BACKGROUND:Cholesterol is closely linked to the occurrence and progression of atherosclerosis. Current approaches to study celular cholesterol dynamics have their own limitations.
OBJECTIVE: To measure the cholesterol efflux rate of RAW 264.7 mouse macrophages by BODIPY-Cholesterol labeling and to explore the effects of extracelular cholesterol and lipopolysaccharide on the cholesterol efflux rate.
METHODS:RAW 264.7 cels were cultured in vitro with DMEM containing 10% fetal bovine serum, and labeled with BODIPY-Cholesterol for 1, 2, 4, 8 hours. Then, the cels were rinsed with serum-free DMEM and inoculated for 6, 12, 24, 48, 96 hours to optimize the labeling time and incubation time. We measured and compared the cholesterol efflux rates after cultured cels were treated with cholesterol, lipopolysaccharide, human sera with high cholesterol or human sera with normal cholesterol.
RESULTS AND CONCLUSION: The best labeling time for BODIPY-Cholesterol was 2-8 hours. Cholesterol efflux rates were gradualy decreased after the cels that were labeled for 2 hours were incubated with increasing concentrations of cholesterol (0.1, 0.5, 2.5 mmol/L,P< 0.01). Treating cels with lipopolysaccharide also decreased the cholesterol efflux rate (P < 0.05). Furthermore, the cholesterol efflux rate was decreased after cels were treated with human sera with high cholesterol (P< 0.05). These findings indicate that BODIPY-Cholesterol can be used to measure celular cholesterol efflux rate and to study the effects of extracelular cholesterol and lipopolysaccharide on the cholesterol efflux rate.

[Objective] The present study aimed to determine the protective role of Melatonin (MT) against Acrylamide (AA)-induced testicular toxicity and the potential molecular mechanism.[Methods] The animals were randomly divided into three groups,the control group (n =12),the AA group (n =12) and the AA+MT group (n =12).The rats in the AA and AA + MT group were gavaged with AA at a dose of 15 mg/(kg · day) for 4 consecutive weeks.After 3 weeks of AA treatment,MT was intraperitoneally injected 30 minutes before AA treatment at 10 mg/(kg· day) for 1 week in the AA + MT rats.Subsequently,the mitochondrial membrane potential measurement,TUNEL assay,Western blot and electron microscopic techniques were applied in the present study.[Results] The results showed that AA could decrease the testis mitochondrial membrane potential (P ＜ 0.05) which could be recovered by MT (P ＜ 0.05).Moreover,MT induced down-regulation of Bax expression and up-regulation of Bcl-2 expression in the testis,compared with AA rats.The amelioration of testicular apoptosis was further confirmed by the TUNEL labeling.Western blot results suggested that the decreased ratios of Bcl-2/Bax and Bcl-xL/Bak in the AA group (both P ＜ 0.05) could be recovered by MT treatment (both P ＜ 0.05).The levels of Cyt-c,Casp-3,p53 and NF-κB in AA group were markedly elevated compared with the control (all P ＜ 0.05),and reduced in MT treatment group (all P ＜ 0.05).MT could relieve abnormal mitochondrial structures in the seminiferous tubule in the electron microscopic level.[Conclusion] MT may exert productive effect through its anti-apoptotic properties associated with mitochondria.

OBJECTIVETo explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout (ApoE(-/-)) mice.

METHODSTotally 24 ApoE(-/-) mice, 7-8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group (drug group) and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7-8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin (0.25 mg/kg) was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), high-sensitive C-reactive protein (hs-CRP), E-selectin, interleukin-6 (IL-6), and stromal metalloproteinase-2 (MMP-2) were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operated calcium channels, including stromal interacting molecule 1 (STIM1), Orail protein, and transient receptor potential channel 1 (TRPC1) were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC, TG and LDL increased (P<0.01), hs-CRP, E-selction, IL-6, and MMP-2 obviously increased (P<0.01, P<0.05) in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased (P<0.01). Compared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group (P<0.05, P<0.01); serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased (P<0.05, P<0.01); expression levels of STIM1, TRPC1, and Orail were significantly down-regulated (P<0.05, P<0.01).

CONCLUSIONHirudin could significantly improve lipids and endothelial functions of ApoE(-/-) mice, down-regulate expression levels of STIM1, Orai1, and TRPC1, and thus delaying the occurrence and development of atherosclerosis.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; C-Reactive Protein ; Cholesterol ; Diet, High-Fat ; Drugs, Chinese Herbal ; E-Selectin ; Hirudins ; metabolism ; Interleukin-6 ; Lipids ; Lipoproteins, HDL ; Lipoproteins, LDL ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; metabolism ; Recombinant Proteins ; metabolism ; Triglycerides

To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats.
Animals ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; pharmacokinetics ; Humans ; Isoenzymes ; genetics ; metabolism ; Liver Cirrhosis ; drug therapy ; enzymology ; genetics ; Male ; Mass Spectrometry ; Rats ; Rats, Wistar

