[Therapeutic effect and mechanism of Jingfang Granules on acute lung injury based on intestinal flora and fecal metabolomics].

This study aims to elucidate the therapeutic effect and mechanism of Jingfang Granules on acute lung injury, and to investigate the regulatory effect of Jingfang Granules on the metabolic disorders of endogenous metabolites in feces and the homeostasis of intestinal microbiota in acute lung injury, mice were randomly divided into a sham group, a model group, and a Jingfang Granules group. After modeling, the mice were continuously administered for 6 days. Using ultra-high performance liquid chromatography quadrupole/electrostatic field orbital trap high-resolution mass spectrometry(UHPLC-HESI-QE-Orbitrap-MS/MS) metabolomics technology and 16S rRNA high-throughput sequencing technology, changes in endogenous small molecule substances and gut microbiota in mouse intestines were determined, and potential biomarkers were identified. The results showed that Jingfang Granules can regulate 11 biomarkers, including L-glutamic acid, succinic acid, arachidonic acid, linoleic acid, linolenic acid, phenylalanine, sphingosine, 2-hydroxy-2-methyl butyric acid, pyruvate, tryptophan, and palmitic acid. Metabolic pathway analysis was conducted on these 11 biomarkers using the online software MetaboAnalyst, identifying potential major metabolic pathways. Among them, a total of 10 metabolic pathways are closely related to the treatment of acute lung injury with Jingfang Granules, including alanine, aspartate and glutamate metabolism, aminoacyl-tRNA biosynthesis, citrate cycle(TCA cycle), alyoxylate and dicarboxylate metabolism, arginine and proline metabolism, linoleic acid metabolism and linolenic acid metabolism, nitrogen metabolism, D-glutamine and D-gluta-matemetabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism. The results of gut microbiota showed significant differences in bacteria, mainly including Bacteroides, Akkermansia, Lachnospiraceae＿NK4A136＿group, Lachnochlostridium, and Klebsiella. Spearman analysis confirms that Akkermansia and Lachnospiraceae＿NK4A136＿group is a significant positive correlation between the abundance of succinic acid, arachidonic acid, linolenic acid, linoleic acid, butyric acid, and pyruvate in the group; Bacteroides, Klebsiella, Lachnochlostrium are significantly positively correlated with the abundance of L-glutamic acid, phenylalanine, and sphingosine. The above results indicate that the therapeutic effect of Jingfang Granules on acute lung injury is achieved by improving the imbalance of gut microbiota in mice with acute lung injury, balancing the metabolism of alanine, biosynthesis of aminoacyl tRNA, aspartic acid, glutamate, tricarboxylic acid cycle, biosynthesis of phenylalanine, tyrosine, tryptophan, and metabolism of linoleic acid.

[Therapeutic effect and mechanism of Mailuo Shutong Pills on posterior limb swelling caused by femur fracture in rats based on intestinal flora and intestinal metabolism].

