| Ding Xin Recipe (DXF) was invented by professor Jia YueHua in Southern Medical University. After long-term clinical and experimental research it had been proven that the recipe was not only effective to prevent and treat tachyarrhythmia, but also to reduce the myocardial ischemia-reperfusion injury (MIRI) . Prohibitin had been proven that it was a pharmacological target of DXF in the study of National Science Foundation "the proteomic mechanism of myocardial ischemia and reperfusion arrhythmia and the effect of DXR" through two-dimensional gel electrophoresis and mass spectrometry experiment. However the mechanism of prohibitin about MIRI was unclear. The problem became start of this study. It had been proven that P38/MAPK was a pharmacological target of DXF. MAPK signaling pathway can be regulated by prohibitin in the study about tumor cells. Prohibitin can be regulated by cytokines in inflammatory cells, for instance TNF-α,NF-κB,IL-6. The hypothesis of this study was prohibitin can regulate the MAPK signaling pathway to reduce MIRI, and it was a pharmacological mechanism of DXF. The experiments in vivo and vitro were carried out to verify the hypothesis.1. Study the expression of prohibitin in rats with MIR and regulated effect of DXF.2. Study the effect of DXF in regulating MAPK signaling pathway.3. Research the relationship between prohibitin and MAPK signaling pathway in reducing myocardial ischemia reperfusion injury in rats induced by DXF.1. Groups:40 male wistar rats that belong to SPF grade and were about 200-250g were randomly divided to 5 groups, shame group, MIR group, DXFH group, DXFM group, DXFL group. Each group contained 8 rats.2. Intervene methods:rats in DXFH, DXFM, DXFL group were drenched by DXF for 7 days. The doses were 37.5g-kg-1·d-1,18.75 g·kg-1·d-1,9.375g·kg-1·d-1. The drug was divided two pieces one day. The rats in shame group and MIRI group were drenched with normal saline. All rats were fasted and openly watered for 12h before last administration. The operation was carried out in 2h after last administration that the left anterior descending coronary artery in the upper 1/3 was ligated for 15min, then it was loosened for 30min.3. Detection index:ⅡECG of rats was recorded and graded as the standard of arrhythmia score during reputation period. Left ventricular was separated at end of experiment and was used to extract protein. The expression of prohibitin, pP38, pJNK, pERK1/2 was detected by western blotting recorded as IOD.4. Statistics methods:Arrhythmia score was ranked data. Multiple groups were analyzed by Kruskal-Wallis H test. The data was expressed as average rank R. IOD ratio of groups was analyzed by Kruskal-Wallis H test, bonferroni test was used in multiple comparion. The data was expressed as mean±SD. The correlation between prohibitin and the protein of MAPK was analyzed by Partial correlation analysis, another ratio of proteins as control variables.1. Arrhythmia score of rats were found significant differences among groups (χ2=29.816, P= 0.000). The average rank of MIR group was highest(34.06), shame group was lowest(4.50), DXFL, DXFM, DXFH were 24.94,22.50 16.50.2. There were significant differences in the expression of prohibitin among groups(χ2= 37.463,P=0.000). Compared with MIR groups, expression of prohibitin in DXFL, DXFM, DXFH groups were lower (3.06±0.12,2.08±0.09,1.65±0.13) and there were significant differences among the three groups (P＜0.05), shame group was lowest (P<0.05). The results indicated expression of prohibitin can be increased by MIRI. The increased expression can be inhibited by DingXin recipe, the effect was positively correlated with the dose.3. There were significant differences in expressions of pERK1/2, pJNK, pP38 among groups (pERK1/2:χ2=32.212,P=0.000, pJNK:χ2= 34.469,P=0.000, pP38:χ2= 34.566. P=0.000). Compared with MIR groups, expression of pERK1/2, in DXFL, DXFM, DXFH decreased orderly (4.39±0.46,5.03±0.67,5.65±0.71) (P＜0.05), shame group with no significant differences (P>0.05). Compared with MIR groups, expression of pJNK and pP38 in DXFL, DXFM, DXFH decreased orderly (pJNK:3.33±0.32,2.86±0.34,2.33±0.41,pP38:2.39±0.25,2.06±0.16,1.74±0.15) (P＜0.05), shame group was lowest (P＜0.05).The results indicated ERK signaling pathway can not be activated by this model, JNK and