AIM:To study the effects of noninvasive delayed limb ischemia preconditioning ( NDLIP) on ani-mal cardiac function , myocardial morphology and myocardial apoptosis after myocardial infarction ( MI ) .METHODS:Healthy SD male rats [n=45, weighing (250 ±10) g] were randomly divided into 3 groups:MI group:the animal model of MI was established by surgical ligation of left anterior descending artery ( LAD) after 2 weeks;NDLIP group:after the success of the MI animal model , NDLIP was carried out every other day until the 4th, 6th and 8th weeks;sham group:as the negative control group , the animals were taken heart LAD threading but no ligation .All rats were fed conventionally .At the end of the 4th, 6th and 8th weeks, all rats were made ventricular intubation , and then the hemodynamic parameters were recorded .The blood samples were withdrawn from the abdominal aorta and the serum was separated via centrifugation . The serum contents of Bcl-2 and Bax were measured by ELISA .Left ventricular anterior wall was homogenized .The mito-chondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲand Ⅳin the myocardial tissues were detected by ELISA .RESULTS:At the end of the 4th, 6th and 8th weeks, compared with MI group, left ventricular systolic pressure in NDLIP group was significantly increased , while left ventricular end-diastolic pressure in NDLIP group was significantly decreased ( both P<0.05).Mitochondrial respiratory chain complexesⅠ, Ⅱ, Ⅲ and Ⅳ in NDLIP group were significantly increased (P<0.05).The serum level of Bcl-2 in NDLIP group was significantly increased and Bax level was reduced remarkably (both P<0.01) .CONCLUSION:NDLIP improves the hemodynamic indexes , promotes the mitochondrial respiratory function and inhibits cell apoptosis , thus improving the prognosis of MI .

Aim To study the preventative effects of noninvasive delayed limb ischemic preconditioning ( NDLIP) on sudden cardiac death in rats with myocar-dial infarction. Methods Thirty healthy SD male rats weighting ( 250 ± 10 ) g were randomly divided into 3 groups:① myocardial infarction ( MI ) group: animal model of MI was established by making surgical ligation of animal LAD. ② MI plus NDLIP group: after the success of the animal model of MI, NDLIP was carried out every other day until 4th week. ③Sham group:as the negative control group, animals were taken heart LAD threading but no ligation. All rats were fed con-ventionally. At the end of 4 weeks, three groups of rats were administered with metaraminol ( 0. 2 mg · min-1 ) . ECG, drug cumulant of sudden death and death onset time were recorded. After sudden death, blood samples were withdrawn from abdominal aorta and serum was separated via centrifugation. ELISA method was used to measure serum caspase-3 , HSP70 and SOD concentration. Results While metaraminol led animal cardiac sudden death, the rats heart rate ( HR) kept declining with the increase of dosage of metaraminol during the administration period. Rat HR of MI+NDLIP group [ ( 479 ± 8 ) vs ( 416 ± 19 ) beat ·min-1 , ( 446 ± 32 ) vs ( 370 ± 20 ) beat · min-1 , (376 ± 53) vs (305 ± 29) beat·min-1, (307 ± 63) vs (244 ± 33) beat·min-1, (283 ± 45) vs (121 ± 35 ) beat · min-1 , P <0. 01 ] was markedly higher than that of MI group at 0 , 5 , 10 , 30 , 50 min before death. Compared with MI group, drugs cumulant to sudden death and death onset time of MI + NDLIP group [ ( 14. 58 ± 3. 03 ) vs ( 10. 76 ± 2. 73 ) mg, (72. 9 ± 15. 2 ) vs ( 53. 8 ± 13. 6 ) min, P <0. 01 ] were significantly increased. Compared with MI group, serum caspase-3 content of MI+NDLIP group was sig-nificantly reduced [ ( 2. 01 ± 0. 52 ) vs ( 2. 34 ± 0. 38 )μg·L-1 , P<0. 01 ]; HSP70 levels were remarkably increased [ ( 3. 01 ± 0. 58 ) vs ( 2. 70 ± 0. 43 ) μg · L-1 , P <0. 05 ]; SOD levels were significantly im-proved [(1. 99 ± 0. 65) vs (1. 70 ± 0. 58) mg·L-1, P<0. 01 ] . Conclusion NDLIP can prevent sudden cardiac death after myocardial infarction in rats, which may be mediated by reducing the myocardial cell apop-tosis, increasing protective protein expression and en-hancing antioxidant capacity.

