ObjectiveTo investigate the effects of overexpression of alpha/beta hydrolase domain-containing protein 5 (ABHD5) on the invasion and migration of human colon cancer cell line HCT116 and the pathway of adenosine monophosphate-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR).MethodsThe expression of ABHD5 in colon cancer tissues and its relationship with clinicopathological features was analyzed by UALCAN database. HCT116 cells were cultured in vitro and transfected with ABHD5 recombinant plasmid, then they were divided into control group, negative transfection group and ABHD5 transfection group. Real time quantitative PCR (qRT-PCR) was used to detect the expression of ABHD5 mRNA in HCT116 cells. The proliferation of HCT116 cells was detected by CCK-8 method. Transwell assay was used to detect the invasion and migration of HCT116 cells. The expression of matrix metalloprotein 9 (MMP-9), E-cadherin, Snail, and AMPK/mTOR pathway proteins p-AMPK, AMPK, p-mTOR and mTOR were detected by Western blot.ResultsThe results of the UALCAN showed that compared with normal colon tissues, the expression of ABHD5 mRNA in colon cancer tissues was decreased (P<0.05), and which in the adenocarcinoma and the N1 stage was lower than that of the mucinous adenocarcinoma (P<0.05) and N0 stage (P<0.05), respectively. Compared with the control group and the negative transfection group, the expression of ABHD5 mRNA in the ABHD5 transfection group was increased (P<0.05), the proliferation inhibition rate of HCT116 cells in the ABHD5 transfection group was increased (P<0.05), the numbers of migration and invasion cells in the ABHD5 transfection group were decreased (P<0.05), the expressions of MMP-9, Snail, p-mTOR and mTOR were reduced, and the expressions of E-cadherin, p-AMPK and AMPK were increased (P<0.05).ConclusionsThe overexpression of ABHD5 can inhibit the invasion and migration of colon cancer HCT116 cells, activate AMPK, and inhibit the expression of mTOR. It suggests that ABHD5 may play a role in inhibiting colon cancer by affecting AMPK/mTOR pathway.
Objective To assess the efficacy and safety of S-adenosyl-l-methionine (SAMe) for outcome improvement of intrahepatic cholestasis of pregnancy. Methods Randomized controlled trials (RCT) and quasi-randomized controlled trials were identified from MEDLINE (1983 to 2003), The Cochrane Library (Issue 4,2003), EMBASE (1980 to 2003), China Hospital Digital Library (CHDL) and Wanfang data (1994 to 2003). We also handsearched the relative references. Two researchers evaluated the quality of the trials and extracted the data independently. RevMan software 4.2 was used for meta-analysis. Results Eight studies involving 424 pregnant women were included. The following data were the results of meta-analysis of SAMe for improvements: ① Reducing cesarean-section ratio: no significant difference was seen between SAMe and placebo groups with OR 1.00, 95%CI 0.23 to 4.33 and P= 1.00; significant differences were seen SAMe versus dexamethasone and SAMe versus Dianglining with OR 0.44, 95%CI 0.23 to 0.85 and P=0.01; OR 0.28 95%CI 0.10 to 0.75 and P=0.01 respectively。② Prolonging the period of pregnancy: SAMe had no significant difference compared with placebo groups with WMD=0.70, 95%CI －0.69 to 2.10, P=0.32. SAMe was more effective than dexamethasone, Ganyinling and Qianglining on prolonging the period of pregnancy with WMD=1.10，95%CI 0.46 to 1.74, P=0.000 07; WMD=2.50，95%CI 1.86 to 3.14, P≤0.000 01; WMD=2.20，95%CI 1.61 to 2.79, P≤0.000 01 respectively；③ Increasing the weight of the newborn: meta-analysis showed that SAMe group had not significant difference compared with placebo group on increasing the weight of the newborn with WMD=-26.27，95%CI -338.35 to 285.82, P=0.87. Significant differences were seen between SAMe and dexamethasone, SAMe and Ganyiling, SAMe and Qiangling with WMD=386.86，95%CI 134.41 to 603.31, P=0.002; WMD=410.00,95%CI 