BACKGROUND: Eucommia ulmoides Oliv. is a medicinal plant native to China, with its bark (Eucommiae Cortex) traditionally being used for medicinal purposes. Previous research has shown that Eucommia male flowers can exert anti-inflammatory, analgesic, antibacterial, and other pharmacological effects, including immune regulation. This study explored the anti-inflammatory effects of the 70% ethanol extract of male flowers (EF) of E. ulmoides in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and LPS-administered mice. METHODS: Cytotoxicity of EF for RAW 264.7 cells was investigated using Cell Counting Kit-8. The production of proinflammatory mediators, nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was determined using enzyme-linked immunosorbent assays. IL-17, IL-23, and IL-10 mRNA levels were determined using quantitative real-time polymerase chain reaction. Activation of the nuclear factor (NF)-κB pathway in RAW 264.7 cells was investigated via Western blotting. In vivo anti-inflammatory effects of EF were studied in an LPS-induced acute inflammation mouse model by analyzing lung tissue histopathology, serum TNF-α and IL-6 levels, and myeloperoxidase (MPO) activity in lung tissue. RESULTS: EF showed no significant cytotoxicity at concentrations from 10 to 60 μg/mL (cell viability > 80%) in the CCK-8 cell viability assay. EF inhibited the RAW 264.7 cell proliferation (EF 60 μg/mL, 120 μg/mL, and 250 μg/mL vs. negative control: 87.31 ± 2.39% vs. 100.00 ± 2.50%, P = 0.001; 79.01 ± 2.56 vs. 100.00 ± 2.50%, P < 0.001; and 64.83 ± 2.50 vs. 100.00 ± 2.50%, P < 0.001), suppressed NO (EF 20 μg/mL and 30 μg/mL vs. LPS only, 288.81 ± 38.01 vs. 447.68 ± 19.07 μmol/L, P = 0.004; and 158.80 ± 45.14 vs. 447.68 ± 19.07 μmol/L, P < 0.001), TNF-α (LPS+EF vs. LPS only, 210.20 ± 13.85 vs. 577.70 ± 5.35 pg/mL, P < 0.001), IL-1β (LPS+EF vs. LPS only, 193.30 ± 10.80 vs. 411.03 ± 42.28 pg/mL, P < 0.001), and IL-6 (LPS+EF vs. LPS only, 149.67 ± 11.60 vs. 524.80 ± 6.24 pg/mL, P < 0.001) secretion, and downregulated the mRNA expression of IL-17 (LPS+EF vs. LPS only, 0.23 ± 0.02 vs. 0.43 ± 0.12, P < 0.001), IL-23 (LPS+EF vs. LPS only, 0.29 ± 0.01 vs. 0.42 ± 0.06, P=0.002), and IL-10 (LPS+EF vs. LPS only, 0.30 ± 0.01 vs. 0.47 ± 0.01, P=0.008) in LPS-stimulated RAW 264.7 cells. EF inhibited the LPS-induced NF-κB p65 (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.78 ± 0.06 vs. 1.17 ± 0.08, P < 0.001; and 0.90 ± 0.06 vs. 1.17 ± 0.08, P =0.002) and inhibitor of kappa B (IκBα) phosphorylation (LPS+EF 20 μg/mL and 30 μg/mL vs. LPS only: 0.25 ± 0.01 vs. 0.63 ± 0.03, P < 0.001; and 0.31 ± 0.01 vs. 0.63 ± 0.03, P < 0.001), LPS+EF 30 μg/mL inhibited IκB kinase (IKKα/β) phosphorylation (LPS+EF 30 μg/mL vs. LPS only, 1.12 ± 0.14 vs. 1.71 ± 0.25, P = 0.002) in RAW 264.7 cells. Furthermore, EF 10 mg/kg and EF 20 mg/kg inhibited lung tissue inflammation in vivo and suppressed the serum TNF-α (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 199.99 ± 186.49 vs. 527.90 ± 263.93 pg/mL, P=0.001; and 260.56 ± 175.83 vs. 527.90 ± 263.93 pg/mL, P = 0.005), and IL-6 (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 41.26 ± 30.42 vs. 79.45 ± 14.16 pg/ ml, P = 0.011; and 42.01 ± 26.26 vs. 79.45 ± 14.16 pg/mL, P = 0.012) levels and MPO (LPS+EF 10 mg/kg and 20 mg/kg vs. LPS only, 3.19 ± 1.78 vs. 5.39 ± 1.51 U/g, P = 0.004; and 3.32 ± 1.57 vs. 5.39 ± 1.51 U/g, P = 0.006) activity in lung tissue. CONCLUSIONS EF could effectively inhibit the expression of inflammatory factors and overactivation of neutrophils. Further investigation is needed to evaluate its potential for anti-inflammation therapy.
Animals ; Anti-Inflammatory Agents ; chemistry ; therapeutic use ; Eucommiaceae ; chemistry ; Flowers ; chemistry ; Inflammation ; blood ; chemically induced ; drug therapy ; Interleukin-1beta ; blood ; Lipopolysaccharides ; toxicity ; Macrophages ; drug effects ; Mice ; NF-KappaB Inhibitor alpha ; blood ; NF-kappa B ; blood ; Nitric Oxide ; blood ; Plant Extracts ; chemistry ; therapeutic use ; RAW 264.7 Cells ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; blood

