Discovery and validation of COX2 as a target of flavonoids in Apocyni Veneti Folium: Implications for the treatment of liver injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Apocyni Veneti Folium (AVF), a popular traditional Chinese medicine (TCM), is known for its effects in soothing the liver and nerves and eliminating heat and water. It is relevant from an ethnopharmacological perspective. Pharmacological research has confirmed its benefits on antihypertension, antihyperlipidemia, antidepression, liver protection, immune system boosting, antiaging, and diabetic vascular lesions. Previous studies have shown that flavonoids, the active ingredients, have a hepatoprotective effect. However, the exact mechanism has not been clarified. AIM OF THE STUDY: This study aimed to identify the active flavonoids in AVF and their corresponding targets for liver injury. Multiple methods were introduced to confirm the targets. MATERIAL AND METHODS: AVF compounds were analyzed using liquid chromatography-mass spectrometry (LC-MS). Then, network pharmacology was utilized to screen potential hepatoprotection targets of the compounds. An enzyme activity assay was performed to determine the effect of the compounds on the targets. Biolayer interferometry (BLI) was applied to confirm the direct interaction between the compounds and the targets. RESULTS: A total of 71 compounds were identified by LC-MS and 19 compounds and 112 shared targets were screened using network pharmacology. These common targets were primarily involved in the TNF signaling pathway, cancer pathways, hepatitis B, drug responses, and negative regulation of the apoptotic process. Flavonoids were the primary pharmacological substance basis of AVF. The cyclooxygenase 2 (COX2) protein was one of the direct targets of flavonoids in AVF. The enzyme activity assay and BLI-based intermolecular interactions demonstrated that the compounds astragalin, isoquercitrin, and hyperoside exhibited stronger inhibition of enzyme activity and a higher affinity with COX2 compared to epigallocatechin, quercetin, and catechin. CONCLUSIONS: COX2 was preliminarily identified as a target of flavonoids, and the mechanism of the hepatoprotective effect of AVF might be linked to flavonoids inhibiting the activity of COX2. The findings can establish the foundation for future research on the traditional hepatoprotective effect of AVF on the liver and for clinical studies on liver disorders.

Reshaping the Cortical Connectivity Gradient by Long-Term Cognitive Training During Development.

The organization of the brain follows a topological hierarchy that changes dynamically during development. However, it remains unknown whether and how cognitive training administered over multiple years during development can modify this hierarchical topology. By measuring the brain and behavior of school children who had carried out abacus-based mental calculation (AMC) training for five years (starting from 7 years to 12 years old) in pre-training and post-training, we revealed the reshaping effect of long-term AMC intervention during development on the brain hierarchical topology. We observed the development-induced emergence of the default network, AMC training-promoted shifting, and regional changes in cortical gradients. Moreover, the training-induced gradient changes were located in visual and somatomotor areas in association with the visuospatial/motor-imagery strategy. We found that gradient-based features can predict the math ability within groups. Our findings provide novel insights into the dynamic nature of network recruitment impacted by long-term cognitive training during development.

Protective Effect of 20(S)-Protopanaxadiol on D-Gal-Induced Cognitively Impaired Mice Based on Its Target Protein Brain-type Creatine Kinase.

Ginseng is an important medicinal herb consumed as dietary supplements. Ginsenosides and their metabolites have been reported to enhance cognitive performance, but their underlying mechanisms remain unclear. Brain-type creatine kinase (CK-BB) was previously screened out as one of the potential targets in brain tissues. In vitro, the strongest direct interaction between 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, and CK-BB was detected using biolayer interferometry (BLI). Drug affinity responsive target stability, cellular thermal shift assay, BLI, and isothermal titration calorimetry were subsequently used, and the binding of PPD to CK-BB was verified. The binding sites of the CK-BB/PPD complex were clarified by molecular docking and site-directed mutagenesis. Enzyme activity assay showed that the binding of PPD to CK-BB in vitro enhanced its activity. In vivo, PPD increased CK-BB activity in D-gal-induced mice. PPD also improved the D-gal-induced cognitive deficits and ameliorated alterations in oxidative stress and hippocampal synaptic plasticity. Therefore, the integration of PPD with its target protein CK-BB may promote CK-BB activity, thereby ameliorating hippocampal synaptic plasticity and cognitive deficits in D-gal-treated mice.

ccTCM: A quantitative component and compound platform for promoting the research of traditional Chinese medicine.

