Objective To investigate the effect of Shenfu injection (SFI) on hypoxia and reoxygenation (H/R)-induced apoptosis and the expression of Bcl-2 and Caspase-3 in cultured neonatal rat cardiomyocytes and to explore the possible molecular protective mechanisms of SFI from hypoxia and reoxygenation injury in cardiacmy-ocytes in vitro. Method The experiment was performed in Research Center for Cardiovascular Regenerative Medicine, Cardiovascular Institute and Fuwai Hospital in Beijing. Ventricular myocytes from the hearts of neonatal Sprague-Dawley rats (1- to 2-day old) were cultured. The model of hypoxia and reoxygenation injury was devel-oped in primary cultured neonatal rat cardiacmyocytes. The cultured cells were randomly divided into four groups: (1) Control group (Con group), without any treatment; (2) Hypoxia and Reoxygenation group (H/R group),4 h hypoxia followed by 16 h reoxygenation; (3) Low-dose SFI group (L-SFI group),cardiacmyocytes were pretreated with a low dose (50 μL/mL) of SFI for 30 min followed by H/R; (4) High-dose SFI group (H-SFI group),car-diacmyocytes were pretreated with a high dose (100 μL/mL) of SFI for 30 min followed by H/R. Apoptosis was quantified by fluoreacence-activated cell sorter (FACS) analysis after staining with Fluorescein isothiocyanate (FITC)-labled Annexin-V (Annexin V-FITC) and propidine iodide (PI). The expressions of Bcl-2 and Caspase-3 were detected by ECL-Western blot analysis. All data are expressed as mean±S.E.M. One-way analysis of vari-ance (ANOVA) was performed followed by Student-Newman-Keul test using SSPS 11.5 software. A p value less than 0.05 were considered as statistically significant. Results The results of FACS analysis indicated that the rate of different apoptotic process in cardiomyocytes was significantly increased after H/R, while after SFI treatment the occurrence of cell apoptosis induced by H/R was decreased significantly. The results of ECL-Western blot analysis showed that cells' exposure to H/R induced proteolytic cleavage of caspases,as revealed by the appearance of the characteristic fragment at 17 000 of Caspase-3 and this proteolytic activation was nearly completed with difference concentration SFI incubation. The anti-apoptotic protein Bcl-2 in cardiomyocytes was decreased after H/R insult and was increased in cells with SFI pretreatment. Conclusions SFI has protective effects on cardiacmyocytes a-gainst apoptosis that could be induced by H/R injury, the mechanisms of which probably involve the inhibition of down-regulation of Bcl-2 protein level and sequential activation of Caspase-3.

Objective To investigate the effect of integrin β4 expression on the metastasis potential of human thyroid follicular cancer cells. Methods Metastasis potential was observed and grouped in human thyroid follicular cancer cell line, CGTHW-1 cells, depending on the cells' penetrating ability in artificial matrigel. The expression of integrin β4 mRNA and protein were determined by immunocytochemistry, Western blot, and semiquantitative RT-PCR. Results The mRNA and protein expressions of integrin β4 were significantly higher in cells with high metastasis potential [0.277 5 ±0.034 0 vs 0. 187 5 ±0.022 2 ( Immunohistochemistry ), 0. 099 7 ± 0.0185 vs 0.039 0±0.010 2(Western blot), 0.555 0±0. 101 2 vs 0.270 0±0.029 9(RT-PCR), all P＜0.01]. Conclusion Integrin β4 may play an important role in human thyroid cancer invasion. It seems probably to be a potential target for human thyroid cancer treatment.

Objective To investigate the effects of adenosine postconditioning (AP) on serum IL-10 and TNF-α concentrations following myocardial ischemia-reperfusion(VR)in rats.Methods Twenty-four SD ratsweighing 180-250 g were randomly divided into 4 groups(n=6 each):group I sham operation (group S);group Ⅱ myocardial I/R;group Ⅲ ischemic postconditioning(group IP)and group Ⅳ AP.Myocardial I/R was induced by 30 rain occlusion of anterior descending branch of left coronary artery followed by 120 min reperfnsion.IP was induced by 3 cycles of 30 s myocardial ischemia followed by 30 s reperfusion at the end of ischemia.In AP group adenosine 1.5 mg/kg was infused at 40μg·kg-1·min-1 before the onset of reperfusion.SP,DP and HR were recorded before ischemia (baseline) at 30 min of ischemia and 30 and 120 min of reperfusion.Arterial bloodsarnples were collected at 120 min of repednsion for determination of serum TNF-α and IL-10 concentrations.Theanimals were then killed.Their hearts were removed for microscopic examination.Myocardial infarct size wasmeasured and myocardial MDA content was determined.Results BP and HR were signilicandy decreased duringreperfusion while myocardial infarct size.MDA content and serum concentrations of IL-10 and TNF-α weresignificantly increased in I/R group compared with group S.Ischemic and adenosine postconditioning significantlyattenuated hypotension,reduced infarct size,myocardial MDA content and serum TNF-α concentration and increased serum IL-10 concentration in group AP and IP as compared with I/R group.There was no significant difference in the above changes between group AP and IP. Myocardial injury was ameliorated in group AP and IP as compared with I/R group. Conclusion Adenosine postconditioning can protect myocardium from I/R injury by increasing IL-10 production and inhibiting TNF-a release.