321.10 to 498.90, P≤0.000 01 respectively. ④ Fetal distress: There was no significant difference compared with dexamethasone and Kuhuang groups on decreasing the fetal distress with OR=0.47, 95%CI 0.14 to 1.16, P=0.23; OR=0.44, 95%CI 0.10 to 1.97, P=0.29 respectively; ⑤ Decreasing pollution of amniotic fluid: no significant differences were seen in SAMe versus dexamethasone, SAMe versus ursoddeoxycholic and SAMe versus Kuhuang with OR=0.46, 95%CI 0.21 to 1.02, P=0.06; OR=0.68, 95%CI 0.20 to 2.31, P=0.53; OR=0.82 95%CI 0.24 to 2.81,P=0.75 recpectively. ⑥ Newborn stifile: SAMe group had no significant difference compared with dexamethasone and Kuhuang groups on decreasing the Newborn stifile with OR=0.19, 95%CI 0.01 to 4.06, P=0.29; OR=0.31, 95%CI 0.08 to 1.13, P=0.08 respectively. Compared with Qianglining group, SAMe group had better effect on reducing ratio of newborn stifile with OR=0.09, 95%CI 0.02 to 0.42, P=0.002. ⑦ Improving Apgar scores: no significant differences were seen between SAMe and placebo, dexamethasone and ursoddeoxycholic with OR=0.25, 95%CI 0.02 to 3.04, P=0.28; OR=2.09, 95%CI 0.70 to 6.27, P=0.19; OR=1.22, 95%CI 0.35 to 4.19, P=0.75 respectively. Six RCTs mentioned the side effects of S-adenosy-l-methionine, only one RCT reported mild gastrointestinal irritation. Conclusions SAMe is partly effective on improving the pregnancy outcomes of intrahepatic choletasis of pregnancy, such as reducting cesarean-section ratio, prolonging the period of pregnancy and increasing the weight of the newborn. The specified efficacy and safety of SAMe require rigorously designed, randomized, double-blind and placebo-controlled trials to offer evidence.
Objective To investigate the changes and roles of myocardial adenosine triphosphate enzyme(ATPase) in the mechanism of cardiac dysfunction after blunt chest trauma（BCT）. Methods Thirtysix rabbits were divided into 6 groups with random number table, control group, 2 h group, 4 h group, 8 h group, 12 h group and 24 h group, 6 in each group. The models of BCT were established with BIMⅡ biological impact machine, catheterization technique was used through the right jugular artery into the left ventricle measure its pressure. The hemodynamics and the activities of ATPase in myocardial cell plasm, homogenate and mitochondria were measured at preinjury(control group), 2 h, 4 h, 8 h, 12 h and 24 h postinjury. Results Left ventricular endsystolic pressure(LVESP), the maximal ascending rate of left intraventricular pressure(+dp/dtmax), isovolemec pressure(IP) and the maximal physiological velocity(Vpm) decreased significantly at 2 h group after BCT(Plt;0.05）, and recovered to preinjury level in 4 h, 8 h and 12 h group during 4-12 h after BCT; isovolumic relaxation phase left ventricular pressure descending time constant (T). Left ventricular enddiastolic pressure(LVEDP) and the maximal descending rate of left intraventricular pressure(-dp/dtmax) were significantly higher （Plt;0.05, 0.01）. The activity of ATPase in homogenate, mitochondria and cell plasm decreased significantly at 2 h group and 4 h group after BCT（Plt;0.05, 001, respectively）, and 8 h group and 12 h group recovered after BCT. There was negative correlations between [CM(159mm]LVEDP and -dp/dtmax and the decrease of the activity of Na+-K+-ATPase in homogenate（r=-0.674, -0.691, Plt;0.05）, the Ca2+-ATPase in homogenate（r=-0.613,-0.642, Plt;0.05）, the Na+-K+-ATPase in mitochondria（r=-0.622,-0.616, Plt;0.05）,the Ca2+-ATPase in myocardial cell plasm（r=-0.672,-0.658, Plt;0.05）, the Na+-K+-ATPase in myocardial cell plasm（r=-0.627,-0.632,Plt;0.05）,and the Mg2+-ATPase in myocardial cell plasm（r=-0.677,-0.661, Plt;0.05）. Conclusion The left ventricular function is impaired obviously after BCT, especially in diastolic phase. The decrease of the activity of ATPase in myocardial cells may be one of the reasons of cardiac dysfunction after BCT.
