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To explore whether epigallocatechin-3-gallate (EGCG) improves renal damage in diabetic db/db mice and high-glucose- (HG-) induced injury in HK-2 cells by regulating the level of Klotho gene promoter methylation. Western blotting was used to detect the protein expression levels of DNA methyltransferase 1 (DNMT1), DNMT3a, DNMT3b, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and Klotho. The methylation level of the Klotho gene promoter was detected by pyrosequencing. Chromatin immunoprecipitation was used to detect the binding of the Klotho gene promoter to DNMT1 and DNMT3a. The expression of oxidative stress markers (reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and 8-hydroxy-2′-deoxyguanosine (8-OHdG)) and inflammatory cytokines (interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)) in kidney homogenates was also measured using ELISA. Klotho and DNMT3b protein expression was upregulated, while DNMT1, DNMT3a, TGF-β1, and α-SMA protein expression was downregulated after EGCG treatment. EGCG treatment also reduced the methylation level of the Klotho gene promoter as well as the binding of DNMT3a to the Klotho gene promoter. In addition, EGCG treatment significantly decreased the levels of ROS, MDA, 8-OHdG, IL-1β, IL-6, and TNF-α and increased the levels of CAT and SOD. Under HG conditions, EGCG regulated Klotho gene promoter methylation via DNMT3a and decreased the methylation level of the Klotho gene promoter, thereby upregulating the expression of the Klotho protein to exert its protective effect.

BACKGROUND The treatment of difficult common bile duct stones (CBDS) remains a big challenge around the world. Biliary stenting is a widely accepted rescue method in patients with failed stone extraction under endoscopic retrograde cholangiopancreatography. Fully covered self-expanding metal stent (FCSEMS) has gained increasing attention in the management of difficult CBDS. AIM To manufacture a drug-eluting FCSEMS, which can achieve controlled release of stone-dissolving agents and speed up the dissolution of CBDS. METHODS Customized covered nitinol stents were adopted. Sodium cholate (SC) and disodium ethylene diamine tetraacetic acid (EDTA disodium, EDTA for short) were used as stone-dissolving agents. Three different types of drug-eluting stents were manufactured by dip coating (Stent I), coaxial electrospinning (Stent II), and dip coating combined with electrospinning (Stent III), respectively. The drug-release behavior and stone-dissolving efficacy of these stents were evaluated in vitro to sort out the best manufacturing method. And the selected stone-dissolving stents were further put into porcine CBD to evaluate their biosecurity. RESULTS Stent I and Stent II had obvious burst release of drugs in the first 5 d while Stent III presented controlled and sustainable drug release for 30 d. In still buffer, the final stone mass-loss rate of each group was 5.19% ± 0.69% for naked FCSEMS, 20.37% ± 2.13% for Stent I, 24.57% ± 1.45% for Stent II, and 33.72% ± 0.67% for Stent III. In flowing bile, the final stone mass-loss rate of each group was 5.87% ± 0.25% for naked FCSEMS, 6.36% ± 0.48% for Stent I, 6.38% ± 0.37% for Stent II, and 8.15% ± 0.27% for Stent III. Stent III caused the most stone mass-loss no matter in still buffer or in flowing bile, which was significantly higher than those of other groups (P < 0.05). In vivo, Stent III made no difference from naked FCSEMS in serological analysis (P > 0.05) and histopathological examination (P > 0.05). CONCLUSION The novel SC and EDTA-eluting FCSEMS is efficient in diminishing CBDS in vitro. When conventional endoscopic techniques fail to remove difficult CBDS, SC and EDTA-eluting FCSEMS implantation may be considered a promising alternative.

The environmental risks posed by heavy metals (HMs) in animal manure are increasing because of the use of trace metals as additives in feedstuffs. Manure samples were collected, and published literature was reviewed in this study to systematically analyze the HMs content in animal manure and compare the results to different sources of animal manures. Results show that the distribution of HMs content in animal manure was skewed. The ranges were between not detected (ND)-147 mg·kg

Pseudomonas aeruginosa strains are potential pathogens that cause respiratory diseases in minks, and caused serious economic loss to mink breeding industry. In this study, we identified antimicrobial resistance and virulence genes in 125 P. aeruginosa isolates from mink in China from 2011 to 2020. The results showed at least one mutation in the gyrA (Thr83Val or Asp87Gly) and parC (Ser87 Leu) genes as well as single mutations in 56 isolates. At least 4-fold reductions in the fluoroquinolone minimum inhibitory concentration values were found when tested in the presence of PAβN in 23 isolates, while 44 isolates were positive for the extended spectrum β-lactamases and 15 antibiotic resistance genes were identified in this population with a prevalence between 1-32%, including qnrA, CTX-M-1G, ermB and C, cmlA, flor, catl, intl1, tetA, B, C, and D as well as sul1, 2, and 3 genes. Interestingly, one isolate carried ten resistance genes. Five virulence genes were detected, where exoS and algD were the most frequently detected (76.8%), which were followed by plcH (76%), lasB (73.6%), and pilB (31.2%). The isolates carrying the antibiotic resistance or virulence genes were genetically variable, suggesting a horizontal spread through the population. Hence, this study provides novel and important data on the resistance and pathogenicity of P. aeruginosa in farmed mink infections. These data provide important insights into the mechanism of fluoroquinolone resistance in P. aeruginosa, highlighting its usefulness in the treatment and control of P. aeruginosa infections in minks.