Objective To evaluate the role of the complement 1 q (C1 q) in hepatic ischemia-reperfusion (I/R) injury in rats.Methods Sixty healthy male Sprague-Dawley rats,aged 3-4 months,weighing 180-200 g,were randomized into 2 groups:sham operation group (S group,n =12) and hepatic I/R group (I/R group,n =48).Hepatic specimens were obtained at 1,3,6 and 24 h of reperfusion and were then cut and stained with haematoxylin and eosin for examination of histological changes of the liver (with light microscope) and for determination of superoxide dismutase (SOD) activity and malondialdehyde (MDA) content (by colorimetric method),expression of Clq mRNA (using real-time PCR) and expression of Clq (by using Western blot).Results Compared with S group,the activity of SOD was gradually decreased,the content of MDA was gradually increased,and the expression of Clq and Clq mRNA was gradually up-regulated and peaked at 3 h of reperfusion with the prolongation of reperfusion time in I/R group (P ＜ 0.05).The pathological changes of the liver were aggravated with the prolongation of reperfusion time in I/R group.Conclusion Activation of C1 q is involved in hepatic I/R injury in rats.

Objective To investigate the effect of different doses of dexmedetomidine (DEX) on myocardial ischemia-reperfusion injury (MIRI) in rats.Methods Seventy-five SD male rats weighing between 250 and 300g were divided into sham group,MIRI group,small-dose DEX group,medium-dose DEX group and high-dose group according to the random number table,with 15 rats per group.Threading the left anterior descending coronary artery was done only in sham group,but the MIRI model was produced in the rest groups by ligation of the artery for 30 minutes followed by 120 minutes of reperfusion.Fifteen minutes before the ligation,small-,medium-and high-dose DEX groups were injected 2.5,5 and 10 μg · kg-1 · h-1 of DEX respectively until the end of reperfusion.Instead,an equal volume of normal saline was given in sham and MIRI groups.At the end of reperfusion,five rats in each group were used to determine the myocardial infarct size,and arterial blood samples and myocardial tissues from ten rats in each group were used to measure serum levels of interleukin-1β (IL-β) and serum tumor necrosis factor-α (TNF-α),expression of myocardial nuclear factor kappa B p65 (NF-κB p65) and change of myocardial pathomorphology.Results Myocardial infract size,degree of myocardial pathomorphology structure damage,serum levels of IL-1 β and TNF-αt and expression of myocardial NF-κB p65 in sham group were significantly lower in sham group than other groups (P ＜ 0.05).Above mentioned parameters in small-,medium-and high-dose DEX groups were all significantly decreased compared to MIRI group (P ＜ 0.05),and the decrease was most significant in medium-dose DEX group (P ＜ 0.05).Conclusions DEX can attenuate the MIRI in rats and the possible mechanism is suppressing the release of NF-κB p65,which can reduce serum pro-inflammatory cytokines like TNF-α and IL-1β.And mediumdose DEX exhibits better protective effect.

Objective To evaluate the efficacy of different doses of dexmedetomidine for prevention of postoperative cognitive dysfunction (POCD) in the elderly patients undergoing hyperthermic intraperitoneal chemotherapy.Methods Sixty ASA physical status Ⅰ or Ⅱ patients of both sexes,aged over 65 yr,weighing 50-70kg,undergoing elective hyperthermic intraperitoneal chemotherapy,were equally and randomly assigned into 4 groups using a random number table:control group (group C),different doses of dexmedetomidine groups (groups D1-D3).In D1-3 groups,a loading dose of dexmedetomidine 0.2,0.4 and 0.8 μg/kg was infused over 30 min before induction of anesthesia,respectively,followed by infusion at a rate of 0.5 μg· kg-1 · h-1 until the end of surgery,while the equal volume of normal saline was given in group C.After induction of anesthesia,the patients were endotracheally intubated and mechanically ventilated.Venous blood samples were collected at 30 min before induction of anesthesia (T0),immediately after intubation (T1),at 0 and 1 h after skin incision (T2,3),at 1 min after abdomen closure (T4) and immediately after onset of extubation (T5) for determination of serum concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by ELISA.The development of POCD was recorded within 7 days after surgery.Results Compared with group C,the serum concentrations of TNF-α and IL6 and incidence of POCD were significantly decreased in D1-D3 groups (P ＜ 0.05).Conclusion Dexmedetomidine pretreatment can decrease the development of POCD in elderly patients undergoing hyperthermic intraperitoneal chemotherapy and inhibition of inflammatory responses may be involved in the mechanism.