Traditional Chinese medicine (TCM) databases play a vital role in bridging the gap between TCM and modern medicine, as well as in promoting the popularity of TCM. Elucidating the bioactive ingredients of Chinese medicinal materials is key to TCM modernization and new drug discovery. However, one drawback of current TCM databases is the lack of quantitative data on the constituents of Chinese medicinal materials. Herein, we present ccTCM, a web-based platform designed to provide a component and compound-content-based resource on TCM and analysis services for medical experts. In terms of design features, ccTCM combines resource distribution, similarity analysis, and molecular-mechanism analysis to accelerate the discovery of bioactive ingredients in TCM. ccTCM contains 273 Chinese medicinal materials commonly used in clinical settings, covering 29 functional classifications. By searching and comparing, we finally adopted 2043 studies, from which we collected the compounds contained in each TCM with content greater than 0.001 %, and a total of 1449 were extracted. Subsequently, we collected 40,767 compound-target pairs by integrating multiple databases. Taken together, ccTCM is a versatile platform that can be used by TCM scientists to perform scientific and clinical TCM studies based on quantified ingredients of Chinese medicinal materials. ccTCM is freely accessible at http://www.cctcm.org.cn.

[Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology].

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

Identification of Adenylate Kinase 5 as a Protein Target of Ginsenosides in Brain Tissues Using Mass Spectrometry-Based Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CETSA) Techniques.

Ginseng is a very famous Chinese herbal medicine with various pharmacological effects. Ginsenosides, the main effective compounds of ginseng, show favorable biological activities in the central nervous system (CNS), but the protein targets of ginsenosides in brain tissues have not been clarified clearly. First, we screened proteins that interact with ginsenosides by mass spectrometry-based drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). Then, we identified and confirmed adenylate kinase 5 (AK5) as a target protein of ginsenosides by biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and molecular docking. Finally, an enzyme activity kit was used to determine the effect of 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, on AK5 activities in vivo and in vitro. We screened out seven overlapping target proteins by proteomics of DARTS and CETSA. The BLI direct action assays showed that the direct interaction of PPD with AK5 was higher compared to the parental ginsenosides. Subsequently, BLI kinetic analysis and ITC assay showed that PPD specifically bound to AK5. Furthermore, key amino acid mutations predicted by molecular docking decreased the affinity between PPD and AK5. Enzyme activity assays showed that PPD increased AK5 activities in vivo and in vitro. The above-mentioned findings indicated that AK5 is a protein target of ginsenoside in the brain and PPD is considered to be a small-molecular activator of AK5, which can improve comprehension of the molecular mechanisms of ginseng pharmacological effects in the CNS and further develop AK5 activators based on the dammarane-type triterpenoid structure.

A strategy for quality evaluation of salt-treated Apocyni Veneti Folium and discovery of efficacy-associated markers by fingerprint-activity relationship modeling.

In this study, a fingerprint-activity relationship between chemical fingerprints and hepatoprotective activity was established to evaluate the quality of salt-treated Apocyni Veneti Folium (AVF). Characteristic fingerprints of AVF samples exposed to different concentrations of salt were generated by ultrafast liquid chromatography tandem triple time-of-flight mass/mass spectrometry (UFLC-Triple TOF-MS/MS), and a similarity analysis was performed based on common characteristic peaks by hierarchical clustering analysis (HCA). Then, the hepatoprotective activity of AVF against CCl4-induced acute liver damage in mice was investigated by assessing biochemical markers and histopathology, which showed that a high dose of AVF exposed to low levels of salt stress produced a marked amelioration of hepatic damage compared with the other salt-treated AVF. Finally, fingerprint-activity relationship modeling, which was capable of discovering the bioactive markers used in the quality evaluation, was investigated by the chemical fingerprints and the hepatoprotective activities utilizing multivariate statistical analysis, gray correlation analysis (GCA) and bivariate correlation analysis (BCA). The results showed that the accumulation of polyphenols, such as flavonoids and phenolic acids, in AVF subjected to low levels of salt stress could result in the effective scavenging of free radicals. Therefore, the present study may provide a powerful strategy to holistically evaluate the quality of salt-treated AVF in combination with chemical fingerprint and bioactivity evaluation.

