[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.

Investigation of anti-aging and anti-infection properties of Jingfang Granules using the Caenorhabditis elegans model.

Jingfang Granule (JFG), a traditional Chinese medicine, is frequently employed in clinical settings for the treatment of infectious diseases. Nevertheless, the anti-aging and anti-infection effects of JFG remain uncertain. In the present study, these effects were evaluated using the Caenorhabditis elegans (C. elegans) N2 as a model organism. The results demonstrated that JFG significantly increased the median lifespan of C. elegans by 31.2% at a dosage of 10 mg/mL, without any discernible adverse effects, such as alterations in the pharyngeal pumping rate or nematode motility. Moreover, JFG notably increased oviposition by 11.3%. Subsequent investigations revealed that JFG enhanced oxidative stress resistance in C. elegans by reducing reactive oxygen species levels and significantly improved survival rates in nematodes infected with Pseudomonas aeruginosa ATCC 9027. These findings suggest that JFG delays reproductive senescence in C. elegans and protects them from oxidative stress, thereby extending their lifespan. Additionally, JFG improves the survival of P. aeruginosa-infected nematodes. Consequently, JFG has potential as a candidate for the development of anti-aging and anti-infection functional medicines.

Gram-Scale Synthesis of Loganetin from S-(+)-Carvone.

Loganetin is the aglycone moiety of loganin that has a 5,6-fused bicyclic framework and exhibits a wide range of interesting biological activities. A gram-scale synthesis of loganetin has been accomplished from the readily accessible S-(+)-carvone. The key reactions of the synthesis are a Favorskii rearrangement to introduce four stereocenters and a sulfuric acid-meditated deprotection/cyclization reaction to assemble the sensitive dihydropyran ring with complete stereoselectivity. This work also enables us to synthesize C1 methoxy loganetin and the enantiomer of loganetin successfully.

Novel MAGL Inhibitors Alleviate LPS-Induced Acute Kidney Injury by Inhibiting NLRP3 Inflammatory Vesicles, Modulating Intestinal Flora, Repairing the Intestinal Barrier, and Interfering with Serum Metabolism.

Acute kidney injury (AKI) is a complication of a wide range of serious illnesses for which there is still no better therapeutic agent. We demonstrated that M-18C has a favorable inhibitory effect on monoacylglycerol lipase (MAGL), and several studies have demonstrated that nerve inflammation could be effectively alleviated by inhibiting MAGL, suggesting that M-18C has good anti-inflammatory activity. In this study, we investigated the effect of M-18C on LPS-induced acute kidney injury (AKI), both in vivo and in vitro, by using liquid chromatography-mass spectrometry (LC-MS), 16S rRNA gene sequencing, Western blot, and immunohistochemistry. The results showed that both in vivo and in vitro M-18C reduced the release of TNF-α and IL-1ß by inhibiting the expression of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and apoptosis-associated speck-like protein containing a CARD (ASC) protein; in addition, M-18C was able to intervene in LPS-induced AKI by ameliorating renal pathological injury, repairing the intestinal barrier, and regulating gut bacterial flora and serum metabolism. In conclusion, this study suggests that M-18C has the potential to be a new drug for the treatment of AKI.

[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.

[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.

[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.

[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-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.

[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.

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.

[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.

A biomimetic fluorescent nanosensor based on imprinted polymers modified with carbon dots for sensitive detection of alpha-fetoprotein in clinical samples.

A biomimetic fluorescent nanosensor based on molecularly imprinted polymers modified with carbon dots (CDs@MIPs) has been prepared for rapid, selective and sensitive detection of alpha-fetoprotein (AFP) in clinical samples. The nanosensor was produced using vinyl-functionalized CDs (V-CDs) as transducer elements and support materials, AFP as the template protein, N-isopropylacrylamide (NIPAM) and 4-vinylphenylbronic acid (VPBA) as the thermo-responsive and pH-responsive monomer, respectively, and ammonium peroxodisulphate (APS) and N,N'-methylene bisacrylamide (MBA) as the initiator and cross-linker, respectively. The newly synthesized nanosensor was characterized by FT-IR, TEM, XRD and elemental analysis, which unambiguously confirmed the successful formation of the nanosensor. The fluorescence quenching degree of CDs@MIPs exhibited a good linear response to AFP in a concentration range of 10 to 100 ng mL-1, the limit of detection (LOD) of 0.474 ng mL-1, and high recoveries at three spiking levels of AFP ranging from 97.05% to 102.00%, with relative standard deviations (RSDs) below 4.2% being obtained. Moreover, the proposed CDs@MIPs were successfully exploited to detect AFP in human serum samples. This study successfully established a novel method for rapid, convenient, and highly sensitive and selective detection of AFP, which provides new ideas for the detection of tumor markers.

Phillygenin exhibits anti-inflammatory activity through modulating multiple cellular behaviors of mouse lymphocytes.

