Experimental And Mechanism Study On The Effect Of Tongguan Capsule Mobilizing Bone Marrow-derived Endothelial Progenitor Cells

BackgroundEndothelial Progenitor cells can proliferate and differentiate into vascular endothelial cells (EPC). EPC derived from bone marrow play an important role in vascular functions and angiogenesis after Ischemia. Tongguan Capsule, a protective effect on vascular endothelial cells, not only promotes the recovery of cardiac function of the patients after percutaneous coronary intervention, but also significantly inhibits the restenosis in stent. The effect of Tongguan Capsule is so similar to EPC biology, which mediated the re-endothelialization and improved the blood supply of ischemic tissue. Some Herbs, in Tongguan capsule prescription, have significantly improved the proliferation, migration, adhesion capacity of EPC.Research PurposesTo explore the effects and Mechanisms of Tongguan Capsul mobilizing bone marrow—derived endothelial progenitor cells.Methodology1 Retrospective analysis of clinical data:60 cases of eligible patients with coronary heart disease, who were treated with percutaneous coronary intervention (PCI), Of which 30 cases of patients were treated with conventional western medicine (the control group) and other 30 cases were treated with Tongguan Capsule and conventional western medicine(the treatment group). The clinical characteristics, coronary angiography and surgical methods were Analysis. Comparative Analysis the left ventricular ejection fraction (LVEF), ventricular wall motion parameters and cardiac function classification of NYHA before the operation and 30 days after the operation. The Major adverse cardiac events (MACE) were Analysis in 30 days after PCI. The level of the serum VEGF before the operation, and 30 days after the operation were Analysis.2 The peripheral blood mononuclear cells were extracted from SD rat, which were given Tongguan Capsule for 9 days. Mononuclear cells were cultivated with endothelial growth medium-2. After being cultured for 7d, the attached cells were identified as EPC by uptaking DiI-acLDL and binding FITC-lectin-1. We measured the number of colony-forming units of EPC. Cell Migration ability was examined by migration assay. The adhesion activity was determined by cell counts. The level of the serum VEGF was measure by ELISA. Isolate and culture the circulating EPC from healthy volunteers. The attached cells were characterized by uptaking Dil-acLDL and binding FITC-lectin-1. And also the attached cells were determined expressing CD34+ and VEGFR-2+ by flow cytometry. Preparation Tongguan Capsule serum by serum pharmacology method. Saline were given to the SD rat as the control. Cultured EPC were incubated with Tongguan Capsule serum and Tongguan Capsule serum with PI3K/Akt inhibitors (wortmannin and LY294002), simvastatin as a positive control. The cell migration ability was examined by migration assay, the adhesion activity was determined by cell counts The proliferation was determined by MTT assay and cell proliferation cycle analysis assay.Research Results1 Clinical Retrospective analysis:Before the operation the clinical characteristics, coronary angiography, surgical methods, LVEF, ventricular wall motion parameters and cardiac function classification of NYHA were no difference in two groups.30 days after the operation, the LVEF, ventricular wall motion parameters of two groups are improved (P<0.05), Meanwhile, compared with the control group, the LVEF and ventricular wall motion scores of the treatment group were further improved. Duing to the severe left ventricular dysfunction, one case in control group was died at 4 weeks after the operation.30 days after the operation, there were on difference in nonfatal myocardial infarction, target vessel revascularization between two groups. The incidence of new heart failure of the treatment group was statistically lower than the control group (P=0.045), thus, the incidence of MACE in the treatment group was statistically lower than the control group (P=0.024). The level of VEGF were decreased significantly at 30 days in two groups. The level of VEGF at 30 days after the operation in the treatment group had significant differences compared with the control group(P<0.01).2 At the fifth day, rat mononuclear cells changed from round to spindle-shaped and grow into a colony. At the seventh day, some of the long spindle-shaped cell showing the distribution of cord-like or tubular. Cells were Identified as EPC that it could uptaking Dil-ac-LDL and binding FITC-lectin-1.3 Result of rat EPC colony-forming units, migration assay, adhesion assay and serum VEGF level assay:compared with the saline group, the numbers of colony-forming units and migration in Tongguan capsule group were significant (P<0.01). The number of adhesion and VEGF level in Tongguan capsule group, were no differences (P>0.05)4 At the fifth day, human mononuclear cells cells changed from round to spindle-shaped and grow into a colony. Cells were Identified as EPC that it could uptaking Dil-ac-LDL and binding FITC-lectin-1. The attached cells positive in expressing both CD34+and VEGFR-2+ were about 15.4±1.8%.5 Result of human EPC migration assay, adhesion assay, proliferation assay and cell proliferation cycle analysis assay:simvastatin as a positive control, compared with other Concentration, 1umol/L simvastatin has a were significant effect on EPC proliferation and proliferation cycle. Compared with the saline group, the numbers of EPC migration, adhesion, proliferation and cell proliferation cycle in Tongguan capsule serum group, lumol/L simvastatin group, and PI3K/Akt inhibitors group were significant (P<0.01). Compared with lumol/L simvastatin group, Tongguan capsule serum group have a lower effect on EPC adhesion and cell proliferation cycle (P<0.01), but have a higher effect on proliferation(P <0.01). Compared with PI3K/Akt inhibitors group, Tongguan capsule serum group have a higher effect on the numbers of EPC migration, adhesion, proliferation and cell proliferation cycle (P<0.01).Conclusion1 Tongguan capsule can significantly improve left ventricular ejection fraction, ventricular wall motion parameters, cardiac function classification of NYHA and serum VEGF levels in patients with coronary heart disease after percutaneous coronary intervention.2 Tongguan capsule can significantly mobilized rat bone marrow EPC to peripheral blood and enhance the biological function of migration. In vitro, Tongguan capsule serum significantly increased in human EPC migration, adhesion and proliferation, but effects were not so as simvastatin. PI3K/AKT inhibitors significantly reduced the biological functions of Tongguan capsule. The mechanisms on the effect of Tongguan Capsule mobilizing bone marrow—derived EPC may not be by increasing the serum VEGF levels and do not rely entirely on the activation of PI3K/AKT signal.