Erythropoietin (EPO) can reduce insulin resistance (IR) in adipocytes; however, it is unknown whether EPO can decrease IR in skeletal muscle. Here we investigated whether EPO could reduce IR in type 2 diabetic mouse skeletal muscle and its possible signaling mechanisms of action. Twelve-week-old db/db diabetic mice were employed in this study. Systemic use of EPO improved glucose profiles in type 2 diabetic mice after 4 and 8 weeks treatment. EPO up-regulated EPOR protein expression in skeletal muscle, and subsequently activated downstream signaling molecules such as JAK2, IRS-1, PI3K, AKT, and eNOS. We next constructed lentivirally-delivered shRNAs against EPOR and transfected skeletal muscle cells to knockdown EPOR. EPOR knockdown inhibited EPO induced JAK2, IRS-1, PI3K, AKT, eNOS signaling transduction, autophagy and Glut 4 translocation, as well as promoted apoptosis in skeletal muscle. Thus, EPO reduces skeletal muscle IR in type 2 diabetic mice via its specific receptor, EPOR. Possible mechanisms involved in its action may include increased autophagy and reduced apoptosis in type 2 diabetic skeletal muscles, which provides a new strategy for the treatment of IR.

A number of miRNAs associated with wound repair have been identified and characterized, but the mechanism has not been fully clarified. MiR-21 is one of wound-related lncRNAs, and the study aimed to explore the functional involvement of miR-21 and its concrete mechanism in wound healing. In this study, the rat model of skin wounds was established. The expression of miR-21, PTEN and related molecules of wound tissues or cells was determined by quantitative real-time PCR and Western blot, respectively. The regulatory role of miR-21 on PTEN was examined by luciferase reporter gene assay. Flow cytometry assay was applied to measure cell number changes. MiR-21 was upregulated at 6, 24, 48, 72 h after model establishment, and the increase reached a maximum at 24 h in wound tissues. MMP-9 expression presented the same tread as miR-21 and was significantly enhanced within 6 h of wound formation, and then remained to be increased to the maximum at 24 h. The increase of miR-21 was accompanied by the increase of cell total number and DCs ratio in wound fluids. MiR-21 overexpression significantly improved the healing of skin wounds and increased the ratio of DCs in rats. The results of using FL confirmed that miR-21 overexpression obviously promoted DCs differentiation. Additionally, miR-21 could activate AKT/PI3K signaling pathway via inhibition of PTEN. MiR-21 contributes to wound healing via inhibition of PTEN that activated AKT/PI3K signaling pathway to increase DCs. J. Cell. Biochem. 118: 3511-3519, 2017. © 2017 Wiley Periodicals, Inc.

The stemness of different side population (SP) cell subtypes in ovarian cancer cells was studied, and the heterogeneity of ovarian cancer stem cells was analyzed. The cisplatin-resistant human serous ovarian cancer cell line C13 was stained with the bisbenzimide Hoechst 33342. A flow cytometry-based fluorescence-activated sorting method was used to obtain lower-SP (LSP) cells, upper-SP (USP) cells, and non-SP cells (NSP) based on their sensitivity to the staining time and Hoechst dye concentration. The sphere-forming capability, expression levels of stem cell markers, resistance to high concentrations of cisplatin, and subcutaneous tumorigenicity in NOD/SCID mice of the different cell subtypes were evaluated. The C13 cells contained SP cells with stemness characteristics, and the LSP cell subtype expressed higher levels of stem cell markers, had higher in vitro sphere-forming capability, higher cisplatin resistance and higher in vivo subcutaneous tumorigenesis than USP cells (P0.05). NSP cells had no stemness. In conclusion, different subtypes of ovarian cancer SP cells have different stemness levels, and ovarian cancer stem cells may be heterogeneous.

Endoplasmic reticulum protein 29 (ERp29), an endoplasmic reticulum (ER) protein, participates in ER stress (ERS), but little is known about the association of ERp29 with ERS in the metastasis and prognosis of cancerous diseases. The present study revealed that ERp29 was important to ERS and interfered with the malignant behaviors of colorectal cancer (CRC). Experiments in in vitro and in animal models revealed that ERS inhibited the cell growth and suppressed the metastatic capacity of CRC cells, but ERp29 counteracted these effects. Furthermore, it was demonstrated that ERp29 recovered the migration and metastatic behaviors of CRC cells suppressed by ERS, mediated only when it combined with cullin5 (CUL5). ERp29 also relied on CUL5 to promote epithelial-mesenchymal transition. From the immunohistochemical examination of CRC tissues, the high expression of ERp29 was revealed to predict the poor prognosis of 457 CRC cases. The retrospective analysis of the clinicopathological data of patients with CRC was consistent with the results of the in vitro and in vivo experiments. Thus, ERp29 protected CRC cells from ERS-mediated reduction of malignancy to promote metastasis and may be a potential target of medical intervention for CRC therapy.

// Chun-Ting Hu 1, 2 , Li-Li Guo 1, 2 , Na Feng 1, 2 , Lei Zhang 3 , Na Zhou 1 , Li-Li Ma 1 , Lan Shen 1 , Gui-Hui Tong 1 , Qian-Wen Yan 1 , Shi-Jie Zhu 1 , Xiu-Wu Bian 4 , Mao-De Lai 5 , Yong-Jian Deng 1, 2 , Yan-Qing Ding 1, 2 1 Department of Pathology, Nanfang Hospital and School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China 2 Guangdong Provincial Key Laboratory of Molecular Tumor Pathology, Guangzhou 510515, China 3 Department of General Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China 4 Department of Pathology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China 5 Department of Pathology, School of Medical Sciences, Zhejiang University, Hangzhou 310006, China Correspondence to: Yong-Jian Deng, e-mail: dengyj@smu.edu.cn Yan-Qing Ding, e-mail: dyq@fimmu.com Keywords: colorectal cancer, hepatic sinusoidal endothelial cell, macrophage migration inhibitory factor, chemotaxis, metastasis Received: March 03, 2015 Accepted: May 20, 2015 Published: June 02, 2015 ABSTRACT Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases.

