Synthesis and evaluation of butylphthalide-scutellarein hybrids as multifunctional agents for the treatment of Alzheimer's disease.

A series of butylphthalide and scutellarein hybrids 3-(alkyl/alkenyl) hydroxyphthalide derivatives were designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease. In vitro bioactivity assays indicated that most of the compounds displayed excellent antioxidant activity and moderate to good inhibition activities of self-induced Aß1-42 aggregation. Among them, compound 7c was demonstrated as a potential and balanced multifunctional candidate displaying the best inhibitory effects on self- and Cu2+-induced Aß1-42 aggregation (90.2 % and 35.4 %, respectively) and moderate activity for disaggregation of Aß1-42 aggregation (42.5 %). In addition, 7c also displayed excellent antioxidant (2.42 Trolox equivalents), metal ions chelating, oxidative stress alleviation, neuroprotective and anti-neuroinflammatory activities. Furthermore, in vivo study demonstrated that 7c could ameliorate the learning and memory impairment induced by sodium nitrite and Aß1-42 in the step-down passive avoidance test. These balanced multifunctional profiles supporting compound 7c as a novel potential candidate for the treatment of AD.

Discovery of novel 5-(2-hydroxyphenyl)-2-phthalide-3(3H)-pyrazolones as balanced multifunctional agents against Alzheimer's disease.

Based on previous work, a series of novel 5-(2-hydroxyphenyl)-2-phthalide-3(3H)-pyrazolones derivatives were identified as potential multifunctional therapeutic agents for Alzheimer's disease. Biological evaluation exhibited that these derivatives had great performance against MAO-B, Aß1-42 aggregation, oxidative stress and metal ion dyshomeostasis. Among them, 10x was selected as the optimal agent for its excellent MAO-B inhibitory activity (IC50 = 0.41 µM, SI > 24.4), good antioxidant activity (1.16 Trolox equivalent) and anti-Aß aggregation activity (56.03% and 57.51% for inhibition of self- and Cu2+-induced Aß1-42 aggregation; 81.91% and 82.40% for disaggregation of self- and Cu2+-induced Aß1-42 fibrils at 25.0 µM). Besides, 10x also exhibited obvious metal-ion chelating ability, anti-neuroinflammation (NO, TNF-α), neuroprotective activity and BBB permeability. More importantly, in vivo behavioral assessment demonstrated 10x could remarkably improve the memory and cognitive impairment in Aß1-42 induced AD mice model. Overall, these test results indicated 10x could serve as a balanced multifunctional anti-AD agent and deserved further research.

SHP2 deneddylation mediates tumor immunosuppression in colon cancer via the CD47/SIRPα axis.

SIPRα on macrophages binds with CD47 to resist proengulfment signals, but how the downstream signal of SIPRα controls tumor-infiltrating macrophages (TIMs) is still poorly clarified. Here, we report that the CD47/signal regulatory protein α (SIRPα) axis requires the deneddylation of tyrosine phosphatase SHP2. Mechanistically, Src homology region 2-containing protein tyrosine phosphatase 2 (SHP2) was constitutively neddylated on K358 and K364 sites; thus, its autoinhibited conformation was maintained. In response to CD47-liganded SIRPα, SHP2 was deneddylated by sentrin-specific protease 8 (SENP8), which led to the dephosphorylation of relevant substrates at the phagocytic cup and subsequent inhibition of macrophage phagocytosis. Furthermore, neddylation inactivated myeloid-SHP2 and greatly boosted the efficacy of colorectal cancer (CRC) immunotherapy. Importantly, we observed that supplementation with SHP2 allosteric inhibitors sensitized immune treatment-resistant CRC to immunotherapy. Our results emphasize that the CRC subtype that is unresponsive to immunotherapy relies on SIRPαhiSHP2hiNEDD8lo TIMs and highlight the need to further explore the strategy of SHP2 targeting in CRC therapy.

The potential mechanism of Bletilla striata in the treatment of ulcerative colitis determined through network pharmacology, molecular docking, and in vivo experimental verification.