Objective To evaluate the clinical value and safety of adenosine monophosphate( AMP)bronchoprovocation test in patients with asthma. Methods Sixty asthmatics, including 19 cases with uncontrolled asthma, 22 with partially controlled asthma, and 19 with controlled asthma were enrolled. Twenty-four healthy volunteers were enrolled as control and 20 patients with upper respiratory tract infection ( URI) were also included. AMP bronchoprovocation test ( AMP-BPT) was performed. PD20 FEV1-AMP lt;40 mg was set as a cut-off value of positive response to AMP. Positive rate, sensitivity, specificity, accuracy and adverse reactions of AMP-BPT were evaluated. Eleven cases with uncontrolled asthma and 12 cases with partially controlled asthma were followed up with AMP-BPT three months and six months after inhaledcorticosteroids treatment. Asthma symptom scores were recorded a week early before each challenge. The correlation between PD20FEV1 -AMP and asthma symptom score was analyzed. Values of PD20 FEV1 -AMP were represented as median and quartile range [ M( QR) ] . Results No positive responses to AMP were found in both healthy and URI subjects. On the other hand, positive responses to AMP were found in all the uncontrolled asthmatics ( 100% ) with PD20FEV1 -AMP as 0. 6 mg ( 0. 4 mg) , in 19 partially controlled asthmatics ( 86. 4% ) with PD20 FEV1 -AMP as 5. 38 mg ( 32. 67 mg ) , and in 5 controlled asthmatics( 26. 3% ) with PD20FEV1 -AMP as 40 mg ( 29. 3 mg) . There were negative correlations between the logarithms of PD20 FEV1 -AMP and logarithms of asthma symptom scores ( r = - 0. 598, P lt; 0. 01) . The sensitivity, specificity and accuracy was 72% , 100%, and 84% , respectively. Percentage of subjects who experienced wheezing, cough, dyspnea, swallows stimulation, chest tightness, expectoration and cyanosis during AMP-BPT were 37. 5%, 21. 2%, 15. 4%,7. 7%, 7. 7%, 4. 8%, and 1. 0%, respectively. No severe adverse reaction was found. Conclusions AMP-BPT is helpful to the diagnosis and differential diagnosis of bronchial asthma. It also can be used to evaluate the severity and control level, and to monitor the therapeutic efficacy in clinical practice. Moreover, AMP-BPT is well tolerated with little adverse reaction.
ObjectiveTo explore the relationship between mitochondrial function and the severity of sepsis by detecting the platelet mitochondrial permeability transition pore, transmembrane potential and adenosine triphosphate (ATP) levels in peripheral blood. MethodsAccording to random number table, 40 male SD rats were randomly divided into three sepsis model groups (group A, B and C) and a sham group (group D). The rats in the model groups received cecal ligation and puncture (CLP) treatment with different percent of ligated length in total length of the cecum (10% in group A, 30% in group B and 50% in group C, respectively). Twenty-four hours later, peripheral blood was collected for TNF-α, IL-1βand IL-6 levels determination, also the mitochondrial permeability transition pore, transmembrane potential and ATP content were tested in the isolated platelet. One-way ANOVA test was used to determine the relevance between above indices and the severity of sepsis. Meanwhile, 29 patients with sepsis were enrolled for clinical study. After APACHEⅡscoring, platelet samples of peripheral blood in the patients were collected for mitochondrial function determination. The relationship between mitonchondrial function and APACHEⅡscore was analyzed by Spearman method. ResultsCalcein fluorescence, membrane potential and ATP synthesis in platelet mitochondria of the rat sepsis model were gradually decreased with the increased severity of CLP, and the difference among these groups were all statistically significant (all P < 0.05). In clinical specimens, APACHEⅡscore was negatively correlated with ATP level of platelet mitochondria(r=-0.895, P < 0.05). ConclusionMitochondrial function of platelet in peripheral blood can be used as an effective indicator for the severity of sepsis.
ObjectiveTo investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD+) levels.MethodsThe bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H2O2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H2O2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H2O2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD+ and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment.ResultsThe rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased (P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H2O2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD+ and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant (P<0.05).ConclusionNMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD+ levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.