Water extract of ginseng and astragalus regulates macrophage polarization and synergistically enhances DDP's anticancer effect.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, supplementing Qi and strengthening body resistance are an important principle of anticancer treatment. Panax ginseng C.A.Mey. (ginseng) and Astragalus membranaceus Bunge (astragalus) are the representative herbs for this therapeutic principle. AIM OF THE STUDY: This study aims to explore the effect of the water extract of ginseng and astragalus (WEGA) on regulating macrophage polarization and mediating anticancer in the tumor microenvironment. MATERIALS AND METHODS: A549 cells were cultured in tumor-associated macrophage (TAM) supernatant with various concentrations of WEGA (0, 5, 10, 20 mg/mL). A549 cell proliferation was determined through methyl thiazole tetrazolium (MTT) assay and real-time cell analysis (RTCA), respectively. In vivo experiments were performed with a Lewis lung cancer (LLC) xenograft mouse model. Forty-eight mice were divided into six groups and treated with saline, WEGA, or cis-diamine dichloro platinum (DDP) with dosage of WEGA (0, 30, 60, 120 mg/kg body weight/day). The different groups were administered with drugs via oral or intraperitoneal injection once a day for 21 consecutive days. Tumor inhibition rate, spleen index, thymus index, cytokine, protein, and mRNA expression levels were detected in mice. RESULTS: In a co-culture system, WEGA remarkably inhibited A549 cell proliferation, promoted the expression of M1 macrophage markers and inhibited M2 TAMs markers. Therefore, WEGA affected the biological behavior of cancer cells by regulating the expression of some markers relevant to macrophage polarization. In addition, the group of WEGA and DDP chemotherapy effectively inhibited the transplanted tumor growth in mice and improved weight loss and immunosuppressive with the cisplatin inducing. CONCLUSIONS: This study provides mechanistic insights into the anticancer effect of WEGA through the regulation of macrophage polarization and highlights that WEGA could be a novel option for integrative cancer therapies.

[Identification of multidrug resistance gene MDR1 associated microRNA of salvianolic acid A reversal in lung cancer].

This paper was aimed to investigate the microRNA associated with multidrug resistance gene MDR1 of salvianolic acid A reversal in lung cance. Human lung cancer A549 cells were divided into normal control group and drug group, and the MDR1 expression levels were determined by real-time quantitative PCR. MicroRNA expression profiling of normal control group and drug group were detected by using the latest microRNA microarray. Quantitative RT-PCR was used to validate the differentially expressed miRNA. Forecast of miRNA associated with MDR1 multi-resistant genes of up-regulated miRNA. Experimental results showed that the dosage of MDR1 expression level significantly lowered compared with control group. The miRNA expression spectrum analyses of human lung cancer A549 cells to drug group and the control group were detected by microRNA microarray, 426 differentially expressed miRNA were screened out. Then target prediction were performed for difference up-expression of miRNA and found that there were four obvious increase of miRNA associated with MDR1 multi-resistant genes. Real-time quantitative PCR for 4 microRNA verification, the results were consistent with the chip. So the author considered that salvianolic acid A down lung cancer multidrug resistance gene MDR1 is likely to be affected by the miRNA expression and regulation of target genes, to further clarify the traditional Chinese medicine to reverse multi-drug resistant mechanism provides the experimental basis.

Pycnogenol(®) treatment inhibits bone mineral density loss and trabecular deterioration in ovariectomized rats.

CONTEXT: Pycnogenol(®) extracted from French maritime pine bark (Pinus pinaster Ait. subsp. atlantica) is functional for its antioxidant activity. OBJECTIVE: To investigate the effects of Pycnogenol(®) on bone mineral density (BMD), trabecular microarchitecture and bone metabolism in ovariectomized (OVX) rats. MATERIALS AND METHODS: Thirty Sprague-Dawley rats were randomized into 3 groups: SHAM group (sham-operated rats), OVX group (OVX rats), and treatment group (OVX rats supplemented with 40 mg/kg Pycnogenol(®) by oral gavage). Serum levels of procollagen type I N-terminal propeptide (PINP), alkaline phosphatase (ALP) and minerals were detected at the end of 9 weeks of gavage. Deoxypyridinoline/creatinine (DPYD/Cr) and N-telopeptide of type I collagen/creatinine (NTX/Cr) rate in urine were also calculated. Left femora were collected for BMD determination, and the right distal femora were made into undecalcified specimens for histomorphometry analysis. RESULTS: At the end of study, PINP level, DPYD/Cr and NTX/Cr rate were significantly increased, and femoral BMD were dramatically decreased in OVX group compared with SHAM group (P < 0.01) while serum minerals and ALP concentrations showed no significant difference. The treatment group had dramatically decreased biomarkers and increased BMD than OVX group (P < 0.01). Histomorphometry analysis showed worse bone microarchitecture parameters in the OVX group compared with the SHAM group which were significantly improved in the treatment group compared with the OVX group (P < 0.01). DISCUSSION AND CONCLUSION: Pycnogenol(®) (40 mg/kg) can inhibit aggravated bone resorption, prevent BMD loss, and restore the impaired trabecular microarchitecture in OVX rats after 9-week-intervention.