Objective of this study was to detect the expression of HtrA2 and WT1 mRNA in acute myeloid leukemia (AML) and investigate the relationship of their expression levels with clinical variates and correlation between them. The expression levels of HtrA2 and WT1 were measured by RQ-PCR in bone marrow cells in 104 newly diagnosed AML patients and leukemia cell lines (K562, HL-60, NB4, Kasumi-1, U937), and the relationship between expression level and clinical parameters (age, sex, WBC count, diagnosis and prognosis) was investigated. The results showed that (1) the expression of HtrA2 gene in newly diagnosed AML was lower than that of the normal controls (P < 0.01), while expression of WT1 gene in newly diagnosed AML was higher than that of the normal controls (P < 0.01), the expression levels of HtrA2 and WT1 genes both did not correlate with age, sex and WBC counts of patients. There were no significant difference of HtrA2 gene expression between different NCCN prognosis group, while WT1 gene expression in better-risk group was significantly lower than that in intermediate-risk group (P = 0.003). The HtrA2 expression level rose after treatment in both CR group and non-CR group (P < 0.05), while WT1 expression level significantly decreased after treatment only in CR group (P < 0.01). Negative correlation between HtrA2 and WT1 expression was also observed (r = -0.249, P = 0.011). It is concluded that the low expression of HtrA2 and high expression of WT1 are closely related with occurrence and development of acute leukemia, so up-regulating expression of HtrA2 and interfering expression of WT1 may become the targets for leukemia therapy in the future.
Adolescent ; Adult ; Aged ; Cell Line, Tumor ; Female ; High-Temperature Requirement A Serine Peptidase 2 ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Mitochondrial Proteins ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; WT1 Proteins ; genetics ; metabolism ; Young Adult

OBJECTIVETo analyze the relationship between body fat and beta-cell function in obesity women of Pi-deficiency with phlegm-dampness type (PDPD) and qi-stagnancy with phlegm-blocking type (QSPB).

METHODSSixty women, who had normal blood glucose level and without family history of diabetes, were enrolled. They were classified into non-obesity group and obesity group depending on their body mass index (BMI), and subjects of obesity group were differentiated into the PDPD type and QSPB type according to Chinese medicine syndrome differentiation. The body fat was detected using double energy X-ray absorptiometry, and the beta-cell function was assessed by measuring the acute insulin response (AIR), the under insulin curve area (AUCins), the under glucose curve area (AUCglu), and their ratio (AUCins/AUCglu), through intravenous glucose tolerance test (IVGTT).

RESULTSBMI, body fat and waist circumference (Wf) were higher in obesity subjects than those in non-obesity subjects, but showed no significant difference between the two obesity types. Comparisons between obesity women of different types showed that the fat content of trunk and total body, the ratio of trunk fat/total mass, AIR, AUCins, and AUCins/AUCglu were all higher in QSPB than those in PDPD. AIR, AUCins, AUCins/AUCglu showed good correlation with BMI, Wf, trunk fat and total body fat contents. Multiple linear regression analysis demonstrated the increasing of trunk fat content was an influencing factor of AIR.

CONCLUSIONObesity women of QSPB type possess higher body fat (especially the trunk fat) content and insulin resistance with high acute insulin response, so clinical intervention should dominantly pay attention to subjects with QSPB type of obesity.

Adipose Tissue ; Adult ; Body Mass Index ; Diagnosis, Differential ; Female ; Humans ; Insulin-Secreting Cells ; metabolism ; Medicine, Chinese Traditional ; methods ; Middle Aged ; Obesity ; diagnosis ; metabolism ; Yang Deficiency ; diagnosis

BACKGROUND:Sepsis has become the greatest threat to in-patients, with a mortality of over 25％.The dysfunction of gut barrier, especially the immunological barrier, plays an important role in the development of sepsis. This dysfunction occurs after surgery, but the magnitude of change does not differentiate patients with sepsis from those without sepsis. Increased intestinal permeability before surgery is of no value in predicating sepsis. The present study aimed to observe the changes of intestinal mucosal immunologic barrier in rat models of sepsis induced by cecal ligation and puncture. METHODS:Sixty Sprague-Dawley rats were randomly divided into a sepsis group (n=45) and a control group (n=15). The rats in the sepsis group were subjected to cecal ligation and puncture (CLP), whereas the rats in the control group underwent a sham operation. The ileac mucosa and segments were harvested 3, 6 and 12 hours after CLP, and blood samples were collected. Pathological changes, protein levels of defensin-5 (RD-5) and trefoil factor-3 (TFF3) mRNA, and lymphocytes apoptosis in the intestinal mucosa were determined. In an additional experiment, the gut-origin bacterial DNA in blood was detected. RESULTS:The intestinal mucosa showed marked injury with loss of ileal villi, desquamation of epithelium, detachment of lamina propria, hemorrhage and ulceration in the sepsis group. The expression of TFF3 mRNA and level of RD-5 protein were decreased and the apoptosis of mucosal lymphocyte increased (P<0.05) in the sepsis group compared with the control group. Significant differences were observed in RD-5 and TFF3 mRNA 3 hours after CLP and they were progressively increased 6 and 12 hours after CLP in the sepsis group compared with the control group (P<0.05, RD-5 F=11.76, TFF3 F=16.86 and apoptosis F=122.52). In addition, the gut-origin bacterial DNA detected in plasma was positive in the sepsis group. CONCLUSION:The immunological function of the intestinal mucosa was impaired in septic rats and further deteriorated in the course of sepsis.