Ulcerative colitis (UC) is a chronic nonspecific intestinal inflammatory disease, which belongs to a subtype of inflammatory bowel disease, but still lacks effective drug treatment. Bletilla striata (B. striata) is one of the most valuable traditional Chinese medicines (TCMs) in China, can stop bleeding, can promote wound healing, and can regulate immunity. Based on data mining, B. striata was found to be a common TCM for the treatment of UC, but the exact therapeutic mechanism is not yet known. This study aims to explore the potential mechanisms of B. striata in the treatment of UC using network pharmacology, molecular docking techniques, and in vivo experimental research. We extracted the active ingredients and targets of B. striata from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and analysis platform. We retrieved and screened the corresponding UC-related target genes in multiple databases. Subsequently, we constructed an herb-ingredient-target-disease-network, generated a protein-protein interaction network, performed Gene Ontology enrichment analysis, and performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify potential treatment mechanisms. After screening for key active ingredients and target genes, we performed molecular docking using AutoDock Vina software to select the best binding target for molecular docking and validate the binding activity. The UC model was established in mice, and the results of network pharmacology and molecular docking were verified by in vivo experiments. In all, 5 compounds were obtained from the TCMSP database, and 74 UC-related pathogenic genes were obtained from GeneCards, DisGeNET, OMIM, TTD, and DrugBank. After KEGG enrichment analysis, pathways in cancer, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway, and metabolic pathways were identified as the top three signalling pathways associated with UC treatment. The results of molecular docking showed that the active components of B. striata have good binding activities to the pivotal targets epidermal growth factor receptor (EGFR) and PIK3CA. In a dextran sulphate sodium-induced colitis model, we found that B. striata can alleviate the symptoms of UC, decrease the secretion of the inflammatory cytokines interleukin-6 and tumour necrosis factor-α, and downregulate the expression levels of EGFR, PIK3CA, and p-AKT. In conclusion, the treatment of UC with B. striata may alleviate the inflammatory response of the colon, and B. striata mainly inhibits the EGFR/PI3K/AKT signalling pathways.

Enhancing the Classification of Aphasia: A Statistical Analysis Using Connected Speech.

Background: Large shared databases and automated language analyses allow for the application of new data analysis techniques that can shed new light on the connected speech of people with aphasia (PWA). Aims: To identify coherent clusters of PWA based on language output using unsupervised statistical algorithms and to identify features that are most strongly associated with those clusters. Methods & Procedures: Clustering and classification methods were applied to language production data from 168 PWA. Language samples were from a standard discourse protocol tapping four genres: free speech personal narratives, picture descriptions, Cinderella storytelling, procedural discourse. Outcomes & Results: Seven distinct clusters of PWA were identified by the K-means algorithm. Using the random forests algorithm, a classification tree was proposed and validated, showing 91% agreement with the cluster assignments. This representative tree used only two variables to divide the data into distinct groups: total words from free speech tasks and total closed class words from the Cinderella storytelling task. Conclusion: Connected speech data can be used to distinguish PWA into coherent groups, providing insight into traditional aphasia classifications, factors that may guide discourse research and clinical work.

Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA.

N6-methyladenosine (m6A) is abundant in the mammalian brain and is considered to have a wide range of effects on learning and memory. Here, we found that the upregulated methyltransferase-like protein 16 (METTL16) in the hippocampal tissues of Morris water maze (MWM)-trained mice contributed to improved memory formation and hippocampal synaptic plasticity. Mechanismly, METTL16 promoted the expression of methionine adenosyltransferase 2A (MAT2A) by the m6A methylation of the MAT2A mRNA-3'UTR-end to increase its stability, and this involved in improving hippocampal global m6A levels, plasticity of dendritic spine, learning and memory. This study provides a new perspective to explore the regulatory mechanisms of m6A for learning and memory.

Early diagnostic value of liver stiffness measurement in hepatic sinusoidal obstruction syndrome induced by hematopoietic stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT)-sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease, is a clinical syndrome characterized by symptoms, such as right upper quadrant pain, jaundice, fluid retention, and hepatomegaly, and is caused by pre-treatment-related hepatotoxicity during the early stages after HSCT. Clinical diagnosis of HSCT-SOS is based on the revised Seattle or Baltimore standards. The revised standard by the European Society for Bone Marrow Transplantation in 2016 has good practicability and can be used in combination with these two standards. Eight studies have shown the value of liver stiffness measurement (LSM) in the early diagnosis of HSCT-SOS. Four studies investigated LSM specificity and sensitivity for the early diagnosis of HSCT-SOS. LSM can distinguish SOS from other post-HSCT complications, enabling a clear differential diagnosis. It has been shown that median LSM of patients with SOS is significantly higher than that of patients with other treatment-related liver complications (e.g., acute cholecystitis, cholangitis, antifungal drug-related liver injury, liver graft-versus-host disease, isolated liver biochemical changes, and fulminant Epstein Barr virus related hepatitis reactivation). Therefore, the above data confirmed the utility of LSM and strongly suggested that LSM improves the positive predictive value of the SOS diagnostic clinical score after allogeneic HSCT. Early diagnosis of SOS is beneficial in preventing severe HSCT complications.

