BMP4 Promoter Targeted Activator Screening From Traditional Chinese Medicines And Their Roles In Activating Smad Signal And Alkaline Phosphatase

In the field of dental restorations,enough alveolar bone is the basis of making denture.No matter which restoration approach we take,it requires the patients to have good bone conditions,including a certain height,width and density,and it should provide enough retention force to guarantee the stability of the effect of the denture.Common dental diseases,such as osteomyelitis,bone malignant tumors can cause the destruction and absorption of the alveolar bone,and affect the denture,finally affect the patient's quality of life.Some systemic diseases,osteoporosis is a major disease which bothers menopausal wommen and the elderly.Because osteoporosis cause bone loss and dyskinesia,it has been become a difficult medical problem.In the metabolism of bone formation,bone morphogenetic proteins(BMPs)is a kind of important protein family which affect bone metabolism and formation.BMP is a kind of transforming growth factor,and they can work in variety of organs including the process of promoting cell proliferation and differentiation,especially obvious promoting in the process of bone reconstruction.BMP has many subtypes,and they participate in many biological processes.BMP4,which is a member of the BMP family,plays a vital role in the bone formation and the process of cell proliferation and differentiation,so it is a key point of osteogenesis signals.Our study concentrates on BMP4,looking for natural Chinese medicine activators that can activate BMP4 and try to reveal the mechanism of traditional Chinese medicines and the synergy of steroid hormones activating Smad and alkaline phosphatase signal.Finally we hope we can find activator of Chinese medicine to promote bone formation.Before the experiment,other members in our team successfully build the pGL2-BMP4 promoter luciferase reporter gene recombinant plasmid by the relevant databases for BMP4 promoter sequences and designing BMP4 promoter primer.The human BMP4 promoter sequence,with length of 1581 bp,is located from upstream 1441th bases of BMP4 promoter transcription start site to downstream 139th base of the transcription start site.PCR products are connected with pGEM-T carrier.After recycling,we transfer products to the competent E.coli DH5 a,and after screening,cultivation,extraction,enzyme identification and sequencing,pGEMT-BMP4p recombinant plasmid is constructed successfully.Finally,the pGEMT-BMP4p recombinant plasmid and pGL-2 carrier both are separately by Sac I and Nhe I enzyme,then via transformation,cultivate and extract,parallel enzyme identification and sequencing,we finanly build the pGL2-BMP4 promoter luciferase reporter gene recombinant plasmid.First of all,we adopt the recombinant plasmid to screen nearly one hundred kinds of natural medicines,we find that the composition such as astragalus root,ginseng and angelica can significantly activate BMP4 promoter report gene,suggesting that three kinds of Chinese native medicine ingredients can activate BMP4 gene transcription.Next,we use the Western blot experiments to detect the role of BMP4 in protein level.As a result we find astragalus root,ginseng and angelica can significantly enhance the BMP4 protein expression.Then we find three kinds of traditional Chinese medicines for the promotion of BMP4 protein expression is dose dependent by Western blot method.BMP-Smad signaling pathway is a vital signal pathway of bone formation.Smad family play a regulated role in many biological behaviors,especially Smad4 and Smadl/5/8.In order to explore the ingredient of BMP-Smad signaling pathways,we take the immunofluorescence method at a cellular level to study the influence of composition such as astragalus root,ginseng and angelica on Smadl/5/8 and Smad4 position,and we discover Smad 1/5/8 in blank control group are relatively weak and Smad4 concentrate in cytoplasm.Smadl/5/8 and Smad4 in drug group turn into increasing trend,and present a phenomenon of positioning in the nucleus.This result show that astragalus root,ginseng and angelica can activate the Smad signal,and promote Smadl/5/8 and Smad4 copolymerization complex formation into the nucleus and regulate downstream related gene transcription.Alkaline phosphatase is an important bone metabolic marker.We adopt immunofluorescence method to detect whether the expression of alkaline phosphatase and Smad4 signal of Mc3T3-El cells.We find that the alkaline phosphatase signal in blank control group is relatively weaker,at the same time,Smad4 signal concentrate in the cytoplasm.In Chinese medicine group we find enhanced expression of alkaline phosphatase,meanwhile,the Smad4 converted into nucleaus significantly.This suggests that Chinese native medicine ingredients activate alkaline phosphatase and Smad4 signaling pathways.In conclusion we find that three traditional Chinese medicine astragalus root,ginseng and angelica can activate BMP4-Smad signaling pathways and bone alkaline phosphatase.Via bioinformatics we know there are a variety of steroid hormone receptor binding sites on BMP4 promoter.In order to reveal the connection of steroid hormone receptor and BMP4 signaling pathways,we adopt the RNA interference and the super expression method to explore and reveal all kinds of steroid hormone receptor pathway of BMP4.We transfection GR,AR,ER-alpha,ER-beta in HEK293T cell.After 16h,we add the related traditional Chinese medicines.After 24h,we detect BMP4 promoter activity applying double fluorescence detection technology.We find the expression of AR expand genetic activation effect of astragalus membranaceus on BMP4 promoter activity.Besides,overexpression of ER-a expand astragalus root,ginseng and angelica BMP4 reports gene activation effect.Finally,we use gene interference experiment to make related steroid hormone receptors silent.We find ER-a silence reverse the activation of BMP4 promoter of astragalus root,ginseng and angelica,while AR silence reverse the activation function of astragalus membranaceus on BMP4 promoter.In a word,astragalus membranaceus activate BMP4 promoter are implemented through the ER-? and AR,American ginseng and angelica activate BMP4 promoter through ER-?.The above results show that astragalus root,ginseng and angelica activate BMP4 promoter through different nonsteroidal androgen receptor signaling pathways.The study proves that the traditional Chinese medicine astragalus root,ginseng and angelica increases BMP4 gene expression through different steroid hormone receptor signal,activate preosteoblast BMP4-Smad signalling pathways,enhance Smadl/5/8 and Smad4 molecules into the cell nucleus,and promote the expression of alkaline phosphatase to regulate bone formation and bone metabolism.Our study research Chinese medicine components and provide the basis for bone defect and osteoporosis drug research.