This study aimed to investigate the protective effect and underlying mechanism of Mailuo Shutong Pills(MLST) on posterior limb swelling caused by femur fracture in rats. The rats were randomly divided into a sham operation group, a model group, a low-dose MLST group(1.8 g·kg~(-1)·d~(-1)), a high-dose MLST group(3.6 g·kg~(-1)·d~(-1)), and a positive drug group(60 mg·kg~(-1)·d~(-1) Maizhiling Tablets). The femur in the sham operation group was exposed and the wound was sutured, while the other four groups underwent mechanical damage to cause femur fracture. The rats were treated with corresponding drugs by gavage 7 days before modeling and 5 days after modeling, while those in the sham operation group and the model group were given an equivalent dose of distilled water by gavage. Hematoxylin-eosin(HE) staining was used to detect the pathological injury of the posterior limb muscle tissues in rats, and the degree of hind limb swelling was measured. The enzyme-linked immunosorbent assay(ELISA) kit was used to detect the expression levels of interleukin-6(IL-6), interleukin-1ß(IL-1ß), and tumor necrosis factor-α(TNF-α) in the serum of rats in each group. The activity of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and glutathione peroxidase(GSH-Px) in rat serum was also measured. Western blot was used to detect the protein expression levels of heme oxygenase 1(HO-1), NAD(P)H quinone oxidoreductase 1(NQO1), and nuclear transcription factor E2-related factor 2(Nrf2) in rat posterior limb muscle tissues. The changes in the intestinal flora and intestinal metabolites in rats were detected by 16S rDNA sequencing and ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), respectively, to explore the underlying mechanism of MLST in treating posterior limb swelling caused by femur fracture in rats. Compared with the model group, MLST significantly improved the degree of posterior limb swelling in rats, reduced the levels of serum inflammatory factors, and alleviated oxidative stress injury. The HE staining results showed that the inflammatory infiltration in the posterior limb muscle tissues of rats in the MLST groups was significantly improved. Western blot results showed that MLST significantly increased the protein expression of HO-1, NQO1, and Nrf2 in rat posterior limb muscle tissues compared with the model group. The 16S rDNA sequencing results showed that MLST improved the disorder of intestinal flora in rats after femur fracture. The UPLC-MS/MS results showed that MLST significantly affected the bile acid biosynthesis and metabolism pathway in the intestine after femur fracture, and the Spearman analysis confirmed that the metabolite deoxycholic acid involved in bile acid biosynthesis was positively correlated with the abundance of Turicibacter. The metabolite cholic acid was positively correlated with the abundance of Papilibacter, Staphylococcus, and Intestinimonas. The metabolite lithocholic acid was positively correlated with Papilibacter and Intestinimonas. The above results indicated that MLST could protect against the posterior limb swelling caused by femur fracture in rats. This protective effect may be achieved by improving the pathological injury of the posterior limb muscle, reducing the expression levels of inflammatory and oxidative stress-related factors in serum, reducing the oxidative injury of the posterior limb muscle, improving intestinal flora, and balancing the biosynthesis of bile acids in the intestine.

[Potentiating effect and mechanism of extract of Jingfang Granules on activation of macrophages].

This study aimed to explore the potentiating effect and mechanism of the extract of Jingfang Granules(JFG) on the activation of macrophages. The RAW264.7 cells were treated with JFG extract and then stimulated by multiple agents. Subsequently, mRNA was extracted, and reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the mRNA transcription of multiple cytokines in RAW264.7 cells. The levels of cytokines in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the intracellular proteins were extracted and the activation of signaling pathways was determined by Western blot. The results showed that JFG extract alone could not promote or slightly promote the mRNA transcription of TNF-α, IL-6, IL-1ß, MIP-1α, MCP-1, CCL5, IP-10, and IFN-ß, and significantly enhance the mRNA transcription of these cytokines in RAW264.7 cells induced by R848 and CpG in a dose-dependent manner. Furthermore, JFG extract also potentiated the secretion of TNF-α, IL-6, MCP-1, and IFN-ß by RAW264.7 cells stimulated with R848 and CpG. As revealed by mechanism analysis, JFG extract enhanced the phosphorylation of p38, ERK1/2, IRF3, STAT1, and STAT3 in RAW264.7 cells induced by CpG. The findings of this study indicate that JFG extract can selectively potentiate the activation of macrophages induced by R848 and CpG, which may be attributed to the promotion of the activation of MAPKs, IRF3, and STAT1/3 signaling pathways.

[Anti-infectious pneumonia target discovery and molecular mechanism study of Jingfang Granules].

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via "target fishing" strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.

[Anti-depression targets and mechanism study of Kaixin San].

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

[Intervention effect of Jingfang Mixture on urticaria mice based on NF-κB/NLRP3/IL-1ß signaling pathway].

This study explored the curative effect of Jingfang Mixture on urticaria mice induced by aluminum hydroxide/ovalbumin, and discussed its mechanism. Sixty male Kunming mice were randomly divided into a normal group, a model group, three Jingfang Mixture(low-dose, medium-dose, and high-dose) groups, and a positive drug(cetirizine hydrochloride) group. The urticarial model in mice was induced by the intraperitoneal injection of the mixed solution of ovalbumin and aluminum hydroxide. The degrees of pruritus were observed after the second immunization. Pathological changes were detected by hematoxylin and eosin(HE) staining. Levels of interleukin 1ß(IL-1ß) and tumor necrosis factor α(TNF-α) in the serum were detected by enzyme linked immunosorbent assay(ELISA). Expressions of NOD-like receptor protein 3(NLRP3) and IL-1ß were detected by immunohistochemistry(IHC). Expressions of nuclear factor kappa-B(NF-κB p65), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate-specific proteases 1(caspase-1), and IL-1ß proteins were detected by Western blot. The results showed that, except for the normal group, the mice in all groups had different degrees of pruritus. Compared with the model group, the Jingfang Mixture groups and the positive drug group prolonged the scratching latency of mice(P<0.05), and significantly reduced the number of scratching(P<0.05). In addition, the Jingfang Mixture groups and the positive drug group improved the pathological morphology of skin tissue. The expression levels of IL-1ß and TNF-α in serum were significantly reduced(P<0.05), and the number of NLRP3 and IL-1ß positive cells was decreased(P<0.01). The expressions of p-NF-κB p65, NLRP3, ASC, cleaved caspase-1, and IL-1ß protein were significantly down-regulated(P<0.05). The results of the above study indicate that Jingfang Mixture inhibit the inflammatory response in urticaria mice, and the mechanism may be related to the inhibition of activating NF-κB/NLRP3/IL-1ß signaling pathway.