OBJECTIVETo investigate the effects of hydrogen sulfide preconditioning on myocardial ischemia reperfusion injury in rats.

METHODSSprague-Dawley male rats were divided into 4 groups with 10 in each group: in S group rats received sham operation; in IR group rats were given with NS (1.0 ml/kg iv) 24 h before ischemia; in H group rats were treated with NaHS (0.05 mg/kg iv) 24 h before ischemia; and in D group, NaHS-treated rats received 5-hydroxydecanoate (5-HD) 15 min before ischemia. Rats in IR group,H group and D group were subjected to ischemia by occlusion of coronary artery for 30 min followed by 2 h of reperfusion. At the end of the reperfusion,myocardial infarct size was measured. SAM-s was measured by Western blotting. Plasma SOD activity and MDA were determined at the end of reperfusion.

RESULTSThe infarct size was significantly lesser in H group (25.40 % ± 3.54%) than that in IR group (38.27% ±5.64%,P<0.05). The SAM-s protein expression in myocardium was significantly lower in H group than that in IR group. The plasma MDA content was significantly lower and SOD activity was higher in H group than those in IR group,but there was no difference between IR group and D group.

CONCLUSIONThe hydrogen sulfide preconditioning attenuates myocardial IR injury possibly through down-regulating SAM-s expression,reducing the production of oxygen free radicals and enhancing anti-oxidize effect in rats.

Animals ; Disease Models, Animal ; Hydrogen Sulfide ; pharmacology ; Ischemic Preconditioning, Myocardial ; Male ; Myocardial Reperfusion Injury ; metabolism ; pathology ; prevention & control ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

Objective To provide anatomical basis for preventing paresthesia that appear in the lateral thoracic wall ,the arm‐pit and the inside of the upper arm during axillary lymph node dissection for breast cancer .Methods The intercostobrachial nerve (ICBN) and its contiguous relationship were observed ,besides the origin ,ramus and branch were measured and recorded by anato‐mizing 30 adult embalmed cadavers (60 sides) .Results Three intercostals nerve (50 sides) and the ICBN (29 sides) were the mainly nerves in lateral thoracic wall ,the medical brachial cutaneous nerve(MBCN) was not observed .Three intercostals nerve , ICBN(42 sides)and MBCN(44 sides) were found in armpit ,but mainly was intercostobrachial nerve (42 sides) .ICBN and the MB‐CN equally distributed in the dorsal and medical of the upper arm ,but the three intercostals nerve was not observed .81 .7% (49 sides) of intercostal nerve and all of the brachial plexus presenced filament .They existenced blood vessel accompanied when the ICBN pierced the chest wall (63 .3% ,38 sides) .Conclusion Identify and intactly preserve the ICBN and relevant nerves and their filament during axillary lymph node dissection of the breast cancer may benefit to prevent paresthesia .

Objective To investigate the dynamics and development trends of drinking water type of endemic fluorosis after water improvement in Xinbaerhuyouqi of Hulunbeir city, Inner Mongolia and to provide a scientific evidence for the development of countermeasures. Methods We mainly selected Adunchulusumu and Kerlunsumu in Xinbaerhuyouqi of Hulunbeir city as the two monitoring points after water improvement in 2000 -2009. Of these, 1 sample of centralized water supply source water and 3 samples of tap water and 5 samples of noncentralized water supply source water according to water well locations of east, west, south, north and center were collected and the levels of water fluoride were tested; the prevalence of dental fluorosis of school children aged 8 to 12 were examined; from 2002 onwards, the urine samples of 30 children aged 8 to 12(five age groups, six urine samples for each age group) were collected, and all urine samples were collected in the case of less than 30, and urine fluoride was tested. Dental fluorosis was diagnosed using Dean method; water fluoride was tested using fluoride ion selective electrode(WS/T 106-1999); urinary fluoride was tested by determination of fluoride in urine using ion-selective electrode(WS/T 89-1996). Results In 2000 - 2009, the mean levels of fluorine in drinking water in Adunchulusumu and Kerlunsumu were 1.79 - 4.35 mg/L and 1.38 - 3.18 mg/L, respectively; the detection rate of dental fluorosis of children aged 8 to 12 were 45.24％(19/42) - 89.78％(123/137) and 40.00％ (28/70) - 74.47％ (70/94), respectively; the median urinary fluoride of them were 2.30 - 4.15 mg/L and 2.73 - 4.55 mg/L, respectively. ConclusionsThe detection rate of children's dental fluorosis remains high in Xinbaerhuyouqi during the past 10 years after changing water. The endemic fluorosis remains a serious disease. Effective prevention and control measures must be taken to control the occurrence of fluorosis in the future.