Exploration Of Panax Notoginseng Saponins's Role In Regulating The Angiogenesis Of Endothelial Progenitor Cells Through Targeted Blockade Of Wnt/?-catenin

Objective:Objective Distraction Osteogenesis(DO)is a dramatic model of endogenous bone tissue engineering technique that promotes bone regeneration by applying controlled gradual mechanical traction between the osteotomy cuts.The rate of osteogenesis is 4× faster than fastest physeal growth in children.This technology has already been widely used in the treatment of various types of malformations of the maxillofacial region,and has achieved satisfactory clinical results.However,this lengthy technique is limited by the undesired long duration of the consolidation phase.Therefore,accelerating callus formation and promoting osteogenic quality are the significant events of shorting consolidation phase during DO.In our previous studying,we found that the traditional Chinese medicine,PNS,can promote osteogenesis.And CD133 and CD34,the surface marker of EPCs,were highly expressed in the neo-formational tissue of osteodistraction.EPCs are reported that it could participate in post-angiogenesis.Meanwhile,preliminary study shows that PNS could promote the angiogenesis;proliferation and migration of EPCs through the classical Wnt/?-cateninsignaling pathway.While,the molecular mechanism during this process remains unclear.To further illuminate the definite mechanism,we investigated EPCs function under conditions of ?-catenin gene inhibition and explored the regulatory of angiogenesis and Wnt signaling pathway.We then investigated whether PNS played a functional role in EPCs angiogenesis by targeting Wnt signaling pathway in vitro.It provides a scientific basis for traditional Chinese medicine in the process of distraction osteogenesis.Methods:1.Isolation,cultivation and identification of EPCs in vitro.2.Immunofluorescence was used to ensure the expression of ?-catenin in EPCs.3-4.After transfer the shRNA-?-catenin-GFP vector,quantitative real-time PCR and western blot were used to confirm whether ?-catenin was inhibited and to determine the expression of angiogenic factor such as VEGF-A,bFGF and VE-Cadherin.5.CCK-8,Transwell and Matrigel tube formation experiments were used to test the proliferation,migration and tube formation ability of different treatments EPCs.6.After add 6.25 mg/L PNS into culture medium to culture Control group;negative group and shRNA-?-catenin group EPCs for 72 h,Matrigel tube formation experiment was used to detect the tube formation ability and qRT-PCR & WB were used to determine the expression of angiogenic cytokines such as VEGF-A,bFGF & VE-Cadherin and the key proteins such as LRP5;DVL;GSK-3?;p-GSK-3? & ?-catenin in Wnt signaling pathway.Results:1.EPCs extracted from the bone marrow of puppies grew adherently.Over time,the peripheral spindle cells finally fused to form a typical "paving stone"-like structure;immunofluorescence detection showed positive expression of CD133,CD34 and VEGFR2;and Matrigel tube formation test shows a capillary-like lumen structure can be formed thereon.2.Immunofluorescence detection showed positive expression of ?-catenin.3.After72 hours of virus transfection of EPCs,the GFP accounted for more than 80% of the total cells.4.qRT-RCR and Western blots were used to detect the silencing efficiency of shRNA-?-catenin virus transfected into EPCs.It was found that shRNA-?-catenin2 had the highest silencing efficiency,so the subsequent experiments were carried out with this designed target.And after Silencing?-catenin,the angiogenesis-related cytokines such as VEGF-A,bFGF and VE-cadherin were down-regulated.5.CCK-8 showed that there was no significant difference in OD values between CTRL group,NC group and shRNA-?-catenin group,indicated that silencing ?-catenin gene had no effect on the proliferation of EPCs.6.Transwell experiments and Matrigel tube formation experiments revealed that silencing ?-catenin gene reduced the ability of migration and tube-like structure formation.While after adding 6.25mg/L PNS in CTRL gruop;NC group and shRNA-?-catenin group,it was found that the tube formation ability of EPCs was enhanced,and had statistical significance(p<0.05).7.qRT-RCR was used to detect the changes of VEGF-A;bFGF;VE-cadherin and related factors of Wnt signaling pathway after induce PNS for72 hours.It reveals that PNS increased the expression of ?-catenin and angiogenesis-related factors.While,LRP5 and DVL,the downstream of?-catenin in Wnt signaling pathway,were increased and GSK-3? was decreased.The result of WB is basically consistent with the PCR and the p-GSK-3?increase.Finally,to detect the expression of ?-catenin in cytoplasm and nucleus.The result shows PNS could promote the ?-catenin access to the nucleus.Conclusion:1.Using RNAi technology to block the Wnt/?-catenin indicates that Wnt/?-catenin regulates the expression of VEGF-A,bFGF and VE-cadherin in EPCs to participate the angiogenic differentiation and neovasculogenesis.2.PNS promote the angiogenesis of EPCs in vitro mainly via Wnt/?-catenin,the functional target is located on the upstream of ?-catenin.