Aim To investigate the effects of dioscin ( Dio) on rat myocardial contractility. Methods Left ventricular contractile function was measured using the Langendorff non-recirculating mode of isolated rat heart perfusion. Effects of low, middle and high concentra-tion of Dio were investigated by measuring left ventricu-lar systolic pressure ( LVSP ) and left ventricular end diastolic pressure ( LVEDP) . Also, peak rates of rise/fall of left ventricular pressure ( ± dp/dtmax ) of isolated rat heart were calculated. Effects of Dio on intracellu-lar free calcium concentration in rat H9 c2 cells were measured by using the confocal microscopy. Mitochon-drial membrane potential was detected with multifunc-tional microplate reader. Results With 0. 1, 1 μmol · L-1 Dio, LVSP were significantly enhanced from (11. 55 ± 0. 52), (10. 53 ± 0. 28) kPa to (13. 08 ± 0. 72), (12. 53 ±0. 64) kPa(P<0. 01); +dp/dtmax were dramatically increased from ( 0. 38 ± 0. 10 ) , (0. 40 ± 0. 07) kPa·ms-1 to (0. 42 ± 0. 11), (0. 43 ± 0. 02) kPa·ms-1(P<0. 05). With the 10μmol· L-1 Dio, LVSP and + dp/dtmax were both decreased from (12. 13 ± 0. 33) kPa and (0. 42 ± 0. 04) kPa· ms-1 to ( 9. 46 ± 0. 77 ) kPa and ( 0. 24 ± 0. 04 ) kPa ·ms-1 (P <0. 01). With 0. 1, 1, 10 μmol·L-1 Dio, the relative fluorescence intensity of intracellular free calcium concentrations was increased significantly from (16. 62 ± 0. 89) to (21. 48 ± 0. 80), (25. 68 ± 0. 69) and (19. 84 ± 0. 66)(P <0. 01)respectively. 0. 1, 1μmol·L-1 Dio showed no significant effects on the mitochondrial membrane potential of rat H9 c2 cells, while with effects of 10 μmol·L-1 Dio, the ra-tio of JC-1 monomer and J-aggregates was changed from (1. 14 ± 0. 03) to (1. 35 ± 0. 06)(P<0. 01), indica-ting a decrease in the mitochondrial membrane poten-tial. Conclusion Low and middle concentrations of Dio show a positive inotropic effect on isolated rat heart, as the LVSP and + dp/dtmax are enhanced, which may concern with the increase of the intracellu-lar concentration of Ca2+. It will not cause the calcium overload while the intracellular concentration of Ca2+ is increased by low and middle concentration of Dio in the myocytes except high concentration of Dio.

Aim To investigate the protective effects of sphingosine 1-phosphate ( S1 P ) postconditioning on hypoxia/reoxygenation( H/R) injury in human umbili-cal vein endothelial cells ( HUVEC ) and its mecha-nisms. Methods HUVECs cells were divided into five groups: normal ( control) group, S1P low concentra-tion group ( L ) , S1 P medium concentration group (M), S1P high concentration group ( H) and H/R group. MTT method was used to measure cell survival. Using flow cytometric analysis, the rate of cell apopto-sis was determined. The activities of total superoxide dismutase ( T-SOD) , copper/zinc superoxide dismuta-se ( CuZn-SOD ) , manganese superoxide dismutase ( Mn-SOD) activity, nitric oxide ( NO) and malondial-dehyde ( MDA ) content in cell culture medium were measured with colorimetry. Mitochondrial membrane potential in cells was observed with fluorescence micro-scope. Bax/Bcl-2, eNOS protein expression levels in HUVECs cells were observed with Western blot. Re-sults Compared with H/R group, S1P low, medium and high concentrations in the intervention group could significantly increase the cell survival rate after H/R injury, and increase activity of T-SOD, CuZn-SOD, Mn-SOD and decrease content of MDA. Moreover, S1 P could significantly increase NO content and in-crease eNOS protein expression, decrease apoptosis rate and inhibit the reduction of mitochondrial mem-brane potential. Conclusions S1P can decrease cell apoptosis rate of HUVECs after H/R injury with a cer-tain concentration dependence. The protection of S1P for cell apoptosis of HUVECs after H/R injury may be related to decreasing the intracellular MDA content and improving intracellular SOD activity, increasing mito-chondrial membrane potential and enhancing expres-sion of Bcl-2, anti-apoptotic protein.

