BACKGROUND: One of important mechanisms underlying myocardial fibrosis is that transforming growth factor β1(TGF-β1) stimulates the proliferation and differentiation of cardiac fibroblasts via Smads signaling pathway.Previous studies have confirmed that tanshinone ⅡA can effectively inhibit myocardial fibrosis.But whether blockage of TGF-β1/Smads signaling pathway is involved in this process remains unclear. OBJECTIVE: To investigate the effects of tanshinone ⅡA on TGF-β1 signal transduction in rat cardiac fibroblasts. METHODS: Neonatal rat cardiac fibroblasts were harvested by trypsin digestion and differential attachment and treated with 5 μg/L TGF-βI and different concentrations of tanshinone Ⅱ A(106,10-5 and 10-4 mol/L).At 6,12,and 24 hours after TGF-β1 application,fibronectin expression was detected by reverse transcription-polymerase chain reaction and Western blot analysis.At 15,30,60,and 120 minutes after TGF-β1 application,Smads protein expression was determined by Western blot analysis. RESULTS AND CONCLUSION: Fibronectin mRNA and protein expression began to increase at 6 hours after TGF-β1 application and was 1.3 and 1.8 times higher than initial level,respectively(P < 0.01),at 24 hours after TGF-β1 application.Phosphorylated Smad2/3 protein expression began to increase at 15 minutes after TGF-β1 application,peaked at 1 hour,decreased at 2 hours,but it was still 3.9 times higher than initial level(P < 0.01).Tanshinone ⅡA(10-5 and 10-4 mol/L)pretreatment downregulated fibronectin and phosphorylated Smad2/3 expression(P < 0.05 or P < 0.01)in a dose-dependent manner.These findings demonstrate that TGF-β1 induced fibronectin protein and mRNA expression and Smad2/3 protein expression in a time-dependent manner.Tanshinone ⅡA against myocardial fibrosis was likely related to its inhibition of TGF-β1-induced Smad2/3 phosphorylation and blockage of TGF-β1/Smads signaling pathways within cardiac fibroblasts.