BACKGROUND: Kidney ischemia-reperfusion (IR) via laparotomy is a conventional method for kidney surgery in a mouse model. However, IR, an invasive procedure, can cause serious acute and chronic complications through apoptotic and inflammatory pathways. To avoid these adverse responses, a Non-IR and dorsal slit approach was designed for kidney surgery. METHODS: Animals were divided into three groups, 1) sham-operated control; 2) IR, Kidney IR via laparotomy; and 3) Non-IR, Non-IR and dorsal slit. The effects of Non-IR method on renal surgery outcomes were verified with respect to animal viability, renal function, apoptosis, inflammation, fibrosis, renal regeneration, and systemic response using histology, immunohistochemistry, real-time polymerase chain reaction, serum chemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and Masson's trichrome staining. RESULTS: The Non-IR group showed 100% viability with mild elevation of serum blood urea nitrogen and creatinine values at day 1 after surgery, whereas the IR group showed 20% viability and lethal functional abnormality. Histologically, renal tubule epithelial cell injury was evident on day 1 in the IR group, and cellular apoptosis enhanced TUNEL-positive cell number and Fas/caspase-3 and KIM-1/NGAL expression. Inflammation and fibrosis were high in the IR group, with enhanced CD4/CD8-positive T cell infiltration, inflammatory cytokine secretion, and Masson's trichrome stain-positive cell numbers. The Non-IR group showed a suitable microenvironment for renal regeneration with enhanced host cell migration, reduced immune cell influx, and increased expression of renal differentiation-related genes and anti-inflammatory cytokines. The local renal IR influenced distal organ apoptosis and inflammation by releasing circulating pro-inflammatory cytokines. CONCLUSION: The Non-IR and dorsal slit method for kidney surgery in a mouse model can be an alternative surgical approach for researchers without adverse reactions such as apoptosis, inflammation, fibrosis, functional impairment, and systemic reactions.
Animals ; Apoptosis ; Blood Urea Nitrogen ; Cell Count ; Cell Movement ; Chemistry ; Creatinine ; Cytokines ; DNA Nucleotidylexotransferase ; Epithelial Cells ; Fibrosis ; Immunohistochemistry ; Inflammation ; Kidney ; Laparotomy ; Methods* ; Mice* ; Nephrectomy* ; Real-Time Polymerase Chain Reaction ; Regeneration*