P38 signaling pathway can be activated. However the ERK signaling pathway can be activated by DXF. The activation of JNK and P38 signaling pathway can be inhibited by DXF.4. There were significantly positive correlations between prohibitin and pJNK or pP38 (r=0.738,P=0.000, r=0.683,P=0.000). There were no significantly correlations between prohibitin and pERK1/2 (r=-0.265,P=0.107). The results indicated the activation of JNK and P38 signaling pathway was positively correlated to prohibitin.1. Prohibitin was inhibited by DXF to reduce myocardial ischemia reperfusion injury.2. ERK1/2 anti-apoptosis signaling pathway can be activated by DXF. P38 and JNK pro-apoptosis signaling pathway can be inhibited by DXF. The above was the mechanism of reducing MIRI.3. The initial mechanism of prohibitin in reducing MIRI maybe activated P38 and JNK signaling pathway induced by DXF.To select preparation method of DXF-containing serum that reduced oxidative stress injury.48 male wistar rats that belong to SPF grade and were about 200-250g were randomly divided to 4 groups. Each group contained 8 rats. The rats were respectively drenched by DXF. The doses were 18.75 g·kg-1·d-1,37.5 g·g·kg-1·d-1,75 g·kg-1·d-1,150 g·kg-1·d-1. The drug was divided two pieces one day. All rats were fasted and openly watered for 12h before last administration.3 rats were respectively executed at 30min,60min,90min,120min after last administration in each groups. Blood was collected from aorta abdominalis to separate serum. The serum was filtered sterilization with 0.22μm microporous membrane, inactived at 56℃for 30min, individually aliquoted and conserved at -20℃.Primary cardiomyocytes were cultured from suckling rats. The cells were cultured in 96 well plates, 1×104/well. The groups were divided as DXF dose and times of blood collection, containing 16 groups and 3 well in a group, so serum of every rat was corresponded to a well. Primary cardiomyocytes were cultured for 24h by DMEM with 12.5% DXF-containing serum. After 24h the wells were washed by DMEM with no serum. Then the wells were added DMEM containing H2O2 200μm/L for 3h. At last, the cells were detected by MTTexperiment, the results were optical density(OD).OD was measurement data. The data fit the normal distribution evaluated by 1-Sample K-S Test. It was expressed as mean±SD (x±s).The experiment containing two factors and four levels was the factorial design. The interaction effects and main effects were analyzed by analysis of variance of factorial design. The differences between main effects were analyzed by bonferroni test, simple effects were analyzed by one-way ANOVA.There was interaction effects between dose effects of DXF and time effects of blood collection (F=2.803, P=0.015). There was significant differences in the effects of dose (F=56.030, P=0.000), with dose increased OD values increased, no differences (P=0.075) between 75 g·kg-1·d-1group (0.956±0.276) and 150 g·kg-1·d-1group (1.020±0.269). The highest average OD values were in 75 g·kg-1·d-1*90min group(1.124±0.139),75 g·kg-1·d-1*120min group (1.209±0.132), 150 g·kg-1·d-1*90min group (1.234±0.132),150 g·kg-1·d-1*120min group (1.020±0.269), no significant differences among the groups.It is the best method to preparate DXF-containing serum for anti-oxidative stress injury that the dose of DXF was 75 g·kg-1·d-1 and time of blood collection was 90min.To analyzed components of DXF-containing serum.10 male wistar rats that belong to SPF grade and were about 200-250g were randomly divided to 2 groups. Each group contained 5 rats. The rats of DXF group were respectively drenched by DXF 75 g·kg-1·d-1. All rats were fasted and openly watered for 12h before last administration. Rats were respectively executed at 90min after last administration in each groups. Blood was collected from aorta abdominalis to separate serum. Control group was drenched by normal saline.2 ml blood sample was added 16ml acetonitrile, shocked for 5min on the vortex mixer. Supernatant lipid was conservated after centrifugal separation with 3000r/min and 20min. The lipid dried by nitrogen at 40℃. The conservated powder was added to 1ml methanol and mixed with vortex mixer. The sample was filtrated by 0.22μm Micro Membrane. Conditions of HPLC-MS, precision, linearity, stability, recovery were selected. At