Danhong injection, a Chinese Materia Medica standardized product extracted from Radix Salviae miltiorrhizae and Flos Carthami tinctorii, is used extensively for the treatment of cerebrovascular diseases such as acutely cerebral infarction in clinic. In this study, we further investigated the mechanisms of Danhong injection on cerebral ischemia/reperfusion (I/R) damage relating to Nrf2/ARE signalling pathway in vivo and in vitro. For in vivo experiment, cerebral I/R injury was induced through middle cerebral artery occlusion. Rats were randomly divided into five groups: sham-operated group, I/R injury group, 6 mg/kg edaravone injection (positive control drug) group, 0.9 ml/kg Danhong injection (DHI-L) group, 1.8 ml/kg Danhong injection (DHI-H) group. The neurological score, cerebral infarction and brain edema were assessed while levels of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) in brain tissue were also evaluated. Transcription levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1) were analysed by real-time polymerase chain reaction. For in vitro experiment, mouse Neuro-2A cells were wounded with H2O2 then cell viability and mRNA transcriptions levels of Nrf2, HO-1, NQO1 were detected. Protein expression level of Nrf2 was assayed by western blotting. The results showed that Danhong injection could ameliorate neurological score, cerebral infarction and brain edema. Also it can increase levels of SOD, GSH and decrease of MDA and upregulate expressions of Nrf2, HO-1, NQO1 in ischemic brain tissue in vivo. What's more, it increased the mRNA transcription of Nrf2 and HO-1 and upregulated protein expression of Nrf2 in vitro. These findings suggested that Danhong injection could prevent I/R-induced brain damage through activating Nrf2/ARE signaling pathway.

This study was designed to investigate the processes underlying the neurotoxicity induced by β-amyloid peptide (Aβ) in the rat brain, as well as to examine whether scutellarin (Scu) can prevent this neurotoxicity. Thirty Wistar rats were randomly divided into 3 groups, i.e., untreated (control), treated with Aβ and treated with both Aβ and Scu. The treated rats were subjected to bilateral intracerebroventricular injection of Aβ(25-35) with or without subsequent dietary exposure to Scu. Learning and memory were assessed with the Morris water maze test; the activities of superoxide dismutase (SOD) and monoamine oxidase (MAO) were assayed biochemically; expression of the interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins was determined by immunohistochemistry; and neuronal apoptosis was detected with Annexin staining followed by flow cytometry. The animals treated with Aβ exhibited impaired learning and memory; reduced SOD and elevated MAO activity, elevated protein levels of IL-1β, IL-6 and TNF-α; and a higher percentage of apoptotic neurons in the brain. Interestingly, all of these effects were ameliorated by administration of Scu. These findings indicate that the deficits in learning and memory demonstrated by the rats receiving Aβ are due to elevated oxidative stress and inflammation, which result in apoptosis and that Scu may prevent these deleterious effects.

Differences in theories, application forms and evaluation standards about curative effect between traditional Chinese medicine and modern medicine lead to not only question safty and effectiveness but also hinder development and internationalization of traditional Chinese medicine. Combination of common problems in traditional Chinese new drug registration with experiences in research on traditional Chinese new drugs of prevention and treatment of coronary heart disease elucidate application value about theory of disease-syndrome combination and multi-objective optimization in several ways such as the indications positioning, preparation process optimization, preclinical efficacy evaluating and clinical assessmenting of efficacy and analysis development prospect.

Coronary heart disease (CHD) with pathological characteristics of atherosclerotic coronary artery and myocardial ischemia is more likely to cause phlegm and blood stasis. The current study showed high morbidity and severity of phlegm and blood stasis syndrome in CHD, which has attracted increasing attention. This paper introduced the knowledge of traditional Chinese medicine (TCM) about the CHD with phlegm and blood stasis syndrome. On the basis of modern medical studies, we found a close relationship between phlegm and blood stasis syndrome and blood fat, hemorheology, oxygen free radical, blood coagulation function, inflammation, sugar metabolism and other related gene expression changes. We reviewed TCM therapies and prescriptions and found that TCM therapies achieved a good clinical effect through the understanding of etiology and pathogenesis and differentiation in organs. Besides, phlegm and blood stasis removing prescription relieved the objective indicators, which would have a good development prospects.

Epigallocatechin-3-gallate (EGCG), extracted from green tea, has been shown to have antioxidative activity. In the present study, we evaluated the effect of EGCG on the kidney function in db/db mice and also tried to investigate the underlying mechanism of the renoprotective effects of EGCG in both animals and cells. EGCG treatment could decrease the level of urinary protein, 8-iso-PGF2a, and Ang II. Moreover, EGCG could also change the level of several parameters associated with oxidative stress. In addition, the protein expression levels of AT-1R, p22-phox, p47-phox, p-ERK1/2, p-p38 MAPK, TGF-β1, andα-SMA in diabetic db/db mice were upregulated, and all of these symptoms were downregulated with the treatment of EGCG at 50 and 100 mg/kg/d. Furthermore, the pathological changes were ameliorated in db/db mice after EGCG treatment. HK-2 cell-based experiments indicated that EGCG could inhibit the expression of MAPK pathways, which is the downstream effector of Ang II mediated oxidative stress. All these results indicated that EGCG treatment could ameliorate changes of renal pathology and delay the progression of DKD by suppressing hyperglycemia-induced oxidative stress in diabetic db/db mice.