Adipose tissue-derived stem cell (ASCs) are an attractive source of stem cells with therapeutic applicability in various fields for regenerating damaged tissues because of their stemness characteristics. However, little has reported on evaluating adverse responses caused by human ASC therapy. Therefore, in the present study, a clinical assessment after human ASC transplantation into dogs was undertaken. A total of 12 healthy male dogs were selected and divided into four groups: saline infusion, saline bolus, ASC infusion, and ASC bolus groups. Physical assessment and blood analysis were performed following ASC transplantation, and the concentrations of angiogenic factors, and pro- and anti-inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). There were no adverse vital sign responses among the dogs. Blood analyses revealed no remarkable complete blood count or serum chemistry results. ELISA results for angiogenic and anti-inflammatory factors including matrix metalloproteinase 9 (MMP9), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were significantly higher in the two ASCs groups than in the controls. In conclusion, this study demonstrated that transplantation of human ASCs produced no adverse effects and could be used safely in dogs. In addition, human ASCs could be involved in modulating secretions of angiogenic factors including MMP9, VEGF, bFGF, and HGF and anti-inflammatory factor IL-10.
Angiogenesis Inducing Agents ; Animals ; Blood Cell Count ; Chemistry ; Cytokines ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Fibroblast Growth Factor 2 ; Hepatocyte Growth Factor ; Humans ; Interleukin-10 ; Male ; Matrix Metalloproteinase 9 ; Stem Cell Transplantation ; Stem Cells ; Transplantation ; Vascular Endothelial Growth Factor A ; Vital Signs

In 2016, external quality assessment trials for urinalysis and faecal occult blood (FOB) were performed with 1,487 participants in Korea. Urine chemistry and FOB tests were performed three and two times, respectively, whereas urine sediment was evaluated once using photography. Urine chemistry tests consisted of pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, and nitrite levels; leukocyte count; specific gravity. The results of the urine chemistry and specific gravity tests showed accuracy rates of >95%. The accuracy rate of urine sediments was low, especially that for transitional epithelial cells and atypical crystals. In the FOB quality test, all reagents showed accuracy rates of >90%, which suggested the improvement of false-positive reaction. In the FOB quantitative test, discrepant results depending on the instrument used was observed. To compensate for the result differences caused by the stool samples, the results should be reported using another unit (µg/g of stool).
Bilirubin ; Chemistry ; Epithelial Cells ; Glucose ; Hydrogen-Ion Concentration ; Indicators and Reagents ; Korea* ; Leukocyte Count ; Occult Blood* ; Photography ; Specific Gravity ; Urinalysis* ; Urobilinogen

Gastroenteropancreatic neuroendocrine neoplam (GEP-NEN) is a rare group of tumors with its incidence rising significantly in recent decades. Because of the late presentation of the disease and limitations in conventional biomarkers, about 50% of GEP-NEN patients manifests advanced disease when diagnosed. Therefore, it is vital to identify circulating biomarkers which can not only be used for early diagnosis but also accurately evaluating the biological behavior of GEP-NEN. This review summarizes the advances of circulating biomarkers in diagnosing and evaluating efficacy of treatment in GEP-NEN. Well-known circulating biomarkers include chromogranin A (CgA), pancreastatin (PST), chromogranin B (CgB), neuron-specific enolase (NSE) and pancreatic peptide(PP). Novel biomarkers including circulating tumor cell(CTC), microRNA and NETest are promising biomarkers with potential clinical benefit, but further researches are needed before their clinical applications.
Biomarkers, Tumor ; blood ; Chromogranin A ; blood ; Chromogranin B ; blood ; chemistry ; Gastrointestinal Neoplasms ; blood ; chemistry ; diagnosis ; genetics ; Humans ; MicroRNAs ; blood ; Neoplastic Cells, Circulating ; Neuroendocrine Tumors ; blood ; chemistry ; diagnosis ; genetics ; Pancreatic Neoplasms ; blood ; chemistry ; diagnosis ; genetics ; Pancreatic Polypeptide ; blood ; Phosphopyruvate Hydratase ; blood