[Anti-inflammatory mechanism of active ingredients in Jingfang Mixture based on network pharmacology and experimental verification].

The present study aimed to explore the regulatory targets and anti-inflammatory mechanism of Jingfang Mixture based on network pharmacology and animal tests. The active ingredients of Jingfang Mixture and the corresponding targets were screened out by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Inflammation-related targets were searched from GeneCards and DisGeNET, and the targets of active ingredients of Jingfang Mixture against inflammation were obtained. The protein-protein interaction(PPI) network was analyzed by STRING and plotted. Gene ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were carried out based on DAVID. The results of network pharmacology showed 159 active ingredients and 276 targets of Jingfang Mixture and 664 inflammation-related targets were screened out, and 90 targets of active ingredients of Jingfang Mixture against inflammation were obtained. As revealed by the PPI network, protein kinase B1(AKT1), caspase-3(CASP3), interleukin-1ß(IL1 B), prostaglandin-endoperoxide synthase 2(PTGS2), and tumor necrosis factor(TNF) might be the key proteins for the anti-inflammatory effect of Jingfang Mixture. KEGG enrichment analysis demonstrated the pathways involved TNF, nuclear factor-kappa B(NF-κB), and mitogen-activated protein kinase(MAPK). The anti-inflammatory effect of Jingfang Mixture was explored through the mouse model of urticaria. The results indicated that Jingfang Mixture could down-regulate the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases(ERK1/2), and NF-κB. The present study revealed the anti-inflammatory effect of Jingfang Mixture with multi-component and multi-target characteristics, which is expected to provide a scientific basis and important support for further research, development, and application.

[Proteomic study of Jingfang Mixture on urticaria based on label-free quantitative proteomics technology].

This study aims to explore the effect of Jingfang Mixture on the protein expression of urticaria in mice and explain the mechanism of Jingfang Mixture in the treatment of urticaria. Twenty-seven male Kunming mice were randomly divided into a normal group, a model group and a Jingfang Mixture group according to body weight. Except for the normal group, mice in the model group and the Jingfang Mixture group were injected with the mixture of ovalbumin and Al(OH)_3 gel for the first immunization, and the second immunization was performed on the 10 th day to induce the urticaria model. Mice in the Jingfang Mixture group started to be administered on the 6 th day after the initial immunization, and was administered continuously for 21 days. The normal group and the model group were replaced with the same amount of purified water. Twenty-four hours after the last administration, an appropriate amount of skin was taken, and label-free quantitative proteomics technology was used to detect the differences in protein expression in skin tissue. The signaling pathways involved in the differential proteins was further analyzed. The results of proteomics indicated that seventy-six proteins were involved in the intervention of Jingfang Mixture on mice with urticaria, and the differential proteins were mainly enriched in biological process(BP), molecular function(MF), and cellular component(CC). Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis showed that the signaling pathways regulated by Jingfang Mixture mainly involved carbon metabolism, metabolic pathways, glucagon signaling pathway, glycolysis/gluconeogenesis, pentose phosphate pathway, hypoxia inducible factor-1(HIF-1) signaling pathway, purine metabolism, adherens junction, calcium signaling pathway, leukocyte transendothelial migration, and inflammatory mediator regulation of transient receptor potential(TRP) channels, which were involved in skin tissue energy metabolism and immune regulation. The findings of this study showed that the protective effect of Jingfang Mixture on mice with urticaria was closely related to the regulation of immune disorders, and the regulatory effect on immune system may be achieved through the regulation of energy metabolism by Jingfang Mixture.

