| Ischemic stroke is the leading cause of mortality, disability and morbility in the world. Once blood flow was occluded for a few minutes, brain cells would be irreversibly damaged. Treatment for ischemic stroke is always awkward. Vascular-neuron unit emphasized the importance of microvessel system for maintaining the neuron function. GSRd, a monomer extracted from San Qi and Ren Shen, has shown some potential effects on enhancing angiogenesis after rats ischemic stroke, the factor of Hif-1? was found participating in angiogenesis by Ginsenoside in our previous works. This study aimed at revealing the mechanism of GSRd in vivo and in vitro with Western blot and immunohistochemistry methods. In this study, we will investigate the rule of factors upstream of Hif-1? in the process of angiogenesis promoted by GSRd, reveal whether or not via signal pathway of AKT- mTOR- Hif-1?- VEGF.Experiment 1 Angiogenesis effect of GSRd on rats after ischemic strokeObjective: To reconfirm the angiogenesis effect of GSRd on rats after ischemic stroke.Methods: Adult male SD rats, weighing 280 ~ 300 g, subjected to MCAO, were randomly divided into 4 groups: Sham + SA group, Sham + GSRd group, MCAO + SA group, MCAO + GSRd group. After 6 h of occlusion, 10 mg/kg/d GSRd was injected intraperitoneally(ip) from PSD 1 to PSD 7. Same volume of saline(SA) was injected in MCAO control group. Rats brains were frozen sliced on PSD 1, PSD 3, PSD 7, PSD 14 and PSD 21, respectively. RECA-1 monocolony antibody was used to marking microvessels on different time point in penumbra area.Results: Microvessel density and branch points in sham groups have no difference(P>0.05, Sham + SA group vs. Sham + GSRd group). Both of them decreased on PSD 1 in MCAO group(P<0.05), then gradually increased on PSD 3, PSD 7 and reached it peak on PSD 14, it decreased on PSD 21(MCAO + SA group vs. Sham groups). With the treatment of GSRd, the total tendency of microvessel density and branch points is basically accordance with MCAO + SA group, but they were higher than MCAO group on each time point(P<0.05, MCAO + GSRd group vs. MCAO + SA group).Conclusion:GSRd has the ability to promote rats angiogenesis after ischemic stroke.Experiment 2 The potential mechanism of GSRd on promoting rats angiogenesis after ischemic strokeObjective: To explore the rule of factors upstream of Hif-1? in the process of angiogenesis promoted by GSRd.Methods:(1) Adult male rats were randomly divided into 4 groups: Sham + SA group, Sham + GSRd group, MCAO + SA group, MCAO + GSRd group. After 6 h of occlusion, 10 mg/kg/d GSRd was intraperitoneally injected(ip) for 3 days. On PSD 3, the expression of VEGF, Hif-1?, p-mTOR and p-AKT in ischemic penumbra area of brains were detected through Western blot.(2) Adult male rats were randomly divided into 3 groups: MCAO + SA group, MCAO + GSRd group, MCAO + GSRd + rapamycin group. Either rapamycin(250 ug/kg/d, ip), injected 30 min before MCAO, or GSRd(10 mg/kg/d, ip), injected 6 h after occlusion, was injected for 3 days, once a day. Then ischemic penumbra area of brains was collected for detecting the expression of VEGF, Hif-1?, p-mTOR and p-AKT through Western blot.