The Effects Of Coumarin On Biological Activity Of Rat Osteoblasts And The Study Of ERK Pathway

Osteoporosis is a system metabolic disease with characters of losing bone mass,reducing bone quality,and bone microstructure damage.This disease can be classified into three kinds,primary osteoporosis including aging osteoporosis and postmenopausal osteoporosis with age increasing,secondary osteoporosis caused by some diseases or drugs,and idiopathic osteoporosis caused by pregnancy,lactation and genetic factors.Osteoporosis is the most common in postmenopausal women.The main pathogenesis of this disease is the decrease of bone metabolism and the excessive of bone resorption for the lack of estrogen,which leads to the reduction of bone mass.In recent years,more and more people fall in osteoporosis,which caused immeasurable loss of individual or the community.At this stage,the method of the prevention and treatment of osteoporosis is Estrogen Replacement Therapy.Clinical research shows that such drugs have strong side effects on patients with long-term use.However,with the continuous development of technology and the development of scientific research,phytoestrogen is widely used for prevention and treatment of osteoporosis.The study reveals the effect of bergapten on activity of osteoblast,and demonstrates osteoblasts proliferation and differentiation mechanism of phytoestrogen BGS3,The study provids effective target and theoretical basis for future treatment of osteoporosis.Part 1:The effect of bergapten on the biological activity of osteoblastsObjective:Research on the effect of bergapten on the proliferation and differentiation of osteoblasts.Methods:1 The culture of primary osteoblasts and observation of their morphological.2 The proliferations of osteoblasts treated by different concentration bergapten for 24,48 and 72 hours were detected by MTT method.3 The alkaline phosphatase（ALP）activity of osteoblasts treated by different concentration bergapten for 48 and 72 hours was detected by microtitration assay.4 The osteocalcin content of osteoblasts treated by different concentration of bergapten for 48 and 72 hours was detected by enzyme-linked immunosorbent assay（ELISA）.5 Different concentrations of bergapten treated cells for 48 and 72 hours.The expression of collagenⅠmRNA was detected by qRT-PCR.Results:1 Morphological observation and identification of osteoblastsObservation with inverted microscope:cells were inoculated in culture flask after 24 hours,the majority of cells attached to the wall and showed a variety of shapes at the early stage,which were polygonal or fusiform,elongated,with 1² nucleus,and nucleolus with clear outline.After 36,48 and72 h’s culture,cell density increases visibly,the elongated cells become oval,and boundary between the cells gradually reduced.After the alkaline phosphatase staining,the active site of the enzyme was changed into purple,and the positive rate was 92%.2 The experiment of proliferationCompared with the control group,osteoblasts were treated by different concentration bergapten for 24,48 and 72 h.The experiments showed that the proliferation of osteoblasts could be promoted by the bergapten of 10^-99 mol/L（P<0.05）,and the proliferation of osteoblasts was the most significant for 48 h and 72 h（P<0.01）.3 The effect of bergapten on alkaline phosphatase activity of osteoblastsCompared with the control group,osteoblasts were treated by different concentration of bergapten for 48 h,the alkaline phosphatase activity of osteoblasts was enhanced by bergapten of 0.3×10^-88 mol/L,1.0×10^-99 mol/L and0.3×10^-99 mol/L concentration（P<0.001）,for 72 h,the alkaline phosphatase activity of osteoblasts was increased by bergapten of 10^-99 mol/L and 0.3×10^-9mol/L concentration（P<0.001）.4 The effect of bergapten on osteocalcin expression of rat osteoblastsCompared with the control group,osteoblasts were treated by different concentration of bergapten for 48 h,the osteocalcin of osteoblasts was promoted by bergapten of 0.3×10^-99 mol/L and 0.3×10^-88 mol/L concentration,0.3×10^-88 mol/L group most significant（P<0.001）,for 72 h,the osteocalcin of osteoblastswaspromotedbybergaptenof1.0×10^-8mol/L concentration（P<0.001）.5 The