Allium sativum L. (DaSuan in Mandarin) is a traditional Chinese herb that has been used to prevent and heal cardiovascular diseases.To study the effects of allitridi (an active constituent of Allium sativum L.) and amiodarone on the conduction system and on reverse use-dependence in the isolated hearts of normal rats and rats with myocardial infarction (MI).Male Sprague Dawley rats, with a ligated left anterior descending coronary artery, were used as myocardial infarction models to investigate the biological effects of the traditional Chinese herb. A single-phase electrode assay and isolated heart perfusion administration methods were employed to study and compare the electrophysiological effects of allitridi and amiodarone on normal and MI rats. Monophasic action potential (MAP) in vitro, effective refractory period (ERP) and monophasic action potential duration (MAPD)/ERP were measured to investigate reverse use-dependence (RUD) with allitridi and amiodarone. Moreover, bundle maps and heart rates were analyzed to evaluate the electrophysiological effects of allitridi on the conduction system of the cardiac muscles. Coronary flow was used to study the beneficial effects of the two drugs on the bundle of His in myocardial infraction.(1) Allitridi and amiodarone can reduce the infarction model of the His bundle (A-H, H-V) conduction and cardiac sinus rhythm in normal rats and isolated rat hearts. After washing in physiological solution (AK-H) for 15 min, the allitridi group partially recovered, but the amiodarone group did not recover. (2) Allitridi and amiodarone had no significant effects on the change of MAPD(90) or ERP in normal and MI rat hearts at different pacing frequencies (200, 250 and 300 beats/min), which indicated no RUD. In addition, the effects of allitridi on prolonging MAPD(90) and ERP were weaker than those of amiodarone (P0.01). The effects of allitridi on myocardial repolarization and its variation rate were also weaker than those of amiodarone (P0.01). However, the prolonged administration of allitridi still did not cause RUD. Allitridi and amiodarone can significantly increase the ERP/APD(90) rate of the isolated heart ventricles of normal rats and rats with MI.We propose that allitridi and amiodarone have similar effects on the cardiac conduction system and on the electrophysiology without RUD, which may be the result of the use of multi-channel blockers, such as calcium channel blockers and IKr and IKs channel blockers. Allitridi may be a promising antiarrythmic drug.

To examine the protective effects of scutellarin (Scu) on rats with learning and memory deficit induced by β-amyloid peptide (Aβ). Fifty male Wistar rats were randomly divided into 5 groups: control, sham operation, Aβ, Aβ+Scu, and Aβ+piracetam groups. Aβ25–35 was injected into the lateral ventricle (10 μg each side). Scu (10 mg/2 mL) or piracetam (10 mg/2 mL was intragastrically administered per day for 20 consecutive days following Aβ treatment. Learning and memory was assessed with Morris water maze test. The protein and mRNA levels of nicotinic acetylcholine receptor (nAChR) α4, α7, and β2 subunits in the brain were examined using Western blotting and real-time PCR, respectively. The activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the brain and plasma were measured using Ellman's colorimetric method. In Aβ group, the escape latency period and first platform cross was significantly increased, and the total number of platform crossings was significantly decreased, as compared with the control and the sham operation groups. Both Scu and piracetam treatment significantly reduced the escape latency period and time to cross platform, and increased the number of platform crosses, but there were no significant differences between Aβ+Scu and Aβ+piracetam groups. In Aβ group, the protein levels of nAChR α4 and α7 subunits in the cerebral cortex were significantly decreased by 42%–47% and 58%–61%, respectively, as compared to the control and the sham operation groups. Scu treatment caused upregulation of α4 and α7 subunit proteins by around 24% and 30%, respectively, as compared to Aβ group, but there were no significant differences between Aβ+Scu and Aβ+piracetam groups. The protein level of nAChR β2 subunit had no significant difference among different groups. The mRNA levels of nAChR α4, α7, and β2 subunits were not significantly changed. In Aβ group, the activities of AChE and BuChE in the brain were significantly increased, but were significantly decreased in the plasma, as compared to the control and the sham operation groups. Scu or piracetam treatment restored the activities in brain and plasma nearly to the levels in the control group. The results suggest that Scu may rescue some of the deleterious effects of Aβ, possibly by stimulating nAChR protein translation and regulating cholinesterase activity.

Sleep deprivation (SD) has been taken as an independent predictor for cardiovascular risks, which was closely related to the increased morbidity and mortality in coronary heart disease (CHD). In this article, after reviewing the impact of modern medical method sleep deprivation on CHD and studies on principle method recipe medicines for preventing and treating CHD, the authors observed the autonomic nerve dysfunction, hormonal metabolism dysfunction, endothelial dysfunction and inflammatory responses after sleep deprivation, which can cause or aggravate CHD. On the basis of the traditional Chinese medicine theories of "heart dominating the blood and vessels and the mind", the authors considered that traditional Chinese medicines can tonify heart and soothe the nerves, reducing all of the risk factors through multi-target and multi-pathway, and improve sleep and decrease the risk factors caused by sleep deprivation, which provides a new idea for the prevention and treatment of CHD.

To explore the effect of Danlou Tablet (DT) on arrhythmia model rats induced by transient myocardial ischemia/reperfusion (I/R).Totally 45 healthy Wistar rats were randomly divided into 3 groups, the sham-operation group, the model group, and the DT group, 15 in each group. Rats in the sham-operation group and the model group were administered with distilled water by gastrogavage at the daily dose of 0.1 mL/kg. Rats in the DT group was administered with 0.53 g/mL DT suspension by gastrogavage at the daily dose of 0.1 mL/kg. All medication was lasted for 10 successive days. The myocardial I/R experiment was performed at 1 h after the last gastrogavage. ECG was performed before ligation and at I/R. The jugular arterial blood pressure of all rats was measured during the whole course. ST segment changes were observed at each time point of I/R. The ventricular fibrillation, the premature ventricular, the number and the duration of ventricular tachycardia within 30 min reperfusion were also observed. Activities of Na(+)-K+ ATPase and Ca2+ ATPase in the myocardium homogenate were detected as well.The jugular arterial blood pressure and the heart rate were slightly lower in the DT group than in the model group, but with no statistical difference (P0.05). Compared with the sham-operation group, the degree of ST segment was obviously elevated in the model group at 0, 5, and 7 min (P0.05). It was significantly lower in the DT group than in the model group (P0.01). ST seg ment was more elevated at 5 min than at 0 min in the model group, but the degree of ST segment elevation was still obviously lower in the DT group than in the model group (P0.05). There was no statistical difference in the degree of ST segment elevation at 7 min between the two groups (P0.05). At 0 min when the decrement of ST segment exceeded one half the ischemia, there was no statistical difference in the degree of myocardial ischemia between the model group and the DT group (P0.05). Compared with the model group, the incidence of fatal and nonfatal ventricular fibrillation, the frequency and duration of ventricular tachycardia and premature ventricular beats were obviously lessened, and activities of Na(+)-K+ ATPase and Ca(2+)-ATPase increased (all P0.05).DT could significantly protect arrhythmias induced by transient I/R. Its effect might be related to lowering the degree of myocardial ischemia, and increasing ion transport channel related enzyme activities.