Effects of membrane androgen receptor binding on synaptic plasticity in primary hippocampal neurons.

Androgens play an important role in the regulation of hippocampal synaptic plasticity. While the classical molecular mechanism of androgen's genomic activity is their binding to intracellular androgen receptors (iARs), they can also induce rapid non-genomic effects through specific membrane androgen receptors (mARs). In this study, we aimed to localize and characterize these mARs in primary rat hippocampal neurons. Specific punctate fluorescent signals on the cell surface, observed by testosterone-fetal bovine serum albumin conjugated fluorescein isothiocyanate (T-BSA-FITC), indicated the presence of mARs in hippocampal neurons. T-BSA-FITC binding to the cell membrane was incompletely blocked by the iAR-antagonist flutamide, and mAR binding site was competitively bound by free testosterone (T). Most neurons expressing androgen membrane binding sites are glutamatergic (excitatory), although several are γ-aminobutyric acid (GABA)ergic (inhibitory). Confocal microscopy and live-cell imaging techniques were used to observe the real-time rapid effects of androgens on hippocampal dendritic spine morphology. Immunofluorescence cell staining was used to observe their effects on the postsynaptic density protein 95 (PSD95) and synapsin (SYN) synaptic markers. While androgens did not cause a short-term increase in dendritic spine density of rat primary hippocampal neurons, they promoted the transformation of dendritic spines from thin to mushroom, promoted dendritic spine maturation, increased dendritic spine surface area, and rapidly increased PSD95 and SYN expression in the primary hippocampal neurons. Hippocampal synaptosomes were prepared using the Optiprep and Percoll density gradient two-step centrifuge methods, and the gene expression profiles of the synaptosomes and hippocampus were compared using a gene chip; PSD95 mRNA expression was detected by reverse transcription-polymerase chain reaction. Several mRNAs were detected at the synaptic site, including PSD95. Finally, the Venus-PSD95 plasmid was constructed and transfected into HT22 cells, which is a mouse hippocampal neuronal cell line. The real-time effect of androgen on synaptic protein PSD95 was observed by fluorescence recovery after photobleaching experiments, which involved the translation process of PSD95 mRNA. In conclusion, our findings increased our understanding of how androgens exert the neuroprotective mechanisms on synaptic plasticity.

Discovery of novel 3-butyl-6-benzyloxyphthalide Mannich base derivatives as multifunctional agents against Alzheimer's disease.

Based on the multitarget-directed ligands strategy, a series of 3-butyl-6-benzyloxyphthalide Mannich base derivatives were designed, synthesized and identified for Alzheimer's disease (AD). Biological activity studies demonstrated that the designed hybrids showed multitarget activities toward AD. Among them, compound 7d was the most potent agent with excellent inhibitory activities on EeAChE (IC50 = 0.087 µM), HuAChE (IC50 = 0.041 µM) and MAO-B (IC50 = 0.30 µM). Furthermore, molecular docking studies were conducted to investigate the interaction mode with enzymes. Besides, 7d also possessed good effects of Cu2+ chelation, ameliorate oxidative stress, and anti-neuroinflammation, desirable BBB permeability and eligible drug-like properties. Altogether, the multifunctional profiles of 7d prove that it deserves further investigation as a novel drug candidate for AD treatment.

Development of novel 2-aminoalkyl-6-(2-hydroxyphenyl)pyridazin-3(2H)-one derivatives as balanced multifunctional agents against Alzheimer's disease.

Based on multitarget-directed ligands approach, through two rounds of screening, a series of 2-aminoalkyl-6-(2-hydroxyphenyl)pyridazin-3(2H)-one derivatives were designed, synthesized and evaluated as innovative multifunctional agents against Alzheimer's disease. In vitro biological assays indicated that most of the hybrids were endowed with great AChE inhibitory activity, excellent antioxidant activity and moderate Aß1-42 aggregation inhibition. Taken both efficacy and balance into account, 12a was identified as the optimal multifunctional ligand with significant inhibition of AChE (EeAChE, IC50 = 0.20 µM; HuAChE, IC50 = 37.02 nM) and anti-Aß activity (IC50 = 1.92 µM for self-induced Aß1-42 aggregation; IC50 = 1.80 µM for disaggregation of Aß1-42 fibrils; IC50 = 2.18 µM for Cu2+-induced Aß1-42 aggregation; IC50 = 1.17 µM for disaggregation of Cu2+-induced Aß1-42 fibrils; 81.7% for HuAChE-induced Aß1-40 aggregation). Moreover, it was equipped with the potential to serve as antioxidant (3.03 Trolox equivalents), metals chelator and anti-neuroinflammation agent for synergetic treatment. Finally, in vivo study demonstrated that 12a, with suitable BBB permeability (log BB = -0.61), could efficaciously ameliorate cognitive dysfunction on scopolamine-treated mice by regulating cholinergic system and oxidative stress simultaneously. Altogether, these results highlight the potential of 12a as an innovative balanced multifunctional candidate for Alzheimer's disease treatment.