BACKGROUND: Preactivation of peripheral blood mononuclear cells (PBMCS) is one of the most important eerly events and facilitating factor for the formation of atherosclerosis. Tanshinone is a lipolytic component extracted from traditional Chinese medicine of denshen, it has definite anti-atherosclerotic effect.OBJECTTVE: To analyze whether PBMCS preactivation existed at early essential hypertension, and investigate the effects of tanshinone on inhibiting the PBMCS activation cultured in vitro by detecting the adhesion and excretory activities of PBMCS.DESTGN: A case-controlled analysis.SETTING: Department of Emergency and Research Room of Traditional Chinese Medicine, Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.PARTTCTPANTS: Thirty patients with untreated essential hypertension or with withdrawal from antihypertensives for at least 2 weeks were selected from the Department of Cardiology, Tongji Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology from January 2003 to October 2004, including 16 males and 14 females, aged (44.6±7.4) years, body mass index of (26.2±4.5) kg/m2, average disease course of (38.5±16.9) months.Informed contents were obtained from all the subjects. Their hypertension was grade Ⅰ-Ⅱ according to the diagnostic standards for hypertension by WHO/ISH in 1999. Secondary hypertension, organic heart disease, hyperglyceridemia,diabetes mellitus, liver and kidney dysfunction, heart, brain, kidney, vessel and other target damaged induced by infection and other clinical conditions and hypertension were excluded by history, physical examination and assistant examination.Another 30 healthy physical examinees with normal blood pressure were enrolled as the normal control group. Human umbilical vein endothelial cells (Species Reserving Center of Wuhan University); Tanshinone injection (Yaan Sanjiu Pharmaceutical, Co., Ltd., batch number: 020724);METHODS: ① Venous blood samples (4.0-5.0 mL) were drawn from all the subjects, and mononuclear cells were separated by means of Ficoll-Hypaque density gradient centrifugation and plastic adhesion, then incubated at 37 ℃ for 40-60 minutes, and the adherent cells were the PBMCS. These cells (viability ＞ 95%, Trypan blue staining) had the characteristics of mononuclear cells (Wright staining). The newly separated adherent PBMCS were resuspended, and then inoculated to the 24-well plate (4×107 L-1). There were 3 wells for each sample: the first was for basic excretion, the second for angiotensin Ⅱ stimulation, and the third for tanshinone pretreatment. The PBMCS were co-incubated with tanshinone for 30 minutes before angiotensin Ⅱ stimulation. The terminal concentration was 1×10-8 mol/L and 1×10-8 g/L for angiotensin Ⅱ andtanshinone respectively, and that of PBMCS was 2×107 L-1. The cells were cultured in the incubator (CO2 of 0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant and cell ingredients were collected respectively. ② The PBMCS suspension was preparedl, and the cellular density was adjusted to 2.5×109 L-1. The human umbilical vein endothelial cells were cultured on the 24-well plate with M199 medium containing fetal bovine serum (0.1 in volume fraction), and spread to monolayer after the cells entered the logarithm phase. Each well was added with PBMCS suspension (100 μL), incubated at 37 ℃ for 2 and 4 hours respectively. The unadherent cells were removed, and the adherent ones were counted after fixed with 20 g/L glutaral, 40 visual sights were counted for each well under high power microscope, and the average value was used. ③ The double-antibody sandwich enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in supernatant of PBMCS, and the expressions of their mRNA.MATN OUTCOME MEASURES: ① Changes of PBMCS adhesion activity; ② Concentrations of cytokines and their mRNA expressions in supernatant of PBMCS.RESULTS: ① At 2 and 4 hours of inoculation, the numbers of PBMCS adhered to endothelial cells under basic conditions were similar between the hypertension group and normal control group (t =1.153-1.577, P ＞ 0.05); After angiotensin Ⅱ stimulation, the adherent cells were obviously more in the hypertension group than in the normal control group (t =3.842-4.536, P ＜ 0.01); The numbers of the adherent cells were decreased to the same levels after tanshinone pretreatment (t =0.855-1.702, P ＞ 0.05). ②Under basic state, the concentrations of TNF-α, IL-1β and IL-6 in supernatant were all lower in both groups (t =0.981-1.829, P ＞ 0.05); The concentrations of the cytokines after angiotensin Ⅱ stimulation were obviously higher in the hypertension group than in the normal control group (t = 2.442, 5.075, P ＜ 0.01,0.01, 0.05); The concentrations of the cytokines after tanshinone pretreatment were all decreased to different extent, and there were no significant differences (t =1.227-1.940, P ＞ 0.05). Similar changes were observed in the mRNA expressions of the cytokines in PBMCS in the two groups.CONCLUSTON: ① The number of PBMCS adhered to endothelial cells, the concentrations and mRNA expressions of the cytokines under basic state in patients with essential hypertension were at the levels of normal subjects, and they were significantly increased after angiotensin Ⅱ stimulation, suggesting that the PBMCS were at preactivation at early essential hypertension. ② Tanshinone could decrease the adhesion and excretory activities of PBMCS in patients with essential hypertension to the normal levels, it is proved that tanshinone can inhibit the further activation of the preactivated PBMCS, and can prevent the occurrence of atherosclerosis to some extent.

Objective To study the factors affecting the activities of daily life (AOL) of patients after the first stroke of cerebral infarction in order to formulate the intervention strategy for improving the capability of ADL of patients. Method A total of 149 patients with the first stroke of cerebral infarction admitted from Oct. 2008 to Dec.2008 were enrolled in this study. The demographics of patients, cerebral infarction risk factors, apopletic score as per National Institute of Health stroke scale (NIHSS), white blood cell count (WBC) and plasma glucose (PG) were recorded on the first day of admission, and many other laboratory examinations were done on the next morning. The occurrences of infection in lung and urinary tract, and atrial fibrillation were recorded during hospi-talization. The NIHSS score and score of Glasgow Coma Scale (GCS) were taken within 24 hours after admission, on the 14th day and at the end of the third month after cerebral infarction respectively. Barthel index (BI) was taken in the second week as well as at the end of third month by follow-up in the OPD or by telephone. The correlation analysis and multiple linear stepwise regression analysis were used to find the risk factors. Results The PG level, WBC count and NIHSS score were independently associated with ADL in the second week as well as at the end of the third month after cerebral infarction. Besides, the urinary tract infection during hospital stay was also independently associated with ADL at the end of the third month after cerebral infarction. Conclusions The plasma glucose level, WBC count, NIHSS score and urinary tract infection are the risk factors. Positive measures should be taken to control these risk factors so as to improve the capability of ADL of the patients after cerebral infarction.

