were used to address the interacting proteins with FKBP25. RESULTS: In the present study, nuclear translocation of FKBP25 was observed following OGD in cultured endothelial cells. Intriguingly, FKBP25 nuclear translocation was further validated in peroxynitrite (ONOO-)-treated endothelial cells. Coimmunoprecipitation and FRET data indicated that FKBP25 translocated into the nucleus, in which it interacted with 60S ribosomal protein L7a, while overexpression FKBP25 protect endothelial cells against OGD injury. CONCLUSION: Our findings reveal that the nuclear import of FKBP25 and binding with 60S ribosomal protein L7a are protective stress responses to ischemia/nitrosaive stress injury.

Endothelial GPR124 Exaggerates the Pathogenesis of Atherosclerosis by Activating Inflammation.

BACKGROUND/AIMS: Endothelial cell dysfunction is the principal pathological process underlying atherosclerotic cardiovascular disease. G protein-coupled receptor 124 (GPR124), an orphan receptor in the adhesion GPCR subfamily, promotes angiogenesis in the brain. In the present study, we explored the role of endothelial GPR124 in the development and progression of atherosclerosis in adult mice. METHODS: Using tetracycline-inducible transgenic systems, we generated mice expressing GPR124 specifically under control of the Tie-2 promoter. The animal model of atherosclerosis was constructed by intravenously injecting AAV-PCSK9DY into tetracycline-regulated mice and feeding the mice a high-fat diet for 16 consecutive weeks. Biochemical analysis and immunohistochemistry methods were used to address the role and mechanism of GPR124 in the pathological process of atherosclerosis. RESULTS: Higher TC (total cholesterol) and LDL-C (low density lipoprotein cholesterol) levels in serum and greater lipid deposition in the aortic sinus were found in atherosclerotic mice with GPR124 overexpression, coincident with the elevated proliferation of smooth muscle cells. We observed an elevation of ONOO- in the aortic sinus in this model by using immunofluorescence, and the experiments showed that the specific overexpression of GPR124 in the endothelium induced the up-regulation of CD68, NLRP3 and caspase-1 levels in the aortic sinus. CONCLUSION: The above results indicate that manipulating GPR124 in the endothelium may contribute to delayed pathological progression of atherosclerosis.

T7 Peptide-Conjugated Lipid Nanoparticles for Dual Modulation of Bcl-2 and Akt-1 in Lung and Cervical Carcinomas.

Expression of Bcl-2 and Akt-1 has been associated with human cancer. G3139 and RX-0201, targeting Bcl-2 and Akt-1, respectively, are antisense oligonucleotides (ASOs) that have shown limited efficacy in clinical trials. Herein, we report a combination of newly designed ASOs based on these agents and was delivered by tumor cell-targeting lipid nanoparticles (LNPs). A "Gapmer" design strategy was applied to these ASOs with the addition of 2'-O-methyl modifications on the nucleotides at 5' and 3' ends. A dual-channel syringe pump-based system was developed for the synthesis of the LNPs. ASO-LNPs composed of DODMA, egg PC, cholesterol, T7-PEG-DSPE, and PEG-DMG at a molar ratio of 35:39.5:20:0.5:5 and carrying either individual ASOs or co-loaded ASO combinations (Co-ASOs) were synthesized and evaluated in both KB and A549 cancer cells and in an A549 murine xenograft model to determine their antitumor effects and biological activities. The ASO-LNPs exhibited excellent colloidal stability and high ASO encapsulation efficiency with relatively small mean particle sizes and moderately positive zeta potentials. Transferrin receptor-targeting T7-conjugated LNPs showed enhanced cellular uptake compared to nontargeted LNPs. In addition, both T7-conjugated Co-ASOs-LNPs and non-T7-conjugated Co-ASOs-LNPs at a molar ratio of (G3139-GAP to RX-0201-GAP at 1:2) showed efficient downregulation of both Bcl-2 and Akt-1 in both A549 and KB cells. Furthermore, T7-conjugated Co-ASOs-LNPs (Co-ASOs-LNPs) produced superior antitumor activity, prolonged the overall survival time, and demonstrated tumor targeting activity in an A549 xenograft model.