Context: Phillygenin (PHI) is an intestinal metabolite of phillyrin from the genus Forsythia. Although the regulatory activity of Forsythia on immune system has been investigated, the effect of PHI on activated lymphocytes is poorly understood. Objective: This study was aimed to discuss the possible anti-inflammation potential of PHI on mitogen-activated stimulated lymphocytes in vitro. Methods: Mice spleen lymphocytes were incubated with PHI for 4 h, and then stimulated with concanavalin A (Con A) or phorbol 12-myristate 13-acetate/ionomycin (PMA + Ion). Cell viability was assayed by cell counting kit-8 (CCK-8). The expression of CD69 and CD25, proliferation, cell cycle, intracellular Ca2+ concentration, apoptosis, mitochondrial inner membrane potential (ΔΨm), mitochondrial permeability transition (MPT), interleukin-2 (IL-2), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were analyzed by flow cytometry. The expression of cyclin B1, cyclin D1, Cyclin E, and the phosphorylation of c-jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 were assayed by western blotting. Results: The results showed that PHI inhibited the proliferation of Con A-activated lymphocytes and induced a G0/G1 phase arrest by suppressing cyclin D1 and cyclin E. Meanwhile, PHI antagonized Con A-induced T cells activation through blocking intracellular Ca2+ overload and suppressing the phosphorylation of JNK and Erk1/2. Both Con A and PMA + Ion-induced secretion of IL-2, IFN-γ, and TNF-α were attenuated by PHI. PHI enhanced Con A-induced lymphocytes apoptosis through decreasing ΔΨm and increasing MPT. Conclusion: These results suggest that PHI exhibits its anti-inflammatory activity through modulating multiple cellular behaviors, leading to the suppression of the adaptive immune response.

Icaritin Improves Antibody-Induced Thrombocytopenia in a Mouse Model by Regulating T-cell Polarization.

Previous studies have shown that icaritin (ICT) has significant protective effects on immune thrombocytopenia (ITP), and the present study aimed to discuss the mechanism of this protective effect from the aspect of regulating T-cell polarization by an antibody-induced ITP mice model. Mice were given rat anti-mouse CD41 antibody (MWReg30) by intraperitoneal injection for 7 d to produce ITP model. At the same time, ICT was administrated at 10 mg/kg/d orally for 9 d. Peripheral blood platelets were counted by hematology analyzer. Spleen index was also tested. Spleen T-helper cell (Th), cytotoxic T-cell (CTL), Th1, Th2, Th17, regulatory T-cell (Treg), and follicular helper T-cell (Tfh) were quantified by flow cytometry. Serum Th1/Th2/Th17 cytokines were tested by mouse Th1/Th2/Th17 cytometric bead array (CBA) kit and transforming growth factor beta (TGF-ß) were analyzed by enzyme-linked immunosorbent assay (ELISA) kit. The results indicated that ICT (10 mg/kg) protected against MWReg30-induced ITP, as evidenced by increased blood platelets and decreased spleen index. In addition, the imbalance of Th/CTL in ITP mice spleen was regulated by ICT. Meanwhile, ICT inhibited Th1, Th17, and Tfh and improved Th2 and Treg in ITP mice spleen. Furthermore, the results of CBA and ELISA suggested that ICT decreased serum Th1- and Th17-related cytokines and increased Th2 cytokines, as well as promoted the release of TGF-ß. These results demonstrated that the protective effect of ICT on ITP was mediated by regulating T-cell polarization.

Improvement of icaritin on hematopoietic function in cyclophosphamide-induced myelosuppression mice.

CONTEXT: Icaritin (ICT), an intestinal metabolite of prenylflavonoids from Herba Epimedii, has been known to regulate many immune processes. But there are little studies of ICT on hematopoietic function. OBJECTIVE: We aimed to investigate the improvement of ICT on hematopoietic function in cyclophosphamide (CTX)-induced myelosuppression mice. METHODS: Mice were given CTX (50 mg/kg) by i.p. for five days to produce bone marrow depression model. 48 h after last treated with CTX, ICT was administrated at 10 mg/kg/d by p.o. for five days. Blood routine, body weight, thymus index and spleen index were tested. The bone marrow hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), cell cycle and apoptosis of HSCs were quantified by flow cytometry. The bone marrow nucleated cells were counted by an automated cell counter. The histology of femoral bone was examined by Haemotoxylin and Eosin (H&E) staining. Serum erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) and thyroperoxidase (TPO) were tested by ELISA kit. RESULTS: ICT (10 mg/kg) protected against CTX-induced myelosuppression, is evidenced by increased blood cell numbers, body weight, thymus index, spleen index and improved femoral bone morphology. ICT corrected the reduction of bone marrow HSCs and HPCs, promoted bone marrow HSCs entering the proliferative cycle phase and prevented cells proceeding to the apoptosis phase. Meanwhile, ICT increased the release of G-CSF and TPO in model mice serum. CONCLUSION: These results demonstrated that ICT improves myelosuppression by improving bone marrow hematopoietic microenvironment, promoting the proliferation and differentiation of HSCs, inhibiting the apoptosis of HSCs and stimulating the expression of G-CSF and TPO.

[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.