[Chemical constituents and mechanism of Chuanzhi Tongluo Capsules based on UPLC-Q-Exactive Orbitrap-MS and network pharmacology].

The chemical constituents of Chuanzhi Tongluo Capsules were analyzed and identified using ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) to clarify the pharmacological substance basis. In addition, network pharmacology was employed to explore the mechanism of Chuanzhi Tongluo Capsules in the treatment of cerebral infarction. Gradient elution was performed using acetonitrile and 1% acetic acid in water as the mobile phase. Mass spectrometry was performed in positive and negative ion modes. Xcalibur 4.2 software was used for compound analysis, including accurate mass-to-charge ratio and MS/MS fragment information, combined with the comparison of reference standards and literature data. A total of 152 compounds were identified, including 32 organic acids, 35 flavonoids and their glycosides, 33 diterpenes, 13 phthalides, 12 triterpenes and triterpene saponins, 23 nitrogen-containing compounds, and 4 other compounds, and their fragmentation patterns were analyzed. SwissTargetPrediction, GeneCards, DAVID, and other databases were used to predict and analyze the core targets and mechanism of Chuanzhi Tongluo Capsules. Protein-protein interaction(PPI) network topology analysis identified 10 core targets, including TNF, VEGFA, EGFR, IL1B, and CTNNB1. KEGG enrichment analysis showed that Chuanzhi Tongluo Capsules mainly exerted their effects through the regulation of lipid and atherosclerosis, glycoproteins in cancer, MicroRNAs in cancer, fluid shear stress, and atherosclerosis-related pathways. Molecular docking was performed between the key constituents and core targets, and the results demonstrated a strong binding affinity between the key constituents of Chuanzhi Tongluo Capsules and the core targets. This study comprehensively elucidated the chemical constituents of Chuanzhi Tongluo Capsules and explored the core targets and mechanism in the treatment of cerebral infarction based on network pharmacology, providing a scientific reference for the study of the pharmacological substance basis and formulation quality standards of Chuanzhi Tongluo Capsules.

Can natural environments enhance acute effects of rehabilitation exercise for older adults? A pilot randomized controlled trial.

The added value of natural environments in rehabilitation exercise is unclear. This study aimed to investigate whether there are more acute health benefits for older adults after a single rehabilitation exercise session performed in an outdoor natural environment compared with an indoor environment. Twenty-two nursing home residents were randomly assigned to the outdoor (n = 11, 79.5 ± 2.1 years) or indoor group (n = 11, 78.8 ± 5.2 years). Performance test outcomes were measured pre- and post-training session. The indoor group had a significantly higher blood pressure, greater increase in heart rate, higher perceived exercise intensity and physiological fatigue than the outdoor group. The combination of rehabilitation exercise with an outdoor natural environment may reduce exercise fatigue and improve cardiovascular health in older adults, with greater acute health benefits compared with an indoor environment. Rehabilitation exercise in the natural environments may be a highly valued environmental intervention for physiotherapy in older adults.

Canan Outdoor Multisurface Terrain Enhance the Effects of Fall Prevention Exercise in Older Adults? A Randomized Controlled Trial.

Walking on complex surface conditions in outdoor environments is important for active aging. This study aimed at examining whether fall prevention exercise integrated with an outdoor multisurface terrain compared with indoor solid ground was more beneficial for older adults. Twenty-two older nursing home residents were randomly assigned to outdoor multisurface terrain (n = 11, 79.5 ± 2.1 years) or indoor solid ground (n = 11, 78.8 ± 5.2 years) groups. Training occurred five times per week (30 min) for 3 weeks. The following performance test outcomes were measured: 10 m walk test (10 mWT), multisurface terrain walk test (MTWT), 2 min walk test (2 MWT), timed up and go test (TUGT), single-leg standing test with eyes open (SLSTEO), single-leg standing test with eyes closed (SLSTEC), and closed cycles test (CCT). Compared with baseline, the outdoor multisurface terrain training significantly improved performance in all tests (p < 0.01). The improvements of the outdoor multisurface terrain group after intervention were significantly higher than those of the indoor solid group in the 10 mWT (p = 0.049), MTWT (p = 0.02), and 2 MWT (p = 0.000). Exercise combined with outdoor multisurface terrain training may be an efficacious approach and a feasible environmental intervention for fall prevention in older adults.

