The Mechanisms Of Osteoking On Promoting Bone Formation Via Regulating TGF-β1/Smads Signal Pathway

BackgroundOsteoporosis(OP)is one of bone metabolic disease,characterized by the loss of bone mass,disruption of skeletal microstructure that leads to fragility fractures.OP is frequently occurred in the middle-aged and older people and postmenopausal women[1].OP has become a serious public health issue as the accelerated aging of population.Particularly,the incidence of OP has risen the third place in chronic diseases that only second to angiocardiopathy and diabetes in China[2].An epidemiological survey indicated that there are about 202 million of people over 60 years in china in 2015,the number will rise to 438 million as well as the patients with OP and low BMD will reach 533 million by 2050.Antiresorptive therapy is still the most predominant way in the treatment of OP up to now.However,some disadvantages are still existed in the therapy of bone resorption inhibition,such as inadeqeunt bone remodeling would be occurred because of the bone loss.As a representative osteotropic drug,although rhPTHl-34 has been approved by FDA,there were many defects existed like expensive price and serious adverse reactions,etc.Some other novel osteotropic drugs,such as insulin-like growth factor 1,bone morphogenetic proteins and Wnt signal pathway antagonists are still in the experimental stage[4].Therefore,it was urgent for searching for efficient,safe,economical and practical drugs for OP therapeutics in future clinical study.It was worthy of noting that traditional Chinese medicine and ethnic-minority medicine play important roles in the prevention and treatment of OP.The first domestic book named "official clinical practice guidelines for evidence-based Chinese medicine in primary osteoporosis" directed by Yongyan Wang and Keji Chen,et al.has been published in 2012[5].As memtioned in the book,the therapeutic principle to the OP was "strengthening bones,nourishing liver and kidney as the mainline assisted with activating blood circulation".As a typical Yi nationality medicine,Osteoking is a compound preparation composed of pseudo-ginseng,safflower,dried tangerine peel,astragalus,ginseng,eucommia ulmoides and turtle shell,etc.And the drug compatibility of Osteoking was followed to the theory of Yi nationality medicine and the principle of traditional Chinese medicine.It has found that Osteoking was not only respond to fracture and osteonecrosis of the femoral head,but also effective to osteoporosis and osteoporotic fractures[6-8].There are a lot of laboratoty studies showed that Osteoking has the abilities of improving the microstructure and bone density,delayed bone loss,increased the contents of serum bone formation markers in OVX treeshrews.In addition,it has also proved that Osteoking could promote the Runx2 and BMP2,which are the key genes of endogenous osteogenesis in OP animal models.Therefore,our studies aimed at exploring the mechanism of Osteoking on promoting bone formation and providing novel drug candidates and scientific evidences for the clinical application of ethnic-minority medicine in the treatment of OP.Excessive bone resorption and inadequete formation are the essence of the pathogenesis of OP.As the most important bone cell in formation,Osteoblast(OB)is derived from osteoprogenitor which differentiated from bone marrow stromal stem cells(BMSCs),[13-14].However,the number of OB,as the ability of proliferation and osteogenesis of it was significantly decreased in OP patients.As the seed cell of OB,BMSCs is an important basis for the treatment of OP depend on its proliferation and osteogenic differentiation potential.As a specific transcriptional factor and key gene,Runx2 can directly activate specific markers of OB,such as OCN and OPG[15],during the differentiation of BMSCs into OB.And it is regulated by multiple signal pathways,in which,the TGF-β1/Smads is important[16-17].TGF-β1 plays an important role in osteogenesis and bone remodeling[18],because it is one of the first growth factors to control the proliferation and osteogenic differentiation of BMSCs.As the key initially transcription factor,Smad2/3 transduce TGF-β1 from the cell surface into nucleus,when they has formed a multiple complex with other factors after being phosphorylated,regulating the transcription of target gene Runx2[19]ObjectiveThis study was to evaluate the promoting effects of osteoking on bone formation via testing the changes