Are scalar implicatures automatically processed and different for each individual? A mismatch negativity (MMN) study.

Scalar implicatures are ordinarily activated in human communication when the speaker uses a weak expression (e.g., some) from a set of stronger alternatives (e.g., many, all). It has been debated whether scalar inferences are generated by default. To clarify this issue and examine whether individual pragmatic ability will affect the mechanism of scalar inference processing, we performed experiment with an MMN paradigm to capture the neurophysiological indicators of automatic processing of spoken sentences and divided participants into high and low pragmatic ability groups. Experimental results showed that compared with the condition that an informative sentence ("Some animals have tails") is the deviant stimuli, when an underinformative sentence ("Some tigers have tails") is the deviant stimuli, the high pragmatic ability group induced mismatch negativity (MMN) and sustained negativity, while the low pragmatic ability group had no ERP effects. These results indicated that at least some people can automatically activate the scalar implicatures when encountering scalar trigger words, even in the inattentive status.

The neural pathway underlying a numerical working memory task in abacus-trained children and associated functional connectivity in the resting brain.

Training can induce significant changes in brain functioning and behavioral performance. One consequence of training is changing the pattern of brain activation. Abacus training is of interest because abacus experts gain the ability to handle digits with unusual speed and accuracy. However, the neural correlates of numerical memory in abacus-trained children remain unknown. In the current study, we aimed to detect a training effect of abacus-based mental calculations on numerical working memory in children. We measured brain functional magnetic resonance imaging (fMRI) activation patterns in 17 abacus-trained children and 17 control children as they performed two numerical working memory tasks (digits and beads). Functional MRI results revealed higher activation in abacus-trained children than in the controls in the right posterior superior parietal lobule/superior occipital gyrus (PSPL/SOG) and the right supplementary motor area (SMA) in both tasks. When these regions were used as seeds in a functional connectivity analysis of the resting brain, the abacus-trained children showed significantly enhanced integration between the right SMA and the right inferior frontal gyrus (IFG). The IFG is considered to be the key region for the control of attention. These findings demonstrate that extensive engagement of the fronto-parietal network occurs during numerical memory tasks in the abacus-trained group. Furthermore, abacus training may increase the functional integration of visuospatial-attention circuitry, which and thus enhances high-level cognitive process.

Neural correlates of serial abacus mental calculation in children: a functional MRI study.

Abacus experts have demonstrated extraordinary potential of mental calculation by using an imaginary abacus. But the neural correlates of abacus mental calculation and the imaginary abacus still remain unclear. Here, we report, respectively, the analysis of fMRI images of abacus experts and non-experts in response to the performance of simple and complex serial calculation by visual stimuli as well as the images of the abacus experts with performance of the same tasks by auditory stimuli. We found that activated areas were quite different between two groups. In experts, enhanced activations were mainly observed in fronto-temporal circuit (lateral premotor cortex (LPMC) and posterior temporal areas) in simple addition, but in fronto-parietal circuit (lateral premotor cortex (LPMC) and posterior superior parietal lobe (PSPL)) in complex one. By contrast, in controls, the activated areas were almost similar in both simple and complex tasks, including bilateral inferior parietal lobule, prefrontal and premotor cortices. Furthermore, visual and auditory stimuli generated almost similar activations in experts. These observations reveal that (1) abacus mental calculation induces special patterns of brain response, and simple and complex tasks are sustained by dissociated brain circuits between the temporal and parietal cortices, respectively; (2) the abacus mental calculation may rely on neural resources of visuospatial representations with a super-modal form of abacus beads; (3) the posterior temporal areas and PSPL may be recruited for imaginary abacus.