Mechanisms of IFNalpha-1a-Induced Apoptosis in a Laryngeal Cancer Cell Line.

BACKGROUND Interferon alpha (IFNalpha) exerts its anti-proliferative effect on many human cancers. Among the 13 subtypes of human IFNalpha, IFNalpha-1 subtype has 2 variants, named IFNalpha-1a and IFNalpha-1b, that differ from each other in only 1 amino acid, at residue 114. However, the mechanism by which IFNalpha-1a mediates growth inhibition is still unclear. MATERIAL AND METHODS Human laryngeal carcinoma HEp2 cells were treated with IFNalpha-1a by either transient transfection or exogenous delivery. Western blot and RT-PCR analysis were carried out to assess apoptotic pathways active in IFNalpha-1a-treated HEp2 cells. Microarray analysis was conducted to uncover the differential gene expressions after IFNalpha-1a treatment. KEGG pathway enrichment analysis was also performed. RESULTS IFNalpha-1a markedly inhibited the proliferation and significantly promoted the apoptosis of HEp-2 cells. Mechanistic studies indicate that IFNalpha-1a-mediated cell apoptosis is directly linked to intrinsic and endoplasmic reticulum (ER) stress-related apoptosis, but is independent of extrinsic apoptosis. The top 40 differentially expressed genes discovered by microarray analysis included 20 upregulated genes (e.g., IFI6, IFI27, IFI44L, and MIR548X) and 20 downregulated genes (e.g., PRKDC, HIST1H3B, DYNC1H1, and HIST1H2AM). KEGG pathway enrichment analysis revealed that 4 out of 6 pathways are TP53-related. CONCLUSIONS We demonstrated a detailed mechanism involved in IFNalpha-1a-mediated anti-proliferation activity in human laryngeal carcinoma cells.

Evaluation of Anti-Inflammatory Activities of a Triterpene ß-Elemonic Acid in Frankincense In Vivo and In Vitro.

The purpose of this research was to extract and separate the compounds from frankincense, and then evaluate their anti-inflammatory effects. The isolated compound was a representative tetracyclic triterpenes of glycine structure according to ¹H-NMR and 13C-NMR spectra, which is ß-elemonic acid (ß-EA). We determined the content of six different localities of frankincense; the average content of ß-EA was 41.96 mg/g. The toxic effects of ß-EA administration (400, 200, 100 mg/kg) for four weeks in Kunming (KM) mice were observed. Compared with the control group, the body weight of mice, the visceral coefficients and serum indicators in the ß-EA groups showed no systematic variations. The anti-inflammatory effects of ß-EA were evaluated in LPS-induced RAW264.7 cells, xylene-induced induced ear inflammation in mice, carrageenin-induced paw edema in mice, and cotton pellet induced granuloma formation in rats. ß-EA inhibited overproduction of tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein 1 (MCP-1), soluble TNF receptor 1 (sTNF R1), Eotaxin-2, Interleukin 10 (IL-10) and granulocyte colony-stimulating factor (GCSF) in the RAW264.7 cells. Intragastric administration with ß-EA (300, 200, and 100 mg/kg in mice, and 210, 140, and 70 mg/kg in rats) all produced distinct anti-inflammatory effects in vivo in a dose-dependent manner. Following treatment with ß-EA (300 mg/kg, i.g.), the NO level in mice ears and PGE2 in mice paws both decreased (p < 0.01). In conclusion, our study indicates that ß-EA could be a potential anti-inflammatory agent for the treatment of inflammatory diseases.

Farnesyl thiosalicylic acid prevents iNOS induction triggered by lipopolysaccharide via suppression of iNOS mRNA transcription in murine macrophages.