[Anti-pneumonia targets and mechanism of Xiaoer Xiaoji Zhike Oral Liquid].

This study aims to identify the anti-pneumonia targets of Xiaoer Xiaoji Zhike Oral Liquid(XXZL) with "target fishing" strategy and investigate the related signaling pathways, thereby clarifying the anti-pneumonia mechanism of XXZL. To be specific, the magnetic nanoparticles cross-linked with XXZL extract were prepared based on the photochemical activity of benzophenone, which were then used to capture the target proteins from the lysate of tissue with lipopolysaccharide(LPS)-induced pneumonia in mice. Then, the target proteins were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS). The signaling pathways and interactions of target proteins were explored with KEGG and STRING analysis on Cytoscape, and the possible biological functions of the target proteins were verified by immunohistochemistry(IHC) and RT-PCR. The result showed that LC-MS/MS identified 62 potential anti-pneumonia targets of XXZL in the lungs. The targets were involved in Ras signaling pathway, mitophagy, leukocyte transendothelial migration, mitogen-activated protein kinase(MAPK) signaling pathway, platelet activation, and actomyosin structure organization, which were closely related to inflammation, pulmonary microcirculation, pulmonary fibrosis, and energy metabolism. XXZL up-regulated the content of CD31, and heat shock protein 60(HSP60) and ATP5 b mRNA expression, down-regulated interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), COL1 A1 content, and alleviated fibrosis, which suggested the obvious effects of XXZL such as anti-inflammation, pulmonary microcirculation improvement, pulmonary fibrosis inhibition, and energy metabolism regulation. This study explained the anti-pneumonia mechanism of XXZL from targets, which can serve as a reference for the clinical application of the prescription.

[Molecular mechanism study of Shouhui Tongbian Capsules on promoting energy metabolism of gastrointestinal stromal cells].

Mechanism study was performed to explore how Shouhui Tongbian Capsules promotes energy metabolism of gastrointestinal stromal cells. In this study, gastrointestinal stromal cells line GIST-882 was used as the model to explore energy metabolism regulation effects of Shouhui Tongbian Capsules extract(10, 20, 50 and 100 µg·mL~(-1)) by measuring the cell proliferation, ATP level, mitochondrial membrane potential, and mitochondrial isocitrate dehydrogenase activity. Meanwhile, Western blot was used to detect the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Our results showed that Shouhui Tongbian Capsules promoted cell proliferation and increased ATP level of gastrointestinal stromal cells. In addition, Shouhui Tongbian Capsules obviously improved mitochondrial structural integrity, and increased mitochondrial membrane potential in GIST-882 cells. Mechanism study revealed that Shouhui Tongbian Capsules increased mitochondrial isocitrate dehydrogenase activity and up-regulated the proteins expression of SCF/c-Kit and CDK2/cyclin A signaling pathways. Collectively, our study indicated that Shouhui Tongbian Capsules promoted the energy metabolism for gastrointestinal stromal cells proliferation by activating mitochondrial isocitrate dehydrogenase to induce ATP production, as well as activating SCF/c-Kit and CDK2/cyclin A signaling pathways.

[Metabonomics study of Shouhui Tongbian Capsules in slow transit constipation based on UPLC-ESI-QE-Orbitrap-MS].

The effect of Shouhui Tongbian Capsules(SHTB) on the endogenous metabolites of colon tissue in mice with slow transit constipation was analyzed by metabolomics methods to explore its mechanism in the treatment of constipation. ICR mice were randomly divided into normal group, model group and SHTB group according to the body weight. The mice were given diphenoxylate to establish the slow transit constipation model. Mouse carbon ink pushing rate, first defecation time and the number of defecation particles in 12 h were observed. The mouse colon tissue was separated and the mucous cells were detected by Periodic acid Schiff and Alcian blue(AB-PAS) staining. Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry(UPLC-ESI-Orbitrap-MS/MS) technology was used to characterize the differences in tissue metabolism to screen out the potential different metabolites and possible metabolic pathways in colon tissue. The results indicated that SHTB could significantly shorten the first defecation time and the number of defecations, and increase the number of intestinal peristalsis and mucous cells in the colonic mucosa compared to the model mice. Metabolomics results showed that, compared with the normal group, a total of 17 potential biomarkers, including L-kynurenine, N6,N6,N6-trimethyl-L-lysine, L-formylkynurenine, N6-acetyl-L-lysine, L-phenylalanine, phenylacetaldehyde, xanthoxin, thymidine, glycyl-L-leucine, cystathionine,(R)-1-aminopropan-2-ol, deoxycytidine, gamma-glutamyl-gamma-aminobutyraldehyde, D-galactose, L-arginine, L-proline and pyruvate, were found and identified in colon tissue. Treated with SHTB, these metabolic differences tended to return to normal levels. Therefore, it could be made a conclusion that the therapeutic effect of SHTB on chronic transit constipation may be related to regulating phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, arginine and proline metabolism, cysteine and methionine metabolism, tyrosine metabolism, arginine biosynthesis, pyruvate metabolism, glycolysis, pyrimidine metabolism, tricarboxylic acid cycle and galactose metabolism.