last analyzed the content of Berberine hydrochloride, Palmatine Hydrochloride, Berberine chloride, tanshinoneⅡA, oxymatrine in DXF-containing serum. The concentration of the explicit material in DXF were that Berberine hydrochloride 1.4141±0.0224μg/ml, Palmatine Hydrochloride 0.3883±0.0082μg/ml, Berberine chloride 0.4889±0.0156μg/ml, oxymatrine 0.4663±0.0129μg/ml. Tanshinone II A were not detected.To choice the optimal concentration of chemosynthesis siRNA mediated by li posomes to transfect siRNA.The transfection can be done when cardiomyocytes grew well and cells fu sion to 80%. Cardiomyocytes were trypsinized by 0.1% trypsin for 3-5 minute s before transfection. Cardiomyocytes were resuspended to adjust 1×105/ml. Set cells to six well plate and 2ml/well. CY3-Negative siRNA mediated by lipid-based agent siPORTTM NeoFXTM to transfect cardiomyocytes, the concentration was 0,5,10,20,30nM. After 24 hours, the transfection efficiency and apo ptotic rate were evaluated by fluorescent microscope and flow cytometer to sel ect an optimal concentration. Based the best concentration, siRNA PHB contai ned ID s129542 (5'-3'GCUUUUAUUGUUACACUUtt) and PHB siRNA ID s2 17968 (5'-3'ACUGUGAAAUUUAAUGAUUtt) was transfected to cardiomyocyt es.48h later, the expression of PHB was tested by western blotting.Transfection efficiency and apoptosis rate were analyzed by Kruskal-Wallis H test, bonferroni test was used in multiple comparion. The IOD ratio was analyzed by Mann-Whitney Test. The data was expressed as mean-SD. 1. With the increased concentration of CY3-Negative siRNA, the number of cells emitted red fluorescence grew under fluorescence microscope. The cells with no CY3-Negative siRNA did not emitted red fluorescence.2. The average transfection effects increased with increased concentration 0.00±0.00%,7.55±0.70%,36.52±4.55%,60.14±3.94%,76.67±4.29,the differences were significant(n=3,χ2=13.597, P=0.009). The average transfection effects in The best concentration to transfect was 30nM. The effects were positively corresponded with concentration.3. The average apoptosis rates increased with increased concentration 3.00±0.87%,3.70±0.53%,3.94±0.52%,4.29±0.62%,4.55±0.43%,with no significant differences (n=3,χ2=7.445, P=0.114). The data indicated the transfection were not toxic.4. The PHB expression of cardiomyocytes transfected siRNA PHB (0.22±0.07) was dropped by 74.11% on average, compared with control group (0.85±0.06). The differences were significant (U=0.000, P=0.0495)The results indicated that lipid based agent siPORTTM NeoFXTM was suitable to transfect chemosynthesis siRNA to primary cardiomyocytes, and the best transfection concentration of siRNA was 30 nmol/L.1. Study the expression of prohibitin in rats with oxidative stress injury and regulated effect of DXF. 2. Study the effect of DXF in regulating MAPK signaling pathway in oxidative stress injury.3. Research the relationship between prohibitin and MAPK signaling pathway in reducing oxidative stress injury of cardiomyocyte induced by DXF.1. There were 7 groups containing Normal, H2O2, H2O2+DXF, PHBsiRNA, H2O2+PHBsiRNA, H2O2+U0126, H2O2+SP600125, H2O2+SB203580. Each group contained 6 wells,3 wells to detecte apoptosis rates, the other to extract protein.24h after transfection, DMEM was changed, the serum of H2O2+DXF contained 12.5% DXF-containing serum, U012610,SP600125,SB203580 were respectively added at 46h, the concentration were 10μM.25μM,5μM.48h after transfection, culture plates were washed with serum-free DMEM, then added DMEM containing 200μM H2O2 for 3h. At last apoptosis and expression were detected.The apoptosis and IOD ratio of groups was analyzed by Kruskal-Wallis H test, bonferroni test was used in multiple comparion. The data was expressed as mean±SD. The correlation between prohibitin and the protein of MAPK was analyzed by Partial correlation analysis, another ratio of proteins as control variables.1. There were significant differences in the apoptosis among groups (x2=18.907,P=0.004). Compared with H2O2 groups (35.04%±2.27%), apoptosis in H2O2+PHBsiRNA (35.04%±2.27%), H2O2+U0126 (42.65%±4.21%) there were no significant differences (P=1.000, P=0.053). There were no significant differences among H2O2+SP600125 (21.22%±1.84%),H2O2+SB203580 (16.67%±2.49%),H2O2+DXF (16.67%±2.49%),their