Angiotensin II (Ang II) is involved in endothelium injury during the development of hypertension. Tribulus terrestris (TT) is used to treat hypertension, arteriosclerosis, and post-stroke syndrome in China. The present study aimed to determine the effects of aqueous TT extracts on endothelial injury in spontaneously hypertensive rats (SHRs) and its protective effects against Ang II-induced injury in human umbilical vein endothelial cells (HUVECs). SHRs were administered intragastrically with TT (17.2 or 8.6 g·kg·d) for 6 weeks, using valsartan (13.5 mg·kg·d) as positive control. Blood pressure, heart rate, endothelial morphology of the thoracic aorta, serum levels of Ang II, endothelin-1 (ET-1), superoxide dismutase (SOD) and malonaldehyde (MDA) were measured. The endothelial injury of HUVECs was induced by 2 × 10 mol·L Ang II. Cell Apoptosisapoptosis, intracellular reactive oxygen species (ROS) was assessed. Endothelial nitric oxide synthase (eNOS), ET-1, SOD, and MDA in the cell culture supernatant and cell migration were assayed. The expression of hypertension-linked genes and proteins were analyzed. TT decreased systolic pressure, diastolic pressure, mean arterial pressure and heart rate, improved endothelial integrity of thoracic aorta, and decreased serum leptin, Ang II, ET-1, NPY, and Hcy, while increased NO in SHRs. TT suppressed Ang II-induced HUVEC proliferation and apoptosis and prolonged the survival, and increased cell migration. TT regulated the ROS, and decreased mRNA expression of Akt1, JAK2, PI3Kα, Erk2, FAK, and NF-κB p65 and protein expression of Erk2, FAK, and NF-κB p65. In conclusion, TT demonstrated anti-hypertensive and endothelial protective effects by regulating Erk2, FAK and NF-κB p65.
Angiotensin II ; metabolism ; Animals ; Antihypertensive Agents ; administration & dosage ; Apoptosis ; drug effects ; Blood Pressure ; drug effects ; Endothelium, Vascular ; drug effects ; metabolism ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Hypertension ; drug therapy ; genetics ; metabolism ; physiopathology ; Male ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Oxidative Stress ; drug effects ; Plant Extracts ; administration & dosage ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Reactive Oxygen Species ; metabolism ; Tribulus ; chemistry

In Korea, external quality assessment trials for urinalysis and faecal occult blood (FOB) were performed for 1,250 participants. Urine chemistry and FOB tests were evaluated three times, whereas urine sediment by photography was evaluated twice. Urine chemistry tests consisted those for pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, nitrite, leukocyte, and specific gravity. The results of the urine chemistry and specific gravity tests showed accuracy rates >95%. In the FOB quality test, all reagents showed false-positive results. These reagents showed positive results in stool specimens containing >11 ng/mL haemoglobin. In the FOB quantitative test, the results were significantly different, based on the instrument used for the measurements. The average accuracy rate of urine sediments was 90.8%, whereas those for renal epithelial cells and cholesterol crystals were 83%.
Bilirubin ; Chemistry ; Cholesterol ; Epithelial Cells ; Glucose ; Hydrogen-Ion Concentration ; Indicators and Reagents ; Korea* ; Leukocytes ; Occult Blood* ; Photography ; Specific Gravity ; Urinalysis* ; Urobilinogen

OBJECTIVETo explore the protective effect and mechanism of total flavones of Bidens pilosa L. (TFB) on IgA1 induced injury of venous endothelial cells in Henoch-Schönlein purpura (HSP) children patients. METHODS Human umbilical venous endothelial cells (HUVECs) were taken as subject. They were intervened by normal IgA1 and HSP children patients' serum IgA1, and added with different concentrations TFB at the same time. Then they were divided into the blank control group, the normal control group, the HSP IgA1 group, and HSP IgA1 plus TFB (1.0, 0.5, 0.25 mg/mL) groups. Levels of TNF-α and IL-8 in supernate were detected by ELISA. The NO level was detected by nitrate reductase method. mRNA and protein expressions of NF-κB and ICAM-1 in HUVECs were detected by fluorescent quantitative PCR and Western blot respectively.

RESULTSCompared with the normal control group and the blank control group, levels of IL-8, TNF-α, and NO all significantly increased in the HSP group (P < 0.05). Compared with the HSP group, levels of IL-8, TNF-α, and NO significantly decreased after intervention of TFB (1.0 and 0.5 mg/mL; P < 0.05, P < 0.01). Results of fluorescent quantitative PCR and Western blot showed, as compared with the blank control group and the normal control group, mRNA and protein expressions of NF-κB and ICAM-1 in HSP children patients' serum IgA1 induced venous endothelial cells significantly increased with statistical difference (P < 0.05, P < 0.01). Compared with the HSP group, mRNA and protein expressions of NF-KB and ICAM-1 were obviously down-regulated after intervention of TFB (1.0, 0.5, 0.25 mg/mL), with statistical difference (P < 0.05, P < 0.01).