Design, synthesis, and in vitro evaluation of 4-aminoalkyl-1(2H)-phthalazinones as potential multifunctional anti-Alzheimer's disease agents.

A series of 4-aminoalkyl-1(2H)-phthalazinone derivatives was designed and synthesized as potential multifunctional agents for Alzheimer's disease (AD) treatment. In vitro biological assay results demonstrated that most synthesized compounds exhibited significant AChE inhibition, moderate to high MAOs inhibitory potencies and good anti-platelet aggregation abilities. Among them, compound 15b exhibited the highest inhibitory potencies towards MAO-B and MAO-A (IC50 = 0.7 µM and 6.4 µM respectively), moderate inhibition towards AChE (IC50 = 8.2 µM), and good activities against self- and Cu2+-induced Aß1-42 aggregation and platelet aggregation. Moreover, 15b also displayed antioxidant capacity, neuroprotective potency, anti-neuroinflammation and BBB permeability. These excellent results indicated that compound 15b could be worthy of further studies to be considered as a promising multifunctional candidate for the treatment of AD.

Pyridoxine-resveratrol hybrids as novel inhibitors of MAO-B with antioxidant and neuroprotective activities for the treatment of Parkinson's disease.

A series of pyridoxine-resveratrol hybrids were designed and synthesized as monoamine oxidase B inhibitors for the treatment of Parkinson's disease. Most of them exhibited potent inhibitory activities on MAO-B with high selectivity. Specifically, compounds 12a, 12g and 12l showed the most excellent inhibition to hMAO-B with the IC50 values of 0.01 µM, 0.01 µM and 0.02 µM, respectively. Further reversibility study demonstrated that 12a and 12l were reversible and 12g was irreversible MAO-B inhibitors. Molecular docking studies of MAO revealed the binding mode and high selectivity of these compounds with MAO-B. In addition, these three representative compounds also exhibited low cytotoxicity and excellent neuroprotective effect in the test on H2O2-induced PC-12 cell injury. Moreover, 12a, 12g and 12l showed good antioxidant activities and high blood-brain barrier permeability. Overall, all of these results highlighted 12a, 12g and 12l were potential and excellent MAO-B inhibitors for PD treatment.

[Acupoint embedding for female functional constipation:a randomized controlled trial].

OBJECTIVE: To compare the effects between acupoint embedding and oral medication for female functional constipation. METHODS: Fifty-six female patients were randomized into an embedding group and a medication group (1:1).There were 22 cases in the embedding group and 26 cases in the medication group with total 8 patients dropped out. The main embedding acupoints were Zhongwan (CV 12), bilateral Tianshu (ST 25) and Guanyuan (CV 4), combined with Xiawan (CV 10), Huaroumen (ST 24), Wailing (ST 26), Zhigou (TE 6) and Shangjuxu (ST 37). It was given once a week. Oral lactulose was applied in the medication group, 3 times a day. The treatment cycle was 8 weeks. The primary outcome was the percentage of patients whose weekly average number of complete spontaneous bowel movement (CSBM) was increased ≥ 2 compared with baseline during the last 6 weeks in the treatment period. The secondary efficacy indices were compared before and after treatment as well as at follow-up at the 12th week, including the weekly average number and increased number of CSBM, the quality of life of patients with constipation (PAC-QOL), the Bristol stool character score, and the assessment of difficulty in defecation. RESULTS: The percentage of patients whose weekly average number of CSBMs were increased ≥ 2 in the embedding group significantly increased compared with that in the medication group (P<0.05). The weekly average number of CSBM in the two groups increased after treatment compared with those before treatment (both P<0.01); the score of Bristol stool character improved (both P<0.01); the scores of PAC-QOL and difficulty in defecation decreased (all P<0.01). The increasing time of weekly average CSBM in the embedding group was higher than that in the medication group (P<0.01); the score of PAC-QOL in the embedding group after treatment was better than that in the medication group (P<0.01); the Bristol stool character and difficulty degree in the embedding group after treatment were superior to those in the medication group (both P<0.05). CONCLUSIONS: The acupoint embedding significantly improve the CSMB, Bristol stool character, the difficulty of defecation, and the quality of life for female patients with functional constipation.