OBJECTIVE:To study the inhibitory effect of dihydrotanshinone(DTS)on human lung cancer GLC-82 cell and its mechanism. METHODS:After treated with 0(blank control),5,10,20,40,80 and 100 μg/ml DTS for 24 and 48 h,MTT as-say was used to measure the inhibition rates and IC50 of cells;cell apoptosis was detected by flow cytometry after treated with 17.85 μg/ml DTS for 12,24 and 48 h to calculate apoptotic rate;Western blot was used to detect the protein expressions of Bcl-2, Bax and Caspase-3. RESULTS:Compared with blank control group,different concentrations of DTS inhibited the proliferation of cells;24 and 48 h maximal inhibition rate were 54.48% and 64.95%,respectively;IC50 were 62.36 and 33.94 μg/ml. DTS could induce cell apoptosis in positive time dependent manner,and the range of inhibition rate was 5.6%-29.6%;Western blot showed DTS could down-regulate the expression of Bcl-2 protein and up-regulate the expression of Caspase-3 protein (P<0.01 or P<0.05). CONCLUSIONS:DTS have significant inhibitory effect on GLC-82 cells and also induce cell apoptosis,by a possible mech-anism of down-regulating the expression of Bcl-2 protein and up-regulating the expression of Caspase-3 protein.

Objective To knock out Asxl2 gene in murine embryonic fibroblast cell line NIH3T3 using CRISPR/Cas9n system. Methods A pair of sgRNAs which targeted exon 5 of Asxl2 gene were designed and subcloned into the pX462 vec?tor. The recombined plasmids were verified by sequencing and transfected into NIH3T3 cell line. Single cells were isolated through serial dilutions, followed by an expansion period to obtain new monoclonal cell lines. The genomic DNA of the new monoclonal cell lines was extracted and a DNA fragment flanked the target site was amplified by genotyping PCR then se?quenced. Lastly, western blotting were applied to confirm whether Asxl2 was successfully knocked out. Results The CRIS?PR/Cas9n plasmids that targeted Asxl2 were successfully constructed. NIH3T3 cells were co-transfected with the two recom?binant constructs. After puromycin selection, subclonal cell lines were obtained and one of them was validated by genotyping PCR-sequencing. Western blotting also confirmed that Asxl2 was completely depleted in the NIH3T3 cell line. Conclu?sion CRISPR/Cas9n plasmids that targeted Asxl2 were successfully constructed therefore a Asxl2 knockout NIH3T3 stable cell line was established via this system.

Aim To study the protective effects of sphingosine 1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation in reperfusion injury salvage kinase ( RISK ) signal path-way. Methods The cultured rat H9c2 cells were ran-domly divided into seven groups: ( 1 ) control group;(2) hypoxia/reoxygenation (H/R) group; (3) S1P group;(4) S1P+LY294002 group(S1P+LY); (5) LY group; ( 6 ) S1 P +PD98059 group ( S1 P +PD );(7) PD group. The viability of H9c2 cells was detec-ted using MTT method. The content of MDA in the cultured medium and the activity of T-SOD and Mn-SOD were measured with colorimetry. The concentra-tion of intracellular free calcium ion was detected by confocal microscopy. The rate of cell apoptosis was de-termined by flow cytometric analysis. Western blot was used to assess phosphorylation of Akt and ERK1/2 in H9c2 cells. Results Compared with the H/R group, S1P significantly increased vaibility of cells, lowered the rate of apoptosis, decreased the content of MDA in the culture medium, increased the activity of T-SOD and Mn-SOD, reduced concentration of intracellular calcium and increased the phosphorylation of Akt and ERK1/2 . When added LY294002 or PD98059 , the effects of S1P above were inhibited. Conclusion S1P protects H9 c2 cells against hypoxia/reoxygenation inju-ry. The protection of S1P was inhibited by LY294002, the inhibitor of PI3 K/Akt and PD98059 , the inhibitor of ERK1/2 . S1 P protects H9 c2 cells against hypoxia/reoxygenation injury via RISK pathway.

