Role Of JAK2/STAT3 In Mediating G-CSF Effect On Myocardial Apoptosis Following Coronary Microembolization

Coronary microembolizaion (CME) is the main reason of coronary no-reflow. CME is a frequent and important event in acute coronary syndromes and during coronary interventions. It is important to reduce the damage induced by CME. In our study on the CME model of rat, we detected the mRNA of Bcl-2, Bax, Fas, Fas-L and the protein expression of caspase-3, caspase-9, cleaved PARP, t-JAK2, p-JAK2, t-STAT3 and p-STAT3. We aimed to investigate the effects of granulocyte colony stimulating factor (G-CSF) on myocardial apoptosis following CME and the role of Janus kinase/singnal transducer and activator of transcription (JAK/STAT) pathway in the process.Part I The effects of CME on myocardial apoptosis and the left ventricular functionObjective To study The effects of CME on myocardial apoptosis and left ventricle function. Methods A total of 56 male Sprague Dawley rats were randomized into control group (n=32) and sham-operated group(n=24). The control group was underwent injection of homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta and the sham-operated group was injected with saline. The rats were sacrificed at day 3,7,14 and 28 after operation. The mRNA of Bcl-2,Bax,Fas,Fas-L and GAPDH which was used as the intracomparison, were evaluated by Real Time PCR. The ratios of Bcl-2/Bax also be compared. The protein expression of Caspase-3, Caspase-9 and the cleaved PARP were detected by western blot. Myocardial apoptosis and left ventricular function were checked by TUNEL staining and hemodynamics. Results In the control group, the histological examination showed that there were multi-focal myocardial ischemia, necrosis with inflammatory cells infiltration and fibrosis in subendocardial region. Compared with the sham-operated group, the mRNA of Bcl-2,Bax,Fas and Fas-L increased whereas the ratio of Bcl-2/Bax markedly decreased in control group (0.18±0.04 vs 2.98±0.49, P< 0.01). Simultaneously, the protein expression of Caspase-3, Caspase-9 and the cleaved PARP enhanced. The myocardial apoptosis index elevated(17.2±1.9 vs 1.2±0.6, P<0.01) and the left ventricular function decreased significantly (P<0.01). Conclusions The CME could promote myocardial apoptosis and depress the left ventricular function by altering the balance of the apoptosis gene and activating the caspase system.Part II Experimental study on the effects of G-CSF on myocardial apoptosis following coronary microembolizationObjective To investigate the effects of G-CSF on myocardial apoptosis and left ventricular function following CME. Methods A total of 88 male Sprague Dawley rats were randomized into control group(n=32), G-CSF-treated group(n=32) and the sham-operated group(n=24). The control and G-CSF-treated group were received CME. RhG-CSF(100ug.kg-1.d-1) were injected subcutaneously for 5 days since 2 hours after operation in the G-CSF-treated group. The control group and the sham-operated group were treated with saline respectively. The rats were sacrificed at day 3,7,14 and 28 after operation. The mRNA of Bcl-2, Bax, Fas, Fas-L and GAPDH which was used as the intracomparison, were evaluated by Real Time PCR. The ratios of Bcl-2/Bax also be compared. The protein expression of Caspase-3, Caspase-9 and cleaved PARP were detected by Western-blot. Myocardial apoptosis and left ventricular function were checked by TUNEL staining and hemodynamics. Results Compared with the control group, the area of micro-infarct in subendocardial region was lessened in the G-CSF-treated group. Compared with the sham-operated group, the mRNA of Bcl-2, Bax, Fas, and Fas-L increased whereas the ratio of Bcl-2/Bax markedly decreased in the control group (P<0.05 or 0.01). Simultaneously, the protein expression of Caspase-3, Caspase-9 and cleaved PARP enhanced, the myocardial apoptosis index elevated and the left ventricular function decreased significantly (P<0.05 or 0.01). All of these changes induce by CME would be lessened when treated with rhG-CSF (P<0.05 or 0.01). Conclusions G-CSF could lighten the myocardial apoptosis and improve the left ventricular function after CME through reduced the mRNA of Bax,Fas and Fas-L, increased the mRNA of Bcl-2 and the ratio of Bcl-2/Bax, decreased the protein expression of Caspase-3, Caspase-9 and cleaved PARP.Part III Role of JAK2/STAT3 in mediating G-CSF effect on myocardial apoptosis following coronary microembolizationObjective To investigate the effects of G-CSF on myocardial apoptosis following CME and the role of Janus kinase/singnal transducer and activator of transcription(JAK2/STAT3) pathway in the process. Methods A total of 152 male Sprague Dawley rats were randomized into CME group(n=32), G-CSF group(treated with G-CSF)(n=32), AG490 group (treated with G-CSF and AG490, the inhibiter of JAK2) (n=32) ,RPM group (treated with G-CSF and rapamycin, the inhibiter of STAT) (n=32) and the Sham-operated group(n=24). All groups were received CME except the Sham-operated group. RhG-CSF(100μg.kg-1.d-1) were injected subcutaneously for 5 days since 2 hours after operation in G-CSF group, AG490 group and RPM group. AG490 group also received AG490(5mg.kg-1.d-1) and RPM group received rapamycin(0.4mg.kg-1.d-1) by intraperitoneal route for 5 days simultaneity. The CME group and Sham group were treated with saline respectively. The rats were sacrificed at day 3, 7, 14 and 28 after operation. The mRNA of Bcl-2, Bax, Fas, Fas-L and GAPDH which was used as the intracomparison, were evaluated by Real Time PCR. The ratios of Bcl-2/Bax also be compared. The protein expression of Caspase-3, Caspase-9 and the cleaved PARP, t-JAK2,p-JAK2,t-STAT3 and p-STAT3 were detected by western blot. Myocardial apoptosis and left ventricular function were checked by TUNEL staining and hemodynamics. Results Compared with the G-CSF group, the area of micro-infarct in subendocardial region was more severe in the AG490 group and the RPM group. In the AG490 group and RPM group, compared with the G-CSF group, the mRNA of Bax, Fas and Fas-L increased whereas the Bcl-2 and the ratio of Bcl-2/Bax markedly decreased (0.57±0.12, 0.31±0.03 vs 2.07±0.29, P<0.05 or 0.01), the protein expression of caspase-3, caspase-9, PARP and the myocardial apoptosis index (14.2±1.5, 15.8±2.1 vs \ 6.1±1.0, P<0.05 or 0.01) enhanced significantly, the left ventricular function also decreased (P<0.05 or 0.01). The protein expression of t-JAK2 and t-STAT3 in the five groups had no significant differences. The p-JAK2 protein expression also had no significant differences in CME group, AG490 group and the sham-operated group, but increased markedly in the G-CSF group and RPM group. The p-STAT3 protein expression had no significant differences in all groups except the G-CSF group which had a more expression. Conclusions Whatever AG490,the inhibiter of JAK2, or rapamycin, the inhibiter of STAT, could inhibited G-CSF to lighten the myocardial apoptosis induced by CME. So, we could make a conclusion that G-CSF lightened the myocardial apoptosis induced by CME throμgh JAK2/STAT3 pathway.