Recruitment of γδ T cells to the lesion via the CCL2/CCR2 signaling after spinal cord injury.

BACKGROUND: Immune cell infiltration and neuroinflammation are heavily associated with spinal cord injury (SCI). C-C motif chemokine ligand 2/C-C chemokine receptor type 2 (CCL2/CCR2) axis has been identified as a critical role player during the invasion of immune cells to lesions in many diseases. γδ T cells, a subgroup of T cells, manage the course of inflammation response in various diseases; however, it remains unknown whether γδ T cells are recruited to injury site through CCL2/CCR2 signaling and exert the regulation effect on neuroinflammation after SCI. METHODS: Basso Mouse Scale (BMS), regularity index, cadence, max contact area, and motor-evoked potential testing (MEP) were measured to determine the neurological function recovery after spinal cord injury. Nissl staining was performed to identify the number of surviving motor neurons at lesion epicenter. Immunofluorescence, Western blot, enzyme-linked immunosorbent assays (ELISA), and quantitative real-time polymerase chain reaction (QRT-PCR) also were employed to evaluate the expression of associated proteins and genes. RESULTS: In this study, we demonstrated that TCRδ-/- mice present improved neurological recovery after SCI. γδ T cell recruitment to the SCI site was significantly reduced and motor functional improvement enhanced in CCL2-/- and CCR2-/- mouse strains. Furthermore, reconstitution of TCRδ-/- mice with γδ T cells extracted from CCR2-/- mice also showed similar results to CCL2 and CCR2 deficient mice. CONCLUSIONS: In conclusion, γδ T cell recruitment to SCI site promotes inflammatory response and exacerbates neurological impairment. CCL2/CCR2 signaling is a vital recruitment mechanism of γδ T cells to the SCI site, and it may be taken as a novel therapeutic target for future SCI.

Arctigenin Inhibits Liver Cancer Tumorigenesis by Inhibiting Gankyrin Expression via C/EBPα and PPARα.

Burdock (Arctium lappa) is a popular vegetable in China and Japan that is consumed for its general health benefits. The principal active component of burdock is arctigenin, which shows a range of bioactivities in vivo and in vitro. Here, we investigated the potential anti-tumor effects of arctigenin using two human hepatocellular carcinoma (HCC) cell lines, HepG2 and Hep3B, and sought to elucidate its potential mechanisms of action. Our results showed that arctigenin treatment inhibited cell growth in both HepG2 and Hep3B cell lines (IC50 of 4.74 nM for HepG2 cells, and of 59.27 nM for Hep3B cells). In addition, migration, invasion, and colony formation by HepG2 cells were significantly inhibited by arctigenin. By contrast, treatment of Hep3B cells with arctigenin did not alter these parameters. Arctigenin also significantly reduced the levels of gankyrin mRNA and protein in HepG2 cells, but not in Hep3B cells. A luciferase assay indicated that arctigenin targeted the -450 to -400 region of the gankyrin promoter. This region is also the potential binding site for both C/EBPα and PPARα, as predicted and confirmed by an online software analysis and ChIP assay. Additionally, a co-immunoprecipitation (Co-IP) assay showed that binding between C/EBPα and PPARα was increased in the presence of arctigenin. However, arctigenin did not increase the expression of C/EBPα or PPARα protein. A binding screening assay and liquid chromatography-mass spectrometry (LC-MS) were performed to identify the mechanisms by which arctigenin regulates gankyrin expression. The results suggested that arctigenin could directly increase C/EBPα binding to the gankyrin promoter (-432 to -422 region), but did not affect PPARα binding. Expression of gankyrin, C/EBPα, and PPARα were analyzed in tumor tissues of patients using real-time PCR. Both C/EBPα and PPARα showed negative correlations with gankyrin. In tumor-bearing mice, arctigenin had a significant inhibitory effect on HCC growth. In conclusion, our results suggested that arctigenin could inhibit liver cancer growth by directly recruiting C/EBPα to the gankyrin promoter. PPARα subsequently bound to C/EBPα, and both had a negative regulatory effect on gankyrin expression. This study has identified a new mechanism of action of arctigenin against liver cancer growth.