Inducible nitric oxide synthase (iNOS) is a molecule critical for the development of inflammation-associated disorders. Its induction should be tightly controlled in order to maintain cellular homeostasis. Upon lipopolysaccharide (LPS) stimulation, iNOS, in most settings, is induced by the activation of inhibitor of κB-α (IκB-α)-nuclear factor κB (NF-κB) signaling. Farnesyl thiosalicylic acid (FTS), a synthetic small molecule that is considered to detach Ras from the inner cell membrane, has been shown to exhibit numerous anti-inflammatory functions. However, it remains unclear whether and how it affects iNOS induction in macrophages. The present study addressed this issue in cultured macrophages and endotoxemic mice. Results showed that FTS pretreatment significantly prevented LPS-induced increases in iNOS protein and mRNA expression levels in murine cultured macrophages, which were confirmed in organs in vivo from endotoxemic mice, such as the liver and lung. Mechanistic studies revealed that FTS pretreatment did not affect IκB-α degradation and NF-κB activation in LPS-treated macrophages. The nuclear transport of the active NF-κB was also not affected by FTS. But FTS pretreatment reduced the binding of NF-κB to its DNA elements, and reduced NF-κB bindings to iNOS promoter inside LPS-treated macrophages. Finally, our results showed that FST pretreatment increased mouse survival rate compared to LPS alone treatment. Taken together, these results indicate that FTS attenuates iNOS induction in macrophages likely through inhibition of iNOS mRNA transcription, providing further insight into the molecular mechanism of action of FTS in inflammatory disorder therapy.

Isopsoralen induces different subchronic toxicities and metabolomic outcomes between male and female Wistar rats.

Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralen-treated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.

The Antiosteoporosis Effects of Zhuanggu Guanjie Pill In Vitro and In Vivo.

We investigated the beneficial effects and underlying mechanisms of Zhuanggu Guanjie (ZGGJ) pill in osteoporosis in vitro and in vivo. Bone marrow macrophages from 4-6-week-old mice were cultured in the presence of macrophage colony-stimulating factor (15 ng/mL) and receptor activator of nuclear factor-κB ligand (30 ng/mL). Osteoclast differentiation was determined by quantification of tartrate-resistant acid phosphatase activity. Gelatin zymography was used to detect the activity of matrix metalloproteinases in osteoclasts. Ovariectomized rats were administered orally with estradiol valerate or ZGGJ for 8 weeks. Blood was collected to measure serum indices. Tibiae were harvested to carry out bone microcomputed tomography scanning, histomorphological analysis, and bone strength determination. ZGGJ inhibited tartrate-resistant acid phosphatase activity, matrix metalloproteinase 9 expression, and bone resorption in vitro. At doses of 0.55, 1.1, and 2.2 g/kg, ZGGJ exerted significant osteoprotective effects including inhibition of bone turnover markers and improved tibia bone strength in ovariectomized rats. Microcomputed tomographic analysis showed that ZGGJ improved the trabecular architecture with increased connectivity density and trabecular thickness and decreased trabecular spacing. These results revealed that ZGGJ prevents bone loss induced by ovariectomy in rats and that inhibition of bone resorption is involved in the bone-protective effects of ZGGJ.

A Study of NMR-Based Hepatic and Serum Metabolomics in a Liver Injury Sprague-Dawley Rat Model Induced by Psoralen.

Psoralen is the main active component of Psoralea corylifolia and is used as a marker to assess its quality. The effects of psoralen on animals have been well characterized. However, the molecular pathway of its toxicity is not fully understood. In this study, the toxic effects of psoralen administration (60 mg/kg) for 7 days in Sprague-Dawley rats were observed. Serum biochemistry and liver histopathology were further investigated. Proton nuclear magnetic resonance was applied to characterize the metabolic profile of liver toxicity induced by psoralen and to find changed metabolites in rat serum and liver. It was revealed that visceral coefficients and serum biochemistry indexes were significantly changed in rats with psoralen-induced liver injury. Furthermore, the histopathological examination exhibited that the liver might be the target organ for psoralen. Metabolic analysis of both serum and liver samples further proved the liver was the target of toxicity of psoralen. Multivariate analysis identified 7 metabolites in serum samples and 15 in liver samples as potential biomarkers in liver injury induced by psoralen. In addition, our results suggest that psoralen can cause a disturbance in amino acid metabolism, especially valine, leucine, and isoleucine biosynthesis in both serum and liver samples. In conclusion, we combined the results of toxicity and metabolomics induced by psoralen and provide useful information about the safety and potential risks of psoralen and Psoralea corylifolia.