of microstructure of lumbar vertebrae and the content of PINP in OVX rats,and to explore the molecular mechanism via testing the levels of related genes and proteins in TGF-β1/Smads pathway of lumbar vertebrae in OVX rats.Meanwhile,it is the first to prove that osteoking can promote the proliferation and osteogenic differentiation of BMSCs via regulating the TGF-β1/Smads signaling pathway at the celluar level.So BMSCs may be the basic cell in promting bone formation by osteoking in this study.Methods1 The effects of osteoking on OVX rats1.1 Making OVX rats model and grouping:SD female rats were bilaterally ovariectomized(OVX),the successful model was divided into two groups after 24 weeks.Osteoking:OVX rats were gavaged osteoking(the dose was 1 time of human equivalent dose(2.25ml/Kg),every other day for 12 weeks,Control:gavageing normal saline,the rest of the conditions are consistent.1.2Evaluation methods:Evaluating qualitatively and quantitatively the microstructure of bone tissue via the effects of bone formation with HE staining and Micro-CT.RT-PCR was used to detect the expression level of TGF-β1,Smad2,Smad 3,Runx2 and OCN in the two groups of lumbar vertebrae.WB was used to detect the protein expression of TGF-β1,p-Smad2/3,Runx2,Osterix and OCN.Evaluating PINP content of in serum of OVX rats by ELISA tests.2 The effects of osteoking on BMSC2.1 The cultivation and generation of rBMSCs:The cell was inoculated into the 25cm2 culture bottle at the density of 1 × 106 cells/cm2,with the complete medium of F12,containing 10%FBS and 1%penicillin-streptomycin,was added to the culture box of 37℃ and 5%CO2.2.2 Preparation of osteoking containing serum:Medicated serum intervents cells to simulate the pharmacological effects of drugs,and avoid the appearance of false positive or false negative results of the production of pigment in Chinese medicine.2.3 Evaluation methods:The MTS assay was used to detect the proliferation of rBMSCs with osteoking containing serum for 48 hours;RT-PCR detects the expression level of TGF-β1,Smad2,Smad3,Runx2 and C-myc after osteoking containing serum intervents rBMSCs for 2 weeks,and WB detects the protein expression level of TGF-β1.3 Statistical treatmentSPSS22.0 was used for statistical analysis.The normal distribution of measurement data was expressed with x±s,and t test was used in the two groups,and P<0.05 was statistical difference.Results1 Osteoking promotes bone formation in OVX rats1.1 Osteoking gavaged for 12 weeks can improve the lumbar bone microstructure in OVX rats.HE staining shows that the bone trabecula was smaller,thinner and the free ends are increased.Between the trabecular bone,the connection is reduced,the distance is widened in group control,comparison with group osteoking.Mirco-CT shows that the BMD,Tb.N and Tb.Sp of osteoking group were all higher than control group,the difference was statistical difference(P<0.05).Tb.Th is higher in number but statistical difference(P>0.05).1.2 In RT-PCR,the expression of TGF-β1,Smad2,Smad 3,Runx2 and OCN in the lumbar vertebra was higher in group osteoking,comparison with control group,and it was statistical difference(P<0.05).1.3 In WB,the expression of TGF-β1,p-Smad2/3,Runx2 and OCN of group osteoking was higher than control group,the difference was statistical difference(P<0.05).OSX is higher but statistical difference(P>0.05).2 Osteoking containing serum promotes the proliferation and osteogenic differentiation of rBMSCs2.1 Containing serum promoted the proliferation of rBMSCs,comparison with non-containing serum(P<0.05),and the proliferation rate was 150%.2.2 In RT-PCR,the expression of TGF-β1 Smad2,Smad 3,Runx2 and C-myc was higher in group containing serum,comparison with control group,and it was statistically significant(P<0.05).2.3 In WB,the expression of TGF-β1,Smad2,Smad3 and Runx2 of group osteoking was higher than control group,the difference was statistically significant(P<0.05).Conclusion:In vivo,we founds that osteoking could promote bone formation in osteoporotic rats,and its molecular mechanism may play a role in regulating TGF-β1/Smads signaling pathway.In vitro,osteoking also regulates the TGF-β1/Smads signaling pathway to promote the proliferation and osteogenic differentiation of BMSCs.It suggests that BMSCs may be one of the target cells for bone formation with osteoking,which also comfires the effect of osteoking on promoting osteoporotic bone formation.It’s a synergy with the experiment in vivo.