(3) Adult male rats were randomly divided into 3 groups: MCAO + SA group, MCAO + GSRd group, MCAO + GSRd + LY294002 group. MCAO was conducted 5 days later after the surgery of a 4.8 mm cannula implanted into right lateral ventricle. LY294002(10 m M, 5 ul) was given to ventricle through the cannula for 3 days and GSRd(10 mg/kg/d, ip) was also injected for 3 days, then protein in ischemic penumbra was collected for detecting VEGF, Hif-1?, p-mTOR and p-AKT levels. LY294002 and GSRd were given for 7 days to conduct immunofluorescent staining.Results:(1) On PSD3, protein levels of VEGF displayed no difference between two different sham groups, the same as Hif-1?, p-mTOR and also p-AKT(P>0.05, Sham + SA group vs. Sham + GSRd group). After operation of MCAO, VEGF, Hif-1?, p-mTOR and p-AKT protein levels in MCAO + SA group were significantly decreased(P<0.05, MCAO + SA group vs. Sham groups). With the treatment of GSRd, the expression of VEGF, Hif-1?, p-mTOR and p-AKT protein levels in MCAO + GSRd group significantly increased(P<0.05, MCAO + GSRd group vs. MCAO + SA group).(2) Rapamycin significantly inhibited the expression of p-mTOR, Hif-1? and VEGF(P<0.05), but not p-AKT(P>0.05, MCAO + GSRd + rapamycin group vs. MCAO + GSRd group).(3) LY294002 significantly inhibited the expression of p-mTOR, Hif-1?, VEGF, as also p-AKT(P<0.05, MCAO + GSRd + LY294002 group vs. MCAO + GSRd group).(4) LY294002 could decrease penumbra area of microvessel density(P<0.01, MCAO + GSRd +LY294002 vs. MCAO + GSRd = 426.56 ± 48.38 vs. 532.03 ± 16.17) and branch point(P<0.01, MCAO + GSRd + LY294002 vs. MCAO + GSRd = 212.50 ± 24.32 vs. 330.47 ± 20.44).Conclusion: GSRd may initially activate AKT and subsequently downstream factors as p-mTOR, Hif-1? and VEGF to promote rats angiogenesis after ischemic stroke.Experiment 3 Effect of GSRd on cultured HUVECs in vitroObjective: To reconfirm whether GSRd promoting angiogenesis via activating AKT-mTOR- Hif-1?- VEGF pathway in vitro.Methods: Experiment model establishing. OGD(oxygen-glucose deprivation/ reperfusion) of cultured HUVECs in vitro was used as experiment model.(1) 5 groups were included: normal cultured HUVECs group, OGD group, OGD + 1 uM GSRd group, OGD + 5 uM GSRd group, OGD + 10 uM GSRd group. After 8 h OGD and 12 h reperfusion, 20 ul supernatant was collected for detecting LDH, adherent cells in 96-well plates was used for detecting cell survival via CCK8 and cell apoptosis analysis via flow cytometry.(2) GSRd mechanism of action exploring. 6 groups were included: group of normal cultured HUVECs, group of OGD only, group of OGD + 1 uM GSRd, group of OGD + 1 uM GSRd + 2-ME2, group of OGD + 1 uM GSRd + rapamycin, group of OGD + 1 uM GSRd + LY294002. After OGD 8 h reperfusion 12 h, total protein was extracted for detecting VEGF, p-mTOR and p-AKT, nucleoprotein was extracted for detecting Hif-1?, respectively.Results:(1) Cells survival largely decreased, cell toxicity and cell apoptosis significantly increased in OGD group(P<0.05, OGD group vs. normal cultured group). GSRd 1 uM could increase cell survival about 1.5 fold higher, cell toxicity and cell apoptosis also decreased(P<0.05, OGD + 1 uM GSRd group vs. OGD group). GSRd 10 uM tended to be harmful to cell survival(P<0.01, OGD + GSRd 10 uM vs. OGD + 1 uM group). No significant difference existed between 1 uM and 5 uM group(P>0.05).(2) After subjected to OGD, the expression of VEGF, Hif-1?, p-mTOR and p-AKT significantly decreased, respectively(P<0.01, OGD group vs. normal cultured HUVECs group), but GSRd 1 uM could increase the expression of them(P<0.05, OGD + 1 uM GSRd group vs. OGD group). After added to Hif-1? inhibitor 2-ME2, mTOR inhibitor rapamycin and AKT inhibitor LY294002, Hif-1? and VEGF expression were both inhibited(P<0.05). Rapamycin could inhibit the expression of p-mTOR but not p-AKT, LY294002 could inhibit both the expression of p-AKT and p-mTOR(P<0.01, OGD + GSRd 1 uM + inhibitors group vs. OGD + GSRd 1 uM group).Conclusion: GSRd 1uM is the best concentration for HUVECs surviving and GSRd may promote angiogenesis via activating AKT- mTOR- Hif-1?- VEGF signaling pathway. |
PDF Full Text Request