Optimization of the Time Window of Interest in Ovariectomized Imprinting Control Region Mice for Antiosteoporosis Research.

This study was performed to determine the optimal window of time during which the properties of osteoporosis are obvious and to explore the best region of interest for microstructural evaluation in antiosteoporosis research in an ovariectomized mouse model by examining changes in micro-computed tomography parameters and serum indices. Ovariectomized mice and sham-operated mice were randomly divided into five groups. At the end of the 4th, 8th, 12th, 16th, and 20th weeks after ovariectomy, the microstructure of the proximal tibia and distal femur was scanned by micro-computed tomography and blood samples were collected to detect serum biochemical indicators including alkaline phosphatase, osteocalcin, N-terminal propeptide of type I procollagen (P1NP), and C-terminal telopeptide fragment of type I collagen (CTX1). The trabecular number and connectivity density decreased while the trabecular thickness and trabecular separation increased, indicating substantial changes in the trabecular microstructure of both the tibia and femur and significant changes in bone turnover after ovariectomy, as indicated by lower levels of serum alkaline phosphatase, osteocalcin, and P1NP and higher level of CTX1 in the ovariectomy than sham group. The proximal tibia from weeks 8 to 16 after ovariectomy was optimal for osteoporosis research in this model.

[Effects of hot shock protein 70 inhibitor PFTµ on inflammatory response in lipopolysaccharide-stimulated RAW264.7 cells and mice underwent myocardial ischemia-reperfusion injury].

OBJECTIVE: To observe the effects of hot shock protein 70 (HSP70) inhibitor (PFTµ) on inflammation response in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and mice underwent myocardial ischemia-reperfusion (I/R) injury. METHODS: RAW264.7 macrophage line of mice was stimulated by LPS as an inflammatory model. These were divided into control (15 min DMSO pretreatment and LPS 2 g/L) and PFTµ treated groups (15 min PFTµ 20 µmol/L pretreatment and LPS 2 g/L). NO concentration was measured by Griess Kit. The expression of iNOS protein and mRNA were detected by Western blot and RT-PCR. Infarct size was determined on mice underwent myocardial ischemia-reperfusion (I/R) injury in the absence or presence (PFTµ 40 mg/kg, intraperitoneal injection). RESULTS: PFTµ significantly blocked the production of NO and protein and mRNA expression of iNOS (P < 0.05 vs. control). PFTµ also significantly reduced the infarct size on mice underwent I/R injury (P < 0.05 vs. control). CONCLUSION: These results suggest that PFTµ could be a potential therapeutic agent for the treatment of inflammatory diseases through inhibiting the production of NO and reducing inflammatory responses.

Current status of diagnosis and treatment of primary breast cancer in beijing, 2008.

OBJECTIVE: To investigate the status of diagnosis and treatment of primary breast cancer in Beijing, 2008. METHODS: All the patients who were diagnosed as primary breast cancer in Beijing in 2008 were enrolled in this study. Information of these patients, including the features of tumors, clinical diagnosis and treatment was collected, and filled in the well-designed questionnaire forms by trained surveyors. The missing data was partly complemented through telephone interviews. RESULTS: A total of 3473 Beijing citizens were diagnosed as primary breast cancer (25 patients with synchronal bilateral breast cancer) in Beijing, 2008. Of them 82.09% were symptomatic. 19.02% and 34.11% were diagnosed using fine needle aspiration biopsy (FNAB) and core needle biopsy (CNB), respectively. 15.92% received sentinel lymph node biopsy (SLNB) and 24.27% received breast conserving surgery (BCS). Among 476 cases with Her-2 positive, only 96 received anti-Her-2 therapy. We found that the standardization level varied in hospitals of different grades, with higher level in Grade-III hospitals. Of note, some breast cancer patients received non-standard primary tumor therapy: 65.63% of the patients with ductal carcinoma in situ (DCIS) received axillary lymph node dissection and 36.88% received chemotherapy; 25.89% of the patients underwent breast conserving surgery without margin status; 12.10% of the patients received chemotherapy less than 4 cycles. CONCLUSION: Although most breast cancer patients received basic medical care, the mode of diagnosis and treatment should be improved and should be standardized in the future in Beijing.