effect of bergapten on collagen I mRNA expression of os-te oblastsCompared with the control group,osteoblasts were treated by different concentration of bergapten for 48 and 72 h,the results of the statistics showed that 48 h,the expression of collagenⅠmRNA was promoted by 0.3×10^-8mol/L bergapten（P<0.001）.For 72 h,the expression of collagenⅠmRNA was promoted by 0.3×10^-99 mol/L bergapten（P<0.001）.Conclusions:1 Certain concentration of bergapten could promote the proliferation of osteoblasts.2 Bergapten could promote the expression of collagenase I mRNA,and the secretion of alkaline phosphatase and osteocalcin,promote the differentiation of osteoblasts.Part 2:The effect of BGS3 on the proliferation and ALP activity of ratosteoblasts through ERK pathway Objective:To study whether BGS3 promote the proliferation and alkaline phos-phatase activity of rat osteoblasts through ERK pathway.Methods:1 The culture of primary osteoblasts and observation of their morphological.2 The osteoblasts were treated by drug for 24,48 and 72 h,the proliferation was detected by MTT method.3 The osteoblasts were treated by drug for 48 and 72 h,the alkaline phosphatase（ALP）activity of osteoblasts was detected by microtitration assay.4 The osteoblasts were treated by drug for 48 and 72 h,the ALP mRNA was detected by qRT-PCR.Results:1 Morphological observation of osteoblastsSame as the first part2 The experiment of proliferationThe treatment for 24 h showed that E₂ group compared with the PD98059+E₂ group,BGS3 group compared with the PD98059+BGS3 group showed no statistical difference（P>0.05）.For 48 h compared with the control group,the proliferation of osteoblasts was promoted in the E₂ and BGS3 group,and the E₂ group most significantly（P<0.01）,E₂ group compared with the PD98059+E₂group（P<0.001）,BGS3groupcomparedwiththe PD98059+BGS3 group（P<0.01）,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s promoted proliferation of osteoblasts.For 72 h also showed that compared with the control group,the proliferation of osteoblasts was promoted in E₂ and BGS3 group（P<0.01）,E₂ group compared with the PD98059+E₂ group,BGS3 group compared with the PD98059+BGS3 group,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s promote the proliferation of osteoblasts（P<0.001）.3 Based on the ERK signal pathway,the effect of BGS3 on osteoblast’s alkaline phosphatase（ALP）activityAccording to the group for drugs treatment,for 48 h,compared with the control,the alkaline phosphatase（ALP）activity of osteoblasts could be enhanced in E₂ and BGS3 group,（P<0.001）,E₂group compared with the PD98059+E₂ group,BGS3 group compared with the PD98059+BGS3 group,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s enhance the alkaline phosphatase activity of osteoblasts（P<0.001）.For 72 h,compared with the control group,the alkaline phosphatase（ALP）activity of osteoblasts could be enhanced in E₂ and BGS3 group（P<0.001）,E₂ group compared with the PD98059+E₂ group,BGS3 group compared with the PD98059+BGS3group,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s enhance thealkaline phosphatase（ALP）activity of osteoblasts（P<0.001）.4 Based on the ERK signal pathway,the effect of BGS3 on osteoblasts expression of alkaline phosphatase（ALP）mRNAAccording to the group for drug treatment,for 48 h compared with the control group,the expression of alkaline phosphatase（ALP）mRNA on osteoblasts could be enhanced in E₂ and BGS3 group,the E₂ group most significant（P<0.01）,E₂ compared with the PD98059+E₂ group,BGS3compared with the PD98059+BGS3 group,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s enhance the expression of ALP mRNA（P<0.001）on osteoblasts.For 72 h,compared with the control group,the expression of alkaline phosphatase（ALP）mRNA（P<0.05）on osteoblasts could be enhanced in E₂ and BGS3 group,E₂ group compared with the PD98059+E₂ group（P<0.05）,BGS3 compared with the PD98059+BGS3group（P<0.001）,ERK inhibitor PD98059 could inhibit estradiol（E₂）and BGS3’s enhance the expression of ALP mRNA on osteoblasts.Conclusions:1 BGS3 promoted the proliferation of osteoblasts via ERK signaling pathway.2 BGS3 promoted the expression of ALP mRNA and the activity of alkaline phosphatase on osteoblasts via ERK signaling pathway.