OBJECTIVE:To observe the effects of different concentration of human urinary kallikrein on blood pressure when used for acute cerebral infarction. METHODS: By a non-randomized historical control study,the blood pressure variation of 34 patients on day one after treatment with low concentration (0.06%) of human urinary kallikrein was observed,the occurrence of hypotension in this group was compared with that of another 47 patients (control) treated with high concentration (0.3%) of human urinary kallikrein. RESULTS: The reduction of blood pressure in low concentration group was less obvious than in the high-concentration group. However,hypotension was noted in both groups when angiotensin-converting enzyme inhibitor (ACEI) hypotensive drugs were used concomitantly. CONCLUSION: Human urinary kallikrein for acute cerebral infarction can transiently down-regulate blood pressure slightly,but its effect on blood pressure can be lowered by suitable reduction of its concentration;at any concentration,it can result in hypotension if used in combination with ACEI.

Objective To analyze the epidemic characteristics of mushroom poisoning incident in order to find the regularity of outbreak and provide the fundamental guidelines of prophylaxis,control,diagnosis and treatment.Methods According to the reported information from the Management Information System of Public Health Emergency in China mainland,the area-time distribution of mushroom poisoning incidents from 2004 to 2014 was analyzed,and the descriptive analysis of mushrooms poisoning incidents including causes,places,occupation of victims and incidents identification were made from 2010 to 2014.Results In China (excluding Hong Kong,Macao and Taiwan),the top five provinces of mushroom poisoning incidents were Yunnan,Guizhou,Sichuan,Guangxi and Hunan.The epidemic peak was reached in summer-autumn season.The major and significant incidents accounted for 76.56％ of overall mushroom poisoning incidents,and the fatality rate of 3 701 patients accounted for 21.24％ (786 deaths).The causes were mistaking poisonous mushrooms as edible mushrooms or purchasing poisonous mushrooms in the market by accident.About 87.50％ incidents happened at home.Farmers,workers,children and students were easily subjected to mushroom poisoning because of their large range of activities,strong curiosity and lacking related ability for distinguishing edible mushroom from poisonous mushrooms.No identification was done in 200 mushroom poisoning incidents from 2010 to 2014,which accounted for 92.59％ of mushroom poisoning incidents in the corresponding period.Standard species identification was carried out only in two poisonous mushroom incidents.Conclusions Mushroom poisoning incident was one of the most important causes of death in per-oral poisoning incidents.It should to cope with surveillance and meticulous management during high prevalence season and in high-risk provinces.At the same time,it should be strengthened to train doctors and health professionals with the knowledge of identification of mushroom poisoning in key areas as well as to develop the health promotion of mushrooms poisoning prevention.

This study examined the effect of tanshinone II A (TSN II A) on the cardiac fibrosis induced by transforming growth factor β1 (TGF-β1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-β1 alone or pretreated with TSN II A at different concentrations (10(-5) mol/L, 10(-4) mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type I (COL I), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLI. The results showed that TGF-β1 induced the expression of CTGF, COL I, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COL I was significantly increased 24 h after TGF-β1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-β1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSN A resulted in a decrease in the expression of p-Smad3, CTGF and COL I (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSN II A as compared with that at 2 h post TGF-β1 stimulation (P<0.05 for low concentration of TSN I IA; P<0.01 for high concentration of TSN II A). It was concluded that TSN II A may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-β1-induced phosphorylation of Smad3 and partially blocking the TGF-β1-Smads signaling pathway.

Objective To observe the effects of functional ambulation training in a realistic environment based on the activities of daily living among stroke patients with hemiplegia.Methods Thirty-two stroke survivors with hemiplegia were randomly divided into an experimental group and a control group,each of 16.Both groups were given routine rehabilitation training,while the experimental group was additionally given functional ambulation training based on the activities of daily living in a realistic environment for 60 min per day,five days a week for six weeks.Both groups were evaluated using the mini-mental state examination (MMSE),the Holden walking functional class assessment (HWFCA),the Berg balance scale (BBS),functional gait assessment (FGA),the 5 times sit to stand test (FTSST),the timed up and go test (TUGT),and rated using the specific activity balance confidence scale (ABC) and the modified Barthel index (MBI).Results Before the training,no significant differences between the two groups were found in terms of any of the measurements.After the six weeks of training,significant improvement was observed in all of the outcome measures except the MMSE and the HWFCA,with the experimental group scoring significantly better,on average,than the control group.Conclusion When combined with routine rehabilitation training,functional ambulation training based on the activities of daily living in a realistic environment can significantly enhance the functional gait,balance and postural control of stroke survivors.This should facilitate their activities in daily life and improve their confidence in maintaining their balance.