Objective To evaluate the efficacy of adductor canal block combined with infiltration anesthesia for postoperative analgesia in the patients undergoing total knee arthroplasty. Methods Sixty pa?tients of both sexes, aged 65-80 yr, weighing 40-80 kg, of American Society of Anesthesiologists physi?cal statusⅠ or Ⅱ, scheduled for elective unilateral total knee arthroplasty, were divided into 3 groups ( n=20 each) using a random number table: single?injection adductor canal block + infiltration anesthesia group ( group A) , single?injection femoral nerve block+infiltration anesthesia group ( group F) , and infil?tration anesthesia group ( group I) . Ultrasound?guided adductor canal block and femoral nerve block were performed with 0.5% ropivacaine 20 ml before induction of anesthesia in A and F groups, respectively. Af?ter completion of the block, all the patients were ventilated through the laryngeal mask airway under general anesthesia. After installation of the knee prosthesis, local infiltration anesthesia was conducted with 0.2%ropivacaine 50 ml around the knee joint. Acetaminophen oxycodone capsule was taken orally one pill every 6 h starting from the morning on 1st day after surgery. When visual analogue scale ( VAS) score > 5, tram?adol 100 mg was injected intramuscularly as rescue analgesic. At 4, 8, 24, 48 and 72 h after surgery, VAS scores at rest and during activity were recorded, the quadriceps strength was measured, and the re?quirement for analgesic drugs and development of adverse reactions were recorded. Results Compared with
group I, VAS scores at rest and during activity were significantly decreased at 4, 8, and 24 h after surger?y, and the consumption of tramadol was significantly decreased after surgery in A and F groups ( P<0.05) . The quadriceps strength at 4 and 8 h after surgery was significantly higher in A and I groups than in group F ( P<0.05) . No patients developed serious adverse reactions in the three groups. Conclusion Adductor ca?nal block combined with infiltration anesthesia provides reliable efficacy for postoperative analgesia with little influence on the quadriceps strength in the patients undergoing total knee arthroplasty.

Objective To evaluate the relationship between the pathomechanism of neuropathic pain (NP)-inducced depression and autophagy in the cortex of the frontal lobe in rats.Methods Forty healthy male Sprague-Dawley rats in which IT catheters were successfully placed,aged 8-10 weeks,weighing 200-220 g,were divided into 4 groups (n =10 each) using a random number table method:sham operation group (group S),group NP,NP plus dimethyl sulfoxide group (group ND) and NP plus autophagy inducer rapamycin group (group NR).The neuropathic pain model was established by ligation of the left fifth spinal nerve of anesthetized rats in NP,ND and NR groups.Rapamycin 0.1 μgwas intrathecally injected via the intrathecal catheter immediately after ligation of the spinal nerve and every day after ligation once a day for 21 consecutive days in group NR.The equal volume of dimethyl sulfoxide was intrathecally injected instead of rapamycin in group ND.The mechanical paw withdrawal threshold (MWT) was measured before ligation and at 1,3,7,10,14 and 21 days after ligation.The forced swimming test was performed at 3 days before ligation and 14 and 21 days after ligation.The rats were sacrificed after the last measurement of the behaviour testing,and the prefrontal cortex was removed for determination of the expression of microtubule-associated protein light chain 3 Ⅰ (LC3 Ⅰ) and LC3 Ⅱ,Beclin-1 and p62 (by Western blot).The ratio of LC3 Ⅱ/LC3 Ⅰ was calculated.Results Compared with group S,the MWT was significantly decreased after ligation,the time of immobility was prolonged,the expression of LC3 Ⅰ was down-regulated,the expression of LC3 Ⅱ,Beclin-1 and p62 was up-regulated,and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in NP and ND groups (P＜0.05).Compared with group NP,the MWT was significantly increased after ligation,the time of immobility was shortened,the expression of LC3 [and p62 was down-regulated,the expression of LC3 Ⅱ and Beclin-1 was up-regulated,and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in group NR (P＜0.05).Conclusion Enhanced autophagy in the cortex of the frontal lobe is involved in the endogenous antidepressant mechanism in rats with NP.