To study the protective mechanism of Huoxue Anxin Recipe (HAR) on peroxidation damage of acute myocardial infarction (AMI) rats.The AMI rat model was established by occluding the left anterior descending coronary artery. Compound Danshen Dripping Pill (CDDP) was used as the positive control. CDDP and HAR were administered to rats for 7 successive days since modeling. The heart function was detected using color Doppler echocardiography. Activities of induced nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), total superoxide dismutase (tSOD) activity, and contents of malondialdehyde (MDA) were detected by ultraviolet spectrophotometer method.Compared with the sham-operation group, ejection fraction (EF) and fraction shortening (FS) rate significantly decreased in the model group (P0.01). Compared with the model group, EF and FS rate significantly increased in the HAR group, showing statistical difference (P0.05). There was no statistical difference in activities of serum iNOS, eNOS, or tSOD among all groups (P0.05). Compared with the sham-operation group, iNOS activities and MDA contents significantly increased in the myocardial tissue of the model group (P0.01), activities of eNOS and tSOD significant decreased (P0.01). Compared with the model group, iNOS activities in the myocardial tissue, MDA contents both in serum and the myocardial tissue significantly decreased (P0.05), activities of eNOS and tSOD significantly increased in the HAR group (P0.05). There was no statistical difference in each index between the CDDP group and the HAR group (P0.05).HAR could significantly improve cardiac functions of AMI rats. Its roles might be associated with regulating imbalanced iNOS/eNOS expressions and alleviating peroxidation damage of the myocardial tissue.

To explore whether a tissue-engineered construct composed of autogenous endothelial cells, osteoblasts and a new bioresorbable nano-hydroxyapatite/recombinant human-like collagen/polylactic acid (nHA/RHLC/PLA) would enhance bone regeneration and repair femoral head defects in canine models.The bone marrow stem cells (BMSCs) were isolated from bone marrow of canine ilium and cultured in Dulbecco's modified eagle medium:nutrient mixture F-12 culture media for 1 week and the second-generation BMSCs were further induced by osteogenic medium (1×10(-8) mol/L dexamethasone, 10 mmol/L B-sodium glycerophosphate and 50 µg/ml vitamin C) and by endothelial cell grow medium (vascular endothelial growth factor and basic fibroblast growth factor) for 14 days in vitro. Thus BMSCs were induced into ECs and OBs. After the second passage, cells were digested and collected.And cell density was adjusted to 1.0×10(6)/ml.The cells and nHA/RHLC/PLA scaffold were co-cultured for 2-4 hours then nHA/RHLC/PLA scaffold composites prepared. Cavity defects of 8 mm in diameter and 10 mm in height were made in femoral heads.The nHA/RHLC/PLA scaffold composited with ECs and osteoblasts (OBs) (group A) and composited with OBs (group B) were inserted into different defects while cell-free nHA/RHLC/PLA scaffold served as controls (group C). New bone formation and defect repair were evaluated at 3 and 6 months by radiographic examination, histology and bone histomorphometry.New bone formation was evident as early as 3 months in groups A, B and C.At 6 months, abundant bone tissue within defects was observed in group A. The control animals with cell-free scaffold showed less bone formation at both timepoints.The scaffold of nHA/RHLC/PLA was degraded and absorbed gradually with the formation of new bone tissues.Histology and bone histomorphometry further revealed significantly increased trabecular bones in group A compared with groups B and C at 6 months postimplantation (P0.01).More abundant new bone tissue may be found in the bone defect areas implanted with osteoblast-endotheliocyte composite than osteoblasts composite and scaffold materials only.ECs and osteoblasts derived from BMSC are ideal seed cells for repairing femoral head defects.

To investigate the microRNAs (miRNAs) expression profile of acute myocardial infarction (AMI) rats and the regulating effects of Huoxue Anxin Recipe (, HAR) on angiogenesis-related miRNAs and genes.Forty-five Wistar rats were randomly assigned to 3 groups according to a random number table: sham, AMI, and AMI+HAR groups (15 in each group). AMI rats were established by ligation of the left descending coronary artery. HAR was intragastrically administered to rats of the AMI+HAR group for successive 21 days since modeling, meanwhile the same volume of 0.9% normal saline was administered to rats of the sham and AMI groups. Doppler echocardiography was used for noninvasive cardiac function test. Hematoxylin and eosin staining was used to observe the histopathological change. miRNAs expression profile was detected by quantitative realtime polymerase chain reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor (VEGF), and a target gene of miR-210 was further detected by qRT-PCR and Western blot, respectively. The microvessels density of myocardium was evaluated by CD31 immunostaining.Compared with the sham group, ejection fraction (EF) and fractional shortening (FS) values were decreased significantly in the AMI group (P0.01), while the infarction area and the interstitial collagen deposition were increased obviously. As for the AMI+HAR group, EF and FS values were increased significantly (P0.05 vs. AMI group), and the infarction area was reduced and the interstitial collagen deposition were alleviated significantly. Total of 23 miRNAs in the AMI group expressed differently by at least 1.5 folds compared with those in the sham group; 5 miRNAs in the AMI+HAR group expressed differently by at least 1.5 folds compared with those in the AMI group. Among them, miR-210 was low in the AMI group and high in the AMI+HAR group. The relative mRNA and protein expressions of VEGF were decreased significantly in the AMI group (P0.05 vs. sham group), and increased significantly in the AMI+HAR group (P0.01 vs. AMI group). CD31 expression area and optical intensity were decreased significantly in the AMI group (P0.05 vs. sham group), and increased significantly in the AMI+HAR group (P0.01 vs. AMI group).HAR could reduce the infarction area, alleviate the interstitial fibrosis and improve the cardiac function of AMI rats. Those effects could be related to promoting myocardium angiogenesis of HAR by up-regulating miR-210 and VEGF.