The antiproliferative effects of somatostatin receptor subtype 2 in breast cancer cells.

AIM: Somatostatin receptor subtype 2 (SSTR2) is the principal mediator of somatostatin's (SST) antiproliferative effects on normal and cancer cells. Therefore, we investigated whether the enhanced expression of SSTR2 could inhibit the proliferation of tumor cells, and, if so, the mechanisms that might be involved. METHODS: SSTR2 expression levels were determined by qRT-PCR in several tumor cell lines. Then, a plasmid pIRES2-EGFP-SSTR2 (pSIG) was constructed and stably transfected into MCF-7 cells (MCF-7/pSIG). After SSTR2 overexpression was identified by qRT-PCR, immunofluorescence staining and a receptor binding assay, the MCF-7/pSIG cells were analyzed by PI staining for apoptosis and cell cycle arrest was tested by flow cytometry for epidermal growth factor receptor (EGFR) expression. The EGF-stimulated proliferation of MCF-7 cells was assayed by MTT. RESULTS: The human breast cancer cell line MCF-7 expresses a lower level of SSTR2, thereby partly accounting for the decreased response to SST. The overexpression of SSTR2 in MCF-7 cells resulted in apoptosis, cytostasis and G(1)/S cell cycle arrest. Furthermore, the expression of EGFR, together with EGF-stimulated proliferation, was markedly decreased in the MCF-7/pSIG cells. CONCLUSION: Enhanced SSTR2 expression played an antiproliferative role in MCF-7 cells through inducing apoptosis and G(1)/S cell cycle arrest, and also by decreasing EGFR expression, thereby counteracting the growth-stimulating effect of EGF. Our data seem to indicate that developing a new therapeutic agent capable of upregulating SSTR expression could potentially be a way to block tumor progression.Acta Pharmacologica Sinica (2009) 30: 1053-1059; doi: 10.1038/aps.2009.59.

Antigen-binding characteristics of AbCD71 and its inhibitory effect on PHA-induced lymphoproliferation.

AIM: To investigate the anti-lymphoproliferative effect of the prepared recombinant chimeric human/murine anti-cluster of differentiation(CD) 71 monoclonal antibody (AbCD71), which is composed of mouse-derived, antigen-binding variable regions and human-derived constant regions. METHODS: After plasmids construction and transfection, the expression of AbCD71 in the transfectoma supernatant was determined by the sandwich ELISA. Indirect immunofluorescence assay was used to measure the antigen-binding characteristic and the percent CD71-expressed peripheral blood mononuclear cells (PBMC). The antibodies were purified from the ascites via diethylaminoethyl(DEAE)-Sephadex A-50 chromatography and then identified by SDS-PAGE. At last, inhibitory effect of AbCD71 on PHA-induced PBMC proliferation was calculated by methyl thiazolyl tetrazolium(MTT) assay. RESULTS: Constant domain of heavy chain (C(H)) and light chain (C(L)) of AbCD71 were in the human Cgamma family and human Ckappa family, respectively. AbCD71 could compete with its original murine mAb to bind with CD71-positive human leukemia cell line CEM cells. AbCD71 could inhibit the peripheral blood mononuclear cell proliferation induced by phytohemagglutinin(PHA) in vitro in a dose-dependent manner, especially at time-points 0 and 12 h after induction. There was no statistical difference when compared with original murine mAb. CONCLUSION: The AbCD71 is a promising immunosuppressant. Our approach to blocking CD71 with the chimeric human/murine mAb provides a novel strategy for prolonging allograft survival.