apoptosis were lower than H2O2 group and upper normal group (35.04%±2.27%) (P＜0.05). The results indicated apoptosis can be increased by OS. The increased apoptosis can be inhibited by DingXin recipe, SP600125 and SB203580,with no significant differences among the three groups,but U0126 can't inhibit increased apoptosis.2. There were significant differences in the expression of prohibitin among groups (x2=18.719, P=0.005). Compared with H2O2 groups(1.88±0.13), H2O2+U0126 (1.90±0.18) was not different (P＞0.05), the expression in H2O2+DXF (2.54±0.12),H2O2+SP600125 (2.27±0.20) and H2O2+SB203580 (2.35±0.09) was increased (P<0.05), with no differences during the three groups, normal and H2O2+PHBsiRNA were lowest with no differences between two groups (P>0.05). The results indicated prohibitin can be increased by OS. The increased expression can be inhibited by PHBsiRNA. However DXF,SB203580 and SP600125 increased the expression of prohibitin.3. There were significant differences in the expression of pERK among groups (x2=18.009, P=0.006). Compared with H2O2 groups(2.55±0.13), H2O2+SB600125 (2.62±0.11) and H2O2+SP600125 (2.62±0.11) was not different (P>0.05),the expression in H2O2+DXF (3.01±0.18) was increased (P<0.05), H2O2+U0126 (1.54±0.05) and H2O2+PHBsiRNA (2.15±0.11) decreased (P＜0.05) but upper normal (1.56±0.13). The results indicated pERK can be increased by OS. The expression can be increased by DXF and inhibited by U0126.SB203580, SP600125 can't affect the expression of pERK.4. There were significant differences in the expression of pJNK among groups (x2=16.398,P=0.012). Compared with H2O2 groups(3.01±0.21), H2O2+ SP600125 (2.00±0.23) and H2O2+DXF (1.76±0.23) was decreased but upper normal (1.53±0.23),in H2O2+PHBsiRNA (3.01±0.16),H2O2+U0126 (2.82±0.07). H2O2+SB203580 (2.84±0.27) there were no differences (P>0.05). The results indicated pJNK can be increased by OS. The expression can be inhibited by DXF and SP600125, it can't affect by SB203580 and U0126.5. There were significant differences in the expression of pP38 among groups (x2=16.277, P=0.012). Compared with H2O2 groups (2.45±0.23), H2O2+ SB203580 (1.25±0.14) and H2O2+DXF (1.78±0.19) was decreased (P＜0.05) but upper normal (1.17±0.10),in H2O2+PHBsiRNA (2.42±0.27),H2O2+U0126 (2.33±0.34),H2O2+SP600125 (2.29±0.14) there were no differences (P>0.05) The results indicated pP38 can be increased by OS. The expression can be inhibited by DXF and SB203580, it can't affect by and U0126 and SP600125.6. There were significantly positive correlations between prohibitin and pERK1/2 (r=0.643,P=0.003). There were no significantly correlations between prohibitin and pJNK or pP38 (r=-0.052,P=0.832, r=-0.031,P=0.900). The results indicated the activation of pERK1/2 signaling pathway was positively correlated to prohibitin.1. Prohibitin was inhibited by DXF to reduce oxidative stress injury.2. ERK1/2 anti-apoptosis signaling pathway can be activated by DXF. P38 and JNK pro-apoptosis signaling pathway can be inhibited by DXF. The above was the mechanism of reducing oxidative stress injury.3. The initial mechanism of prohibitin in reducing oxidative stress injury maybe activated ERK signaling pathway induced by DXF.4. The initial mechanism of prohibitin in reducing MIRI maybe regulated MAPK signaling pathway induced by DXF, with regulated two-way signaling pathway.1. MIRI can be reduced by DXF with regulating MAPK signaling.2. Prohibitin can be inhibited by DXF during MIR, but can be increased by DXF during oxidative stress.3. The initial mechanism of prohibitin in reducing MIRI maybe regulated MAPK signaling pathway induced by DXF, with regulated two-way signaling pathway.4. It is the best method to preparate DXF-containing serum for anti-oxidative stress injury that the dose of DXF was 75 g·kg-1·d-1 and time of blood collection was 90min.5. The concentration of the explicit material in DXF were that Berberine hydrochloride 1.4141±0.0224μg/ml, Palmatine Hydrochloride 0.3883±0.0082μg/ml, Berberine chloride 0.4889±0.0156μg/ml, oxymatrine 0.4663±0.0129μg/ml. TanshinoneⅡA were not detected.6. Lipid based agent siPORTTM NeoFXTM was suitable to transfect chemosynthesis siRNA to primary cardiomyocytes, and the best transfection concentration of siRNA was 30 nmol/L.
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