[Efficient discovery and capturing of nNOS-PSD-95 uncouplers from Trifolium pratense].

Magnetic molecularly imprinted polymers(MMIPs) were prepared with ZL006 as template, acrylamide(AA) as the functional monomer, and acetonitrile as pore-forming agent; then Fourier transform infrared spectroscopy(FT-IR) and scanning electron microscopy(SEM) were used to characterize their forms and structures. Simultaneously, the MMIPs prepared previously were used as sorbents for dispersive magnetic solid phase extraction(DSPE) to capture and identify potential nNOS-PSD-95 uncouplers from extracts of Trifolium pratense and the the activities of the screened compounds were evaluated by the neuroprotective effect and co-immunoprecipitation test. The experiment revealed that the successfully synthesized MMIPs showed good dispersiveness, suitable particle size and good adsorption properties. Formononetin, prunetin and biochanin A were separated and enriched from Trifolium pratense by using the MMIPs as artificial antibodies and finally biochanin A was found to have higher cytoprotective action and uncoupling action according to the neuroprotective effect and co-immunoprecipitation test.

Inhibitory effect of arctigenin on lymphocyte activation stimulated with PMA/ionomycin.

This study investigated the effect of arctigenin (Arc) on the cell activation, cytokines expression, proliferation, and cell-cycle distribution of mouse T lymphocytes. Mouse lymphocytes were prepared from lymph node and treated with Phorbol-12-myristate-13-acetate (PMA)/Ionimycin (Ion) and/or Arc. CD69, CD25, cytokines, proliferation and cell cycle were assayed by flow cytometry. The results showed that, at concentrations of less than 1.00 micromol x L(-1), Arc expressed non-obvious cell damage to cultured lymphocytes, however, it could significantly down-regulate the expression of CD69 and CD25, as well as TNF-alpha, IFN-gamma, IL-2, IL-4, IL-6 and IL-10 on PMA/Ion stimulated lymphocytes. At the same time, Arc could also inhibit the proliferation of PMA/Ion-activated lymphocytes and exhibited lymphocyte G 0/G1 phase cycle arrest. These results suggest that Arc possesses significant anti-inflammatory effects that may be mediated through the regulation of cell activation, cytokines expression and cell proliferation.

[Efficacy comparison of different points combination in the treatment of menopausal insomnia: a randomized controlled trial].

OBJECTIVE: To compare the efficacy of different points combination in the treatment of menopausal insomnia. METHODS: Ninety-six cases of menopausal insomnia were randomized into 3 groups, Xinshu (BL 15), Shenshu (BL 23), Sishencong (EX-HN 1), Shenmen (HT 7), Sanyinjiao (SP 6) were chosen in the restore interaction between the heart and the kidney group (group A, 32 cases); Zhaohai (KI 6), Jiaoxin (KI 8), Shenmai (BL 62), Pucan (BL 61) were chosen in the acupuncturing qiao mai group (group B, 32 cases); auricular Shenmen (TF4) and sensitive spot at the distribution area of auricular vagus nervus were chosen in the ear acupuncture group (group C, 32 cases). Six days made one session and the treatments were finished after 4 courses. The polysomnography (PSG) and Pittsburgh sleep quality index (PSQI) were employed before and after treatment to evaluate the alleviation of insomnia. RESULTS: The parameters of the sleep latency (SL), rapid wave sleep latency (RL) and sleep efficiency (SE) were significantly improved in the three groups, and the differences were statistically significant (P < 0.05, P < 0.01). The SL and awaking time (AT) in group C [SL (401.08 +/- 16.54) min and AT (4.87 +/- 2.64) times] were significantly superior to those in the other two groups [SL (50.36 +/- 18.47) min, (54.87 +/- 20.92) min, AT (5.98 +/- 2.11) times, (6.13 +/- 3.04) times, all P < 0.05]. The S(3+4) (%) in group C was also significantly higher than those in the other two groups (both P < 0.05). It was indicated by PSQI that the sleep quality of group C (0.78 +/- 0.12) was significantly superior to that in group B (1.32 +/- 0.29), the total score and cured and markedly effective rate in group C [(4.34 +/- 1.43), 68.8% (22/32)] were superior to those in group A [(7.48 +/- 3.09), 53.1% (17/32), both P < 0.05]. CONCLUSION: Ear acupuncture has a better curative effect than the restore interaction between the heart and the kidney group and acupuncturing qiao mai group, it is worth of being promoted.