Aim To investigate the antiarrhythmic mechanism of taurine-magnesium coordination com-pound on abnormal sodium current channel ( INa ) in-duced by hypoxia-reoxygenation in ventricular myocytes of rats. Methods Single ventricular myocytes were i-solated from each rat heart using enzymatic dissociation through Langendorff retrograde aortic perfusion. Whole-cell patch clamp was applied in voltage clamp mode to record INa both in normal ventricular myocytes and single ventricular myocytes of arrhythmia induced by hypoxia-reoxygenation. Results The peak density of INa was changed from ( 56. 89 ± 2. 07 ) pA/pF to (35. 05 ± 1. 52) pA/pF( n=6, P <0. 01 vs control) by hypoxia-reoxygenation with the INa I-V curve shifting upward. TMCC(200, 400 μmol·L-1)was able to re-store the reduction caused by H/R to (35. 78 ± 1. 95) pA/pF, (41. 52 ± 0. 86) pA/pF, (n=6,P <0. 01) and (48. 34 ± 0. 99) pA/pF(n=6,P<0. 01) respec-tively, but not at 100 μmol·L-1(n=6, P>0. 05), in a concentration-dependent manner, while amioda-rone restored it to (39. 44 ± 1. 24) pA/pF (n=6,P<0. 01 ) . Both high concentration of TMCC and amioda-rone could shift the I-V curve downward. In addition, TMCC and amiodarone could restore the INa inactivation curve and slow down its inactivation, whereas the acti-vation curves showed no significant differences among groups. Conclusion TMCC(200,400 μmol·L-1) could restore the H/R induced INa reduction and shift the I-V curve downward by inhibiting steady-state inac-tivation, which is suggested to be one of the mecha-nisms of the antiarrhythmic effects of TMCC in hypoxia-reoxygenation model.

Aim To investigate the protective effects of nicorandil, glutamine and metoprolol used in single and combination on the antiapoptosis ability of cardio-cytes and the expression of HSP70 after myocardial is-chemia/reperfusion injury in rats. Methods Male Wistar rats were divided randomly into seven groups:( 1 ) Sham group:in which the coronary artery was not roped;(2) I/R group:in which only 30 min ischemia and 120 min reperfusion of LAD was executed; ( 3 ) Nicorandil group; ( 4 ) Glutamine group; ( 5 ) Meto-prolol group; ( 6 ) Nic + Glu + Met ( NGM ) group;(7) low dose of Nic+Glu+Met ( NGML) group. In the pharmacological precondition groups, correspond-ing drugs were administered 30 min before I/R proto-col, and the initial drug dose in each group was 1 mg ·kg-1 . Myocardial apoptotic rates were measured with TUNEL ( terminal dexynucleotidyl transferase-mediated dUTP nick end labeling) , and the expression of apop-tosis related proteins-Bcl-2/Bax and protective pro-tein-HSP70 was detected by Western blot. ResultsIn I/R group, the apoptotic rate in ischemic region was very high ( 36.9% ± 10.3%) , and Bcl-2/Bax was very low ( 0.14 ±0.08 ) . Compared with I/R group, the apoptotic rate of pharmacological precondition groups was decreased significantly ( P <0.01 ) , and the ratio of Bcl-2 to Bax was raised ( P<0.01 ) . Com-pared with Sham group, the expression of HSP70 in-creased significantly ( P<0.01 ) in I/R group. Com-pared with IR group, the expression of HSP70 of phar-macological precondition groups was elevated ( P <0.01 ) , and the expression in NGM group was higher than in the single drug group ( P<0.01 ) . Conclusion Compared with single therapy after I/R in rat, the combination therapy of nicorandil, glutamine, meto-prolol can decrease the apoptotic rate, and the expres-sion of apoptosis related proteins is developed. In the mean time, the expression of protective protein HSP70 is elevated.

Aims To investigate the protective effects of noninvasive limb ischemic preconditioning (LIPC) on myocardial ischemia-reperfusion (I /R)injury,and to explore the mechanism.Methods Healthy male Wistar rats were divided randomly into I /R,I /R +LIPC,I /R +5-Hydroxydecanoate (5-HD)and I /R +LIPC +5-HD groups.The I /R +LIPC and I /R +LIPC-5-HD groups of rats were subjected to three cy-cles of LIPC induction per day with 5 min of reperfu-sion after occlusion for 5 min at the left hind limb for 3 days.All rats were subjected to myocardial I /R injury on the fourth day.The I /R +5-HD and I /R +LIPC-5-HD groups of rats were given the inhibitor of ATP-sen-sitive potassium channel 5-HD before and during myo-cardial I /R injury. Results Compared with I /R group,LIPC reduced myocardial infarct size (P <0.05),lowered cardiocyte apoptosis index and Fas, FasL positive cell number (P <0.01 ),increased the reduced nitric oxide (NO)/endothelin (ET)-1 ratio (P <0.05)in serum in I /R +LIPC group.5-HD a-bolished the protective effects induced by LIPC in I /R+LIPC-5-HD group.Compared with normal myocardi-al tissue,expression of mir-30a-3p was increased in I /R group (P <0.01 )and was decreased in LIPC group (P <0.01 ).Conclusion LIPC alleviates myocardial I /R injury and improves endothelial function. The mechanism may be related with the opening of ATP-sensitive potassium channel,regulating the balance be-tween NO and ET-1 and decreasing the expression of myocardial mir-30a-3p.