Multimodal Rehabilitation Program Promotes Motor Function Recovery of Rats After Ischemic Stroke by Upregulating Expressions of GAP-43, SYN, HSP70, and C-MYC.

BACKGROUND: Despite the intense efforts devoted to preventing and treating cerebral ischemia, some individuals will continue to have completed infarctions. Failure of prevention or intervention does not, however, preclude therapeutic approaches to enhance recovery. Our study aims to explore the effect of multimodal rehabilitation program on the motor function recovery of rats with ischemic stroke. METHODS: Rat models of ischemic stroke were established using clean-grade adult male Sprague-Dawley rats. Motor function of rats was scored by the Bederson neurological function, balance beam test, and screen test. Nissl staining was conducted for morphological and structural changes of nerve cells in the arteriae cerebri anterior zone. Immunohistochemistry was applied to detect the expressions of growth-associated protein (GAP-43), synaptophysin (SYN) and Caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out in the corpus striatum 21 days after operation; reverse transcription quantitative polymerase chain reaction and Western blot analysis were conducted for testing messager RNA (mRNA) and protein expressions of heat shock protein 70 (Hsp70) and MYC proto-oncogene (c-Myc). RESULTS: Rats receiving multimodal rehabilitation program had lower Bederson neurological function, balance beam, and screen test scores on the 7th, 14th and 21st days after operation; more number of neurons surviving in the arteriae cerebri anterior zone at each time point after operation, higher GAP-43 expression on the 7th and 14th days after operation, and higher SYN expression on the 14th and 21st days after operation, on the 7th, 14th and 21st days after operation, higher mRNA and protein expressions of HSP70 and C-MYC, lower Caspase-3 positive expression and TUNEL positive stained cells. CONCLUSIONS: Multimodal rehabilitation program could promote motor function recovery of rats after ischemic stroke by upregulating GAP-43 and SYN expressions at arteriae cerebri anterior zone and upregulating HSP70 and C-MYC expressions in the brain tissues.

Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of cytochrome P450 3A4 and pregnane X receptors.

Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids, FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays, respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity, and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXRCYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with other drugs.

Lipid Nanoparticles Loaded with an Antisense Oligonucleotide Gapmer Against Bcl-2 for Treatment of Lung Cancer.

PURPOSE: Bcl-2 is an anti-apoptotic gene that is frequently overexpressed in human cancers. G3139 is an antisense oligonucleotide against bcl-2 that has shown limited efficacy in clinical trials. Here, we report the synthesis of a new antisense oligonucleotide containing additional chemical modifications and its delivery using nanoparticles. METHODS: An oligonucleotide G3139-GAP was synthesized, which has 2'-O-methyl nucleotides at the 5' and 3' ends based on a "gapmer" design. Furthermore, G3139-GAP was incorporated into lipid nanoparticles (LNPs) composed of DOTAP/egg PC/cholesterol/Tween 80. The LNP-loaded G3139-GAP was evaluated in A549 lung cancer cells both in vitro and in a murine xenograft model for biological activity and therapeutic efficacy. RESULTS: The LNPs showed excellent colloidal and serum stability, and high encapsulation efficiency for G3139-GAP. They have a mean particle diameter and zeta potential of 134 nm and 9.59 mV, respectively. G3139-GAP-LNPs efficiently downregulated bcl-2 expression in A549 cells, as shown by 40% and 83% reduction in mRNA and protein levels, respectively. Furthermore, G3139-GAP-LNPs were shown to inhibit tumor growth, prolong survival, and downregulate tumor bcl-2 expression in an A549 murine xenograft tumor model. These data indicate that G3139-GAP-LNPs have excellent anti-tumor efficacy and warrant further evaluation.