[Change characteristics of soil moisture and nutrients in rain-fed winter wheat field under different fertilization modes in Southern Shanxi of China during summer fallow period].

In 2009-2011, a field experiment was conducted in a rain-fed winter wheat field in Southern Shanxi of China to study the effects of different fertilization modes on the change characteristics of soil moisture and nitrate-N contents in 0-200 cm layer and of soil available phosphorus (Oslen-P) and potassium contents in 0-40 cm layer during summer fallow period (from June to September). Three fertilization modes were installed, i. e., conventional fertilization (CF), recommended fertilization (RF), and ridge film furrow planting (RFFP) combined with straw mulch. The results showed that the rainfall in summer fallow period could complement the consumed water in 0-200 cm soil layer in dryland wheat field throughout the growth season, and more than 94% of the water storage was in 0-140 cm soil layer, with the fallow efficiency ranged from 6% to 27%. The rainfall in summer fallow period caused the soil nitrate-N moving downward. 357-400 mm rainfall could make the soil nitrate-N leaching down to 100 cm soil layer, with the peak in 20-40 cm soil layer. Straw mulching or plastic film with straw mulch in summer fallow period could effectively increase the Oslen-P and available K contents in 0-40 cm soil layer, and the accumulative increment in three summer fallow periods was 16-45% and 36-49%, respectively. Among the three modes, the binary coverage mode of RFFP plus furrow straw mulching had the best effect in maintaining soil water and fertility. The accumulative water storage and mineral N in 0-200 cm soil layer in three summer fallow periods were up to 215 mm and 90 kg x hm(-2), and the accumulative Oslen-P and available K contents in plough layer were increased by 2.7 mg x kg(-1) and 83 mg x kg(-1), respectively, being significantly higher than those in treatments CF and RF. There were no significant differences in the change characteristics in the soil moisture and nutrients between treatments CF and RF.

[Treatment of peripheral facial paralysis with acupuncture at Renying (ST 9) mainly cooperated with stellate ganglion block: a randomized controlled trial].

OBJECTIVE: To explore the better therapy for peripheral facial paralysis. METHODS: One hundred and twenty patients were randomized into three groups: a common acupuncture group: acupuncture at Yangbai (GB 14), Sibai (ST 2) and Yingxiang (LI 20) as main acupoints, a ST 9 group: acupuncture at Renying (ST 9) as main and a ST 9 plus SGB group: acupuncture at Renying (ST 9) as main cooperated with stellate ganglion block (SGB). Once daily, 7 treatments made one session. After three sessions of treatment, the latency period and amplitude of evoked potential in ENoG, R1 value and R2 value of blink reflex were compared before and after the treatment in different groups separately. The total therapeutic effect was evaluated after treatment. RESULTS: All the treatments shortened the latency period of ENoG, and elevated the amplitude evoked potential significantly. After treatment, the latency period in ST 9 plus SGB group was reduced significantly as compared with common acupuncture group (P < 0.05). The amplitude of evoked potential in ST 9 group was increased significantly as compared with the other two groups (both P < 0.05). After treatment, in each group, R1 and R2 values were shortened significantly. The difference values of R1 and R2 in ST 9 group and ST 9 plus SGB group were all significantly higher as compared with common acupuncture group (both P < 0.05). Additionally, the difference value of R1 in ST 9 plus SGB group was higher significantly than that in ST 9 group (P < 0.05). The clinical cured and remarkably effective rate was 87.5% (35/40) in ST9 plus SGB group, which was higher than 77.5% (31/40) in ST 9 group, and 65.0% (26/40) in common acupuncture group (P < 0.05). CONCLUSION: As compared with common acupuncture group, ST 9 group and ST 9 plus SGB group achieve the much superior efficacy on peripheral facial paralysis. The treatment with ST 9 acupuncture and SGB can better repair the early reflex induced by the injury of facial nerve.