Objective: To explore the expression level of serum interleukin (IL)-7 in patients with acute coronary syndrome (ACS) and analyze the relationship between IL-7 level and prognosis. Methods: A total of 130 ACS patients [ACS group, including 70 cases with acute myocardial infarction (AMI) and 60 cases with unstable angina pectoris (UAP)], 33 cases with stable angina pectoris (SAP，SAP group) and 89 healthy subjects (healthy control group) were selected. IL-7 level was measured using enzyme linked immunosorbent assay (ELISA) and compared among all groups. The 130 ACS patients were followed up, and Logistic regression analysis was used to analyze the relationship between IL-7 level and prognosis. Results: Compared with healthy control group and SAP group, there was significant rise in IL-7 level in UAP group and AMI group [(1.84±0.47) pg/ml, (2.11±0.63) pg/ml vs. (4.87±0.52) pg/ml, (5.15±0.71) pg/ml, P<0.05 or P<0.01]. There were no significant difference in IL-7 level between healthy control group and SAP group, UAP group and AMI group (P>0.05 both); Logistic regression analysis indicated that expression level of serum IL-7 was an independent risk factor for adverse cardiovascular events in ACS patients (OR=1.212, 95%CI:1.061-1.418). Conclusion: Interleukin-7, as an important inflammatory cytokines, its serum level abnormally elevated in patients with acute coronary syndrome, it may have important prognostic value.

Objective To establish acute hepatotoxic model induced by Amanita exitialis and to study the characteristics of acute toxic liver failure induced by mushrooms containing peptide toxins,in hope for providing some help to experimental research on poisoning induced by mushrooms containing peptide toxins.Methods UPLC-MS/MS (Ultra performance liquid chromatography-tandem mass spectrometry) method was used to detect peptide toxins in Amanita exitialis.To establish acute toxic liver hepatic failure model,the beagles were fed with 60 mg/kg of lyophilized powder of Amanita exitialis fungus which encapsulated in starch capsules.Toxic sighs were observed,coagulation function,hepatic and renal function,liver histopathological morphology,peptide toxin concentration in plasma and urine were detected during the experiment.Results Total peptide toxins in Amanita exitialis was (3 482.6 ± 124.94) mg/ kg.All the beagles had toxic signs including vomiting and diarrhea in 12-48 h after ingestion.On 24 h after ingestion,the beagles' ALT,AST,TBIL,ALP,PT and APTT levels increased obviously.On 36 h after ingestion,the beagles' ALT,AST,PT and APTT values reached their peaks (ALT:283.2 ± 112.9 Kallmann unit;AST:223.9 ±93.8 Kallmann units;PT:132.9 ± 152.6 s;APTT:131.4 ± 153.9 s).On 48 h after ingestion,the beagles' TBIL and ALP levels reached their peaks (TBIL:23.3 ± 14.6 mol/L;ALP:274.5 ± 115.5 U/L).The beagles' TBIL,TP and APTT returned to normal 1 week after ingestion,their ALT,AST and ALP levels returned to normal 3 weeks after ingestion.Three dogs died during 24-72 h after ingestion.Liver histopathological morphology study showed hemorrhagic necrosis of hepatocytes.Peptide toxins can be detected in plasma within 24 h after ingestion.Peptide toxins can be detected in urine within 96 h after ingestion.Conclusion Amanita peptide toxins can cause hemorrhagic necrosis of liver cells and lead to acute liver failure.This model is consistent with clinical pathophysiological process of acute toxic liver failure induced by mushrooms containing peptide toxins,and it can be applied to the study of diagnosis and treatment of poisoning induced by mushrooms containing peptide toxins.

Summary: The effects of tanshinone Ⅱ A (TSN) on transforming growth factor β1 (TGFβ1) signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively.The mRNA levels of fibronectin (FN) were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively (P<0,01, P<0.01), and the protein expression of phosphorylated Smad2/3 (p-Smad2/3) increased by 7 times at the end of TGFβ1 stimulation (P<0.01). TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression (both P0.05). After pretreatment with 105 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively (P<0.05, P<0.01), the FN protein levels were decreased by 40% and 44% respectively (P<0.05, P<0.05), and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively (P<0.05, P<0.01). The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβl-Smads signal pathway in renal interstitial fibroblasts.