CONCLUSIONTFB could protect vascular damage by inhibiting in vivo high expression of NF-κB, reducing the production of IL-8, TNF-α, and NO in vascular endothelial cells of HSP children patients.

Bidens ; chemistry ; Child ; Flavones ; pharmacology ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Immunoglobulin A ; blood ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-8 ; metabolism ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Purpura, Schoenlein-Henoch ; blood ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVEThis study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish.

METHODSThe in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels.

RESULTSR1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs.

CONCLUSIONR1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

Animals ; Blood Vessels ; pathology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Collagen ; pharmacology ; Disease Models, Animal ; Drug Combinations ; Ginsenosides ; chemistry ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; enzymology ; physiology ; Humans ; Laminin ; pharmacology ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Proteoglycans ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism ; Zebrafish

BACKGROUNDPremature ovarian failure (POF) is a disease that affects female fertility but has few effective treatments. Ovarian reserve function plays an important role in female fertility. Recent studies have reported that hydrogen can protect male fertility. Therefore, we explored the potential protective effect of hydrogen-rich water on ovarian reserve function through a mouse immune POF model.

METHODSTo set up immune POF model, fifty female BALB/c mice were randomly divided into four groups: Control (mice consumed normal water, n = 10), hydrogen (mice consumed hydrogen-rich water, n = 10), model (mice were immunized with zona pellucida glycoprotein 3 [ZP3] and consumed normal water, n = 15), and model-hydrogen (mice were immunized with ZP3 and consumed hydrogen-rich water, n = 15) groups. After 5 weeks, mice were sacrificed. Serum anti-Müllerian hormone (AMH) levels, granulosa cell (GC) apoptotic index (AI), B-cell leukemia/lymphoma 2 (Bcl-2), and BCL2-associated X protein (Bax) expression were examined. Analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software.

RESULTSImmune POF model, model group exhibited markedly reduced serum AMH levels compared with those of the control group (5.41 ± 0.91 ng/ml vs. 16.23 ± 1.97 ng/ml, P = 0.033) and the hydrogen group (19.65 ± 7.82 ng/ml, P = 0.006). The model-hydrogen group displayed significantly higher AMH concentrations compared with that of the model group (15.03 ± 2.75 ng/ml vs. 5.41 ± 0.91 ng/ml, P = 0.021). The GC AI was significantly higher in the model group (21.30 ± 1.74%) than those in the control (7.06 ± 0.27%), hydrogen (5.17 ± 0.41%), and model-hydrogen groups (11.24 ± 0.58%) (all P < 0.001). The GC AI was significantly higher in the model-hydrogen group compared with that of the hydrogen group (11.24 ± 0.58% vs. 5.17 ± 0.41%, P = 0.021). Compared with those of the model group, ovarian tissue Bcl-2 levels increased (2.18 ± 0.30 vs. 3.01 ± 0.33, P = 0.045) and the Bax/Bcl-2 ratio decreased in the model-hydrogen group.

CONCLUSIONSHydrogen-rich water may improve serum AMH levels and reduce ovarian GC apoptosis in a mouse immune POF model induced by ZP3.

Animals ; Anti-Mullerian Hormone ; blood ; Apoptosis ; drug effects ; Female ; Granulosa Cells ; cytology ; Hydrogen ; chemistry ; pharmacology ; Mice ; Mice, Inbred BALB C ; Ovarian Reserve ; drug effects ; physiology ; Ovary ; drug effects ; metabolism ; Primary Ovarian Insufficiency ; blood ; metabolism ; prevention & control ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Water ; administration & dosage ; chemistry ; pharmacology ; Zona Pellucida ; drug effects ; physiology ; bcl-2-Associated X Protein ; metabolism