Objective:To construct and identify the lentiviral vector of adenosine RNAi-adenosine A2a receptor (A2aR) in rats.Methods:Three pairs of short hairpin RNA(shRNA)-A2aR sequences (shRNA-A2aR 1, shRNA-A2aR 2, shRNA-A2aR 3) were designed, and three pairs of double-stranded shRNA oligos were respectively inserted into the shRNA virus vector to gain three kinds of shRNA lentiviral recombinant plasmid.The recombinant plasmid, packaging vector, and shuttle vector were co-transfected into 293T cells to obtain virus liquid.The experiment was performed in two parts.Part Ⅰ The rat primary cardiomyocytes were divided into 3 groups ( n= 6 each) by a random number table method: vehicle group (V group), shRNA-A2aR 1 group and shRNA-A2aR 3 group.Each group was transfected with virus solution of MOI 10 for 48 h. The expression of A2aR was detected by Western blot to select the most efficient lentivirus vector.Part Ⅱ The cardiomyocytes were randomly divided into 6 groups ( n=36 each): vehicle group (V group), MOI5 group, MOI10 group, MOI15 group and MOI20 group.Each group was transfected with the corresponding MOI virus liquid (the most effective lentivirus vector). At 24, 48, and 72 h of transfection, the cell viability and cell death were observed with a fluorescent microscope, and the A2aR expression was detected by Western blot to determine the interference efficiency. Results:Part Ⅰ Two types of shRNA-A2aR lentiviral vectors (shRNA-A2aR 1, 3) were successfully constructed, among which shRNA-A2aR 3 virus solution with a titer of 3.5×10 8 TU/ml had the best effect.Compared with group V and group shRNA-A2aR 1, the expression of A2aR in cardiomyocytes was significantly down-regulated ( P<0.01), and the interference efficiency of shRNA-A2aR 3 was 73% in shRNA-A2aR 3 group.Part Ⅱ shRNA-A2aR 3 was selected to screen out the transfection plan.The cell survival rate in each group was more than 85% at 24 h of transfection, the cell survival rate was more than 80% at 48 h of transfection in MOI5 and MOI10 groups; the cell survival rate in each group was less than 70% at 72 h of transfection.Under an inverted fluorescent microscope, a slightly lower fluorescence density was found in MOI5 group, the fluorescent density was higher and the cell condition was better at 48 h of transfection in MOI10 group and at 24 h of transfection in MOI20 group, and the cardiomyocyte viability was significantly decreased, and dead cells were increased at 72 h of transfection in each group.The results of Western blot showed that the interference efficiency at 48 h of transfection in MOI10 group, 48 h in MOI15 group, 24 and 48 h in MOI20 group was all > 70%. Conclusion:MOI of 10, transfection for 48 h or MOI of 20, transfection for 24 h is the optimal transfection protocol.

Objective:To evaluate the effect of valproic acid on the expression of M1/M2 microglia in the prefrontal cortex of rats with neuropathic pain (NP).Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6-7 weeks, weighing 200-230 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (group S), group NP, and valproic acid group (group V). The NP model was established by ligation of the L 5 spinal nerve (SNL) of anesthetized rats.Valproic acid 300 mg/kg was intraperitoneally injected immediately after SNL and every day after ligation, once a day, for 3 consecutive days in group V, while the equal volume of normal saline was given instead of valproic acid in S and NP groups.The mechanical paw withdrawal threshold (MWT) was measured before ligation and at 1, 3, 7, 14, 21 and 28 days after ligation.Sucrose preference test and forced-swim test were performed on day 28 after ligation.After the end of the behavior test, the prefrontal cortex was removed for determination of the expression of cluster of differentiation (CD) 16 and CD206 by Western blot.The ratio of CD206/CD16 was calculated. Results:Compared with group S, the MWT at each time point after ligation and rate of preference for sucrose were significantly decreased, the duration of immobility in forced-swim test was prolonged, the expression of CD16 and CD206 was up-regulated, and the ratio of CD206/CD16 was decreased in group NP ( P<0.05). Compared with group NP, the MWT at each time point after ligation and rate of preference for sucrose were significantly increased, the duration of immobility in forced-swim test was shortened, the expression of CD16 was down-regulated, the expression of CD206 was up-regulated, and the ratio of CD206/CD16 was increased in group V ( P<0.05). Conclusion:The mechanism by which valproic acid improves depression may be related to promoting the expression of M2 microglia and inhibiting the expression of M1 microglia in the prefrontal cortex of rats with NP.