125I seeds coated with titanium are considered a safe and effective interstitial brachytherapy for tumors, while the cost of 125I seeds is a major problem for the patients implanting lots of seeds. The aim of this paper was to develop a novel silicone coating for 125I seeds with a lower cost. In order to show the radionuclide utilization ratio, the silicone was coated onto the seeds using the electro-spinning method and the radioactivity was evaluated, then the anti-tumor efficacy of silicone 125I seeds was compared with titanium 125I seeds. The seeds were divided into four groups: A (control), B (pure silicone), C (silicone 125I), D (titanium 125I) at 2 Gy or 4 Gy. Their anti-tumour activity and mechanism were assessed in vitro and in vivo using a human extrahepatic cholangiocarcinoma cell line FRH-0201 and tumor-bearing BALB/c nude mice. The silicone 125I seeds showed higher radioactivity; the rate of cell apoptosis in vitro and the histopathology in vivo demonstrated that the silicone 125I seeds shared similar anti-tumor efficacy with the titanium 125I seeds for the treatment of extrahepatic cholangiocarcinoma, while they have a much lower cost.

To study the protection effects and the mechanisms of Huoxue Anxin Recipe (HAR) on acute myocardial infarction (AMI) rats.The AMI Wistar rat model was prepared by ligating the left anterior descending coronary artery. By taking elantan long as the positive control drug, HAR was extracted from Chinese herbs by modern pharmacological methods and composed according to theories of Chinese medicine (CM). The medication time started from the day of modeling to the 21st day of the modeling. The heart function, the morphological changes of the heart, changes in the mRNA and protein levels of toll like receptor 4 nuclear factor kappa B tumor necrosis factor alpha (TLR4-NFkappaB-TNFalpha) pathway were observed.Compared with the sham-operation group, the ejection fraction (EF) and left ventricular fractional shortening (FS) significantly decreased (P0.01), the left ventricular intemal dimension end-diastolic (LVIDd) and left ventricular internal dimension end-systolic (LVIDs) significantly increased (P0.01), the mRNA and protein levels of TLR4-NFkappaB-TNFalpha channel significantly increased in the model group (P0.01). The infarction in the front wall of the left ventricle was obviously seen, accompanied with severe inflammatory cell infiltration and collagen deposition in model group. Compared with the model group, the EF and FS significantly increased, LVIDd and LVIDs significantly decreased in the positive control group, the high and low dose HAR groups (P0.05, P0.01). The infracted area of the front wall of the left ventricle was obviously contracted. The inflammatory cell infiltration and collagen deposition were obviously alleviated. In the high and low dose HAR groups, the mRNA and protein expression levels of TLR4-NFkappaB-TNFalpha significantly decreased (P0.05, P0.01), but no inhibition was found in the positive control group.HAR could significantly improve the morphological structures and functional abnormality induced by myocardial ischemia in AMI rats. Its effects was correlated with inhibiting TLR4-NFkappaB-TNFalpha pathway.

MicroRNAs (miRNAs) regulate gene expression in a post-transcriptional sequence-specific manner. Expression profile analysis of miRNAs in liver is necessary to understand the possible roles of miRNAs in hepatitis B virus (HBV) infection.We obtained HBV-positive liver samples from mice, using HBV transgenic mouse model. MicroRNA expression was analyzed with Agilent microRNA Array and validated using quantitative real-time PCR analysis.RNA samples from liver of HBV transgenic mouse liver exhibited notably different miR profiles from negative controls for both maternities and fetuses. Significant differences in expression profile of miRNAs were also found in fetal and maternal group of HBV transgenic mouse model. Expression of miR-1892 and miR-1187 decreased by approximately 2-fold (p0.01), whereas expression of miR-92a increased by more than 6-fold (p0.001) in the fetal group, as validated by qPCR.Deregulation of miRNAs expression in fetal livers could be implicated in HBV intrauterine infection. Further study is warranted to identify the target genes of these miRNAs and their function. Besides, these data might offer new ideas for blocking HBV intrauterine infection.

Haemophilus parasuis is the etiological agent of Glässer’s disease, which is characterised by fibrinous polyserositis, meningitis and polyarthritis, causing severe economic losses to the swine industry. In this study, a loop-mediated isothermal amplification (LAMP) test was developed to improve the specificity, facility and speed of diagnosis of H. parasuis isolates. The LAMP assay rapidly amplified the target gene within 50 min incubation at 63 °C in a laboratory water bath. The LAMP amplicon could be visualised directly in the reaction tubes following the addition of SYBR Green I dye. The detection limit of this LAMP method was 10 CFU/mL, which was 10 times more sensitive than the earlier 16S rRNA polymerase chain reaction (PCR) test conducted by Oliveira, Galina and Pijoan (2001), and no cross-reactivity was observed from other non-H. parasuis strains. This LAMP test was evaluated further on 187 clinical specimens from pigs suspected of being infected with H. parasuis. Forty-three were found positive by bacterial isolation of H. parasuis, as well as by the 16S rRNA PCR and LAMP tests. The 43 H. parasuis isolates were classified into 9 serovars and had 37 genetic patterns when analysed by pulsed-field gel electrophoresis (PFGE). This displayed that various H. parasuis serovars and genotypes were widely distributed in South China. Therefore, the speed, specificity and sensitivity of the LAMP test, the lack of a need for expensive equipment, and the visual readout showed great potential for a correct clinical diagnosis of H. parasuis in favour of controlling Glässer’s disease.

To investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).Wistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).(1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P0.01, P0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P0.05 and P0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P0.05). The concentration of HSP70 did not show anystatistical differences in all groups (P0.05).The TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

To investigate the influence of Scutellarin on the oxidative stress and neuronal apoptosis in rats with dementia and to reveal therapeutic mechanism of the drug to Alzheimer's disease.Wistar rats were randomly divided into 5 groups: normal control group, sham group, model group,scutellarin group and positive control group. The animal model with dementia was by bilateral ventricle injection with beta-amyloid peptide (AP) 25-35 and intraperitoneal injection with D-galactose. Learning and memory ability of rats were detected by Morris Water Maze test; Histopathology of the cerebral cortex and hippocampus were observed under light microscope; Changes of Nissl's body in the hippocampus were survey by Nissl staining; The activities of SOD and MAO were detected by xanthinoxidase method and chemical method, respectively, and neuronal apoptosis was measured using flow cytometry.Compared with control group and sham group, the ability of learning and memory of the rats in model group was decreased; Neuropathological changes were observed in the hippocampus; The activity of SOD was decreased while the activity of MAO increased in brain tissues; Neuronal apoptosis percentage was increased. After treated with Scutellarin, learning and memory ability of rats with dementia was improved; The pathologic changes were alleviated; The activity of SOD was up-regulation and the activity of MAO was decreased; Neuronal apoptosis percentage was declined. No significant difference between the scutellarin group and positive drug control group was found.Scutellarin might play an therapeutic role by inhibiting oxidative stress and apoptosis in AD treatment.

To observe the effect of Scutellarin (Scu) on expressions of nicotinic acetylcholine receptor (nAChR) subunit protein and mRNA in dementia rats, and to study its possible mechanism on dementia.Forty-two Wistar rats were randomly divided into 5 groups, i.e., the normal control group (n=6), the sham-operative group (n=6), the memory deficit model group, the Scu treatment group (n=10), and the positive drug (piracetam) control group (n=10). The dementia rat model was established by bilateral ventricle injection with beta-amyloid peptide (Abeta)(25-35) and abdominal cavity injection with D-galactose. Rats in the Scu treatment group or the piracetam control group were treated with Scu or piracetam by gastrogavage. The learning and memory ability of rats were detected by Morris water maze test, nAChR alpha4, alpha7, and beta2 subunits at protein and mRNA levels were detected by Western blot and Real-time PCR respectively.Compared with the normal control group and the sham-operative group, the learning and memory ability decreased in rats of the model group (P0.05). nAChR alpha4 and alpha7 subunit protein expressions were obviously lowered (P0.05), but changes of beta2 were not obvious. No obvious change of mRNA expressions in all three nAChR subunits was seen (P0.05). After treatment of Scu, the learning and memory ability was greatly improved, nAChRs alpha4 and alpha7 subunit protein expressions increased in rats with dementia (all P0.05). No obvious change of mRNA expressions in all three nAChR subunits was seen (P0.05). No obvious difference of each index was shown between the Scu treatment group and the positive drug (piracetam) control group.Scutellarin could improve the learning and memory ability of dementia rats. Its mechanism might be associated with its up-regulation of nAChR expressions.

To investigate the in vivo effects of nanosized titanium dioxide (TiO2) on the main organs, particularly the reproductive system, of male mice.Forty-five male ICR mice aged 6 weeks were equally and randomly divided into 2 experimental groups and a control group, intraperitoneally injected with 200 and 500 mg/kg nanosized TiO2 and equal volume of saline, respectively, every other day for 5 times. One week after drug cessation, we obtained the coefficients of the main organs, serum biochemical parameters and the levels of gonadal hormones (T and E2), analyzed the pathological changes of the main organs by HE staining, observed sperm count, motility and abnormality under the microscope, and detected germ cell apoptosis by TUNEL.Compared with the control, the low-dose (200 mg/kg) group showed no significant changes in the above parameters, while the high-dose (500 mg/kg) group exhibited a decrease in the coefficients of the liver, heart and kidneys, and a significant increase in such serum biochemical parameters as ALT, ALT/AST and BUN (P0.05). The high-dose group also showed significantly reduced sperm density and motility, increased sperm abnormality and germ cell apoptosis (P0.05), but no obvious pathological changes in the liver, kidney spleen, testis and epididymis.Low-dose nanosized TiO2 has no obvious effect on male mice, but high-dose may significantly reduce their sperm count and function and induce germ cell apoptosis, although its damage on the liver and kidney function is slight.

To investigate the effects of effective components extracted from medicines compatibility on ischemic myocardial remodeling.The animal models of ischemic myocardial remodeling was copied by ligating the left anterior descending branch of coronary artery in rat. And then decoction and effective components extracted from the same medicines were administrated to rats. The heart function was detected by ultrasound diagnostic apparatus and the heart morphology was examined by Hematoxylin and Eosin, picrosirius staining respectively on the 3rd and 56rd day.Both concentrated decoction and effective components compatibility showed good restrained effects on ventricle malignant remodeling, such as improved heart function, reduced the area of myocardial infarction, lowered heart index, mitigated the fibrosis level and decreased apoptosis cell numbers in infarction and non-infarction areas. The difference were statistically significant when compared with the control group (P0.01, P0.05), and effective components treated group had better effects than concentrated decoction group (P0.05). Concentrated decoction group showed little effects on the 3rd day, while effective components group showed significant effect (P0.05).Effective components compatibility has better effects on inhibiting the process of ischemic myocardial remodeling than concentrated decoction. Effective components compatibility has better effects on ventricle malignant remodeling by eliminating the interfrence non-pharmacodynamic elements of the medicine.