Reduced neurotrophic factor level is the early event before the functional neuronal deficiency in high-fat diet induced obese mice.

Neurodegeneration is considered one of the possible complications of high fat diet (HFD) induced obesity. Much evidence has shown the close relationship between HFD and dementia at comparatively later stage of neuronal injury. It is so far not clear that the initial events of neuronal injury resulting from HFD and obesity. In the present research, obese mouse model achieved by 3-month HFD was applied for the investigation of the possible neuronal deficiency before the obvious cognitive decline. We found that 3-month HFD has already increased the average level of body weight of mice. But almost no obvious cognitive defect was observed. At such time point, we detected the cleavage of amyloid precursor protein (APP), including the expression and maturation level of α- and ß-secretase and proteolytic fragment soluble APP. Results showed similar readout between HFD and normal diet (ND) mice. Besides, neuronal inflammation and brain-blood barrier permeability were also detected. No obvious changes could be observed between HFD and ND mice. Surprisingly, the first detectable neuronal changes was showed to be the downregulation of some neurotrpic factors, like neuronal growth factor ß and brain derived neurotrophic factor, together with the activity of specific receptors, like Trk receptor phosphorylation. All the data piled up indicated that the early neuronal change in HFD induced obese mice was the downregulation of some neurotrophic factors. The results may provide the potential clue to therapeutic and preventive strategy for HFD induced cognitive decline.

Two new cassane-type diterpenes from the seeds of Caesalpinia crista.

Two new cassane-type diterpenes, phangininoxys D and E (1 and 2), together with five known compounds were isolated from the seeds of Caesalpinia crista Linn. The structures of these compounds were elucidated by means of various spectroscopic analyses. All the isolated compounds were evaluated for cytotoxicity activities against HeLa, HT-29 and KB cell lines, and compound 7 showed moderate selective activities against KB cell line with an IC50 value of 17.1 µM.

N-butanol fraction of Entada phaseoloides ethanol extract inhibits hepatocellular carcinoma HepG2 cell proliferation by inducing apoptosis.

PURPOSE: To screen for substances with inhibitory effects on the proliferation of hepatocellular carcinoma (HCC) HepG2 cell line from extracts of traditional Chinese medicinal plants including Heliciopsis lobata (Merr.) Sleum, Bidens pilosa, Entada phaseoloides, Plantago major, and Smilax, and unveil their mechanism of action. METHODS: 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the inhibition of HepG2 cell proliferation by plant extracts. Cell apoptosis was evaluated by Hoechst 33342 staining and mitochondrial transmembrane potential (ΔCgr;m) dissipation was measured using JC-1 probe by fluorescence microscopy. RESULTS: Heliciopsis lobata, Bidens, Plantago, and Smilax extracts showed reduced inhibitory effects on HepG2 cell proliferation compared with Entada phaseoloides (all p<0.05). The n-butanol fraction of Entada phaseoloides ethanol extract exhibited the highest inhibition rate. Treatment of HepG2 cells with 500, 250, and 100 µg/ml n-butanol extract resulted in 89.92±0.58%, IC50 81.66±0.42%, 68.85±0.57% decrease in cell viability, respectively, indicating an IC50 of 9.27 µg/ml. In the presence of 100 µg/ml entada phaseoloides n-butanol extract for 24h, apoptotic nuclei and hyperchromatic, dense fluorescent massive granules were observed in the cytoplasm, effects that increased with extract concentrations in HepG2 cells. Finally, we showed that Entada phaseoloides n-butanol extract induced depolarization of mitochondrial membrane potential. CONCLUSIONS: Entada phaseoloides n-butanol extract inhibits HepG2 cell proliferation by inducing cell apoptosis likely through mitochondrial apoptotic pathway. This extract is therefore a potential natural source towards the discovery for a new drug-candidate for the treatment of HCC.