There are many differences between traditional Chinese medicine (TCM) and western medicine, and the use of TCM has its feature under the guide of TCM theory. Determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs is the base of TCM treatment, the main form of TCM apply is compatibility, formula corresponding differentiation of symptoms and signs is the key for TCM to get better effects, integrated adjustment is the TCM mode of action. All above determined the methods and standards to evaluate the pharmacodynamic effect of TCM cannot completely accord to Western medicine. Use modern technology and combine the feature of TCM to estabilish the method for effects evaluation and standard system of TCM, which is the key point to correctly appraise the effects of TCM. Stabilized quality control is the precondition of pharmacodynamic action appraisement, animal models combined disease and symptoms are the necessary tools for effect appraisement, integrated adjustment need mutiple index to appraise pharmacodynamic action, the method to avoid subjective error in TCM reseach is using mathematical model and parameter to scientificly analyse data, also the explaination of mechanism of action need standard clinical pharmacology experimental date.

AIM: To investigate the activity of vesicular glutamate transporter-3 (VGLUT3) in a visceral hyperalgesia rat model of irritable bowel syndrome, and the role of mast cells (MCs). METHODS: Transient intestinal infection was induced by oral administration of Trichinella spiralis larvae in rats. On the 100th day post-infection (PI), the rats were divided into an acute cold restraint stress (ACRS) group and a non-stressed group. Age-matched untreated rats served as controls. The abdominal withdrawal reflex was used to measure the visceromotor response to colorectal distension (CRD). The expression levels of VGLUT3 in peripheral and central neurons were analyzed by immunofluorescence and western blotting. RESULTS: VGLUT3 expression in the L6S1 dorsal root ganglion cells was significantly higher in the PI group than in the control group (0.32 ± 0.009 vs 0.22 ± 0.008, P < 0.01), and there was no significant difference in the expression of VGLUT3 between MC-deficient rats and their normal wild-type littermates. Immunofluorescence showed that the expression levels of VGLUT3 in PI + ACRS rats were enhanced in the prefrontal cortex of the brain compared with the control group. CONCLUSION: VGLUT3 is involved in the pathogenesis of visceral hyperalgesia. Coexpression of c-fos, 5-hydroxytryptamine and VGLUT3 after CRD was observed in associated neuronal pathways. Increased VGLUT3 induced by transient intestinal infection was found in peripheral nerves, and was independent of MCs. Moreover, the expression of VGLUT3 was enhanced in the prefrontal cortex in rats with induced infection and stress.

The zinc-finger motif found in many transcription factors is thought to be important for human heart development and diseases. This study reports cloning and expression analysis of a novel human zinc finger protein ZNF18 cDNA. The ZNF18 cDNA is 2,767 bp in length encoding a 549-amino-acid protein that contains a SCAN-box, a KRAB box and five consecutive C2H2 zinc finger motifs. ZNF18 shows high similarity with mouse Zfp535 (identity 77%). The ZNF18 gene is located on human chromosome 17p12-p13 with 9 exons and 8 introns. ZNF18 was ubiquitously expressed in adult mouse tissues except heart as revealed by Northern blot. Whole-mount in situ hybridization showed that expression of ZNF18 in mouse embryos was very dynamic. Expression was predominantly in extraembryonic tissues of E7.5 mouse embryo. By E8.5, ZNF18 began to express in anterior of trunk, and became abundant in tail and heart at E9.0, especially in the embryo heart at E10.5. These expression results suggest that ZNF18 may play an important role in the development of embryo heart.

The adrenergic agonist phenylephrine (PE) induced typical [Ca2+]i oscillations in freshly isolated rat hepatocytes, and the photodynamic effect of Victoria Blue BO on induced calcium oscillations was investigated. PE induced calcium oscillations disappeared after Victoria Blue BO photodynamic action (in 20 out of 26 experiments), or were present with a decreased amplitude (in 1 out of 26 experiments) or frequency (in 5 out of 26 experiments). After VBBO photodynamic action, the basal calcium level remained at pre-stimulatory level. In comparison, after treatment with the mitochondrial uncoupler CCCP, calcium oscillations disappeared in all experiments, with a significant increase in basal [Ca2+]i. After washing out of CCCP, basal [Ca2+]i returned to pre-stimulation level and calcium oscillations recovered. The photodynamically-induced blockade of calcium oscillations was irreversible, and this effect was present independent of the time interval between VBBO incubation and light illumination. Calcium oscillation blockade was not seen with VBBO in the dark at the concentration used, nor was it seen with light illumination alone. Taken together, it is concluded that VBBO photodynamic action could irreversibly block calcium oscillations in rat hepatocytes, possibly due to blockade of mitochondrion calcium release.

To identify the physiological concentration ranges of norepinephrine (NE), vasopressin (VP), and ATP in the rat liver.Rat hepatocytes were isolated by collagenase perfusion. Isolated cells were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester (Fura-2 AM). The effects of different concentrations of norepinephrine, vasopressin, and ATP on intracellular calcium concentration ([Ca2+]i) increases in the freshly isolated rat hepatocytes were investigated. [Ca2+]i was measured by microfluorometry and recorded as fluorescence ratios (F340/F380).NE, VP, and ATP induced increases in [Ca2+]i in a concentration-dependent manner. At lower concentrations, [Ca2+]i tended to show an oscillatory increase; with increasing concentrations, [Ca2+]i in more cells tended to show phasic or plateau increases. NE, VP, and ATP concentrations likely to induce an oscillatory [Ca2+]i response were 100 - 500 nmol/L, 50 - 100 pmol/L, and1 micromol/L respectively.Physiological concentrations of NE, VP, and ATP are 100 - 500 nmol/L, 50 - 100 pmol/L, and1 micromol/L respectively in the rat liver.