| Background&Objective: postmenopausal osteoporosis(PMOP)is caused by the loss of ovarian dysfunction and the decreasement of estrogen levels in postmenopausal women.It's liable to cause a dynamic imbalance between bone formation and bone resorption,fracture and health lesion in patients.At present,osteoporosis-targeting drugs are increasing.Research and development of anti-osteoporosis drugs have been developed from cell to signaling pathway.It opens up a new path for accurate treatment of osteoporosis.Polygonatum sibiricum polysaccharide(PSP)is the main component of herbal Polygonatum sibiricum Red.and has the functions of antioxidation,anti-inflammation,reducing blood sugar,blood fat and delaying senescence.The preliminary study has found the anti-osteoporosis effect of PSP,the specific mechanism remains a further study.MicroRNAs(miRNAs)is one kind of endogenous non-coding RNA which is highly conservative with the functions ofregulating cell proliferation,differentiation,lipornetabolism and hormone secretion.Studies have shown that miRNAs can regulate the processes of osteoporosis,including the differentiation of osteoblasts,osteoclasts and chondrocytes,and it has been one of the important regulatory factors for certain bone metabolic diseases.Therefore,this study aims to explore the effects of PSP to mi RNAs expression on ovariectomized rats,process of rat bone marrow mesenchymal stem cells(rBMSCs)differentiation into osteoblasts and process of bone marrow-derived mononuclear macrophages(BMMs)differentiation into osteoclasts,which provide the basis for accurate anti-osteoporosis effects of PSP.Contents: 1.Remove the bilateral ovaries to establish a ovariectomized-osteoporosis rat model,and the effect of PSP on the expression of mi RNA was observed in bone marrow stromal cell derived from rat model.2.Induce rBMSCs differentiate into osteoblasts,and observe the effect of PSP on miRNA expression of osteogenic differentiation.3.Induce rat BMMs differentiate into osteoclasts,and observe the effect of PSP on miRNA expression of osteoclast differentiation.4.Predict the target gene of significantly down-regulated rno-miR-1224,and observe the effect of PSP on osteoclast differentiation based on Hippo signaling pathway.Methods: 1.Established a postmenopausal osteoporosis rat model by extripating bilateral ovaries.(1)Body weight was measured weekly.(2)Bone mineral density(BMD)was measured by dual-energy X-ray absorptiometry(DEXA).(3)Alkaline phosphatase(ALP),osteocalcin(OC),tartrate resistant acid phosphatase(TRAP),serum calcium(Ca)and serum phosphorus(P)were detected by Elisa Kit and spectrophotometry.(4)Microstructure of tibial metaphysis in rats were observed by micro-CT scanning.(5)Primary BMSCs ofOVX group and H-PSP group were isolated by whole bone marrow method,and RNA was detected for miRNA microarray detection after extraction.2.Established a osteoblastic differentiation cell model induced by BMSCs.(1)Culture of BMSCs and observed cellular morphology of P3 cells under the microscope.(2)Observe cell proliferation of BMSCs treated with different concentrations of PSP by the method of MTT assay.(3)After induction for 21 d,ALP staining was observed in each group.(4)After induction for28 d,mineralization was observed by alizarin red staining.(5)After induction for7/14/28 d,mRNA expression of osteogenesis related genes of ALP,Runx2 and OCN was detected by real-time quantitative PCR(qRT-PCR).(6)RNA was extracted for miRNA microarray detection after induction for 21 d when treated with the concentration of PSP of 25mg/L.3.Established a osteoclastic differentiation cell model induced by BMMs.(1)Primary BMMs were cultured and observed cellular morphology under the microscope.(2)After induction for 7d,TRAP staining was used to identify the proliferation of osteoclasts.(3)After induction for 7d,RNA was extracted for miRNA microarray detection.4.(1)Predict the target genes regulated by rno-miR-1224 using the software of mirPath v.3.(2)Protein expression of Limd1,Mst1,Lats1,TAZ,TEAD3,CTGF was detected at the point of 0,15,30,60 min when treated with PSP using western-blot methods.Results:1.(1)Body weight of OVX group was significantly increased than Sham group.After treated with different concentrations of PSP for 4 weeks,body weight of H-PSP group was highly decreased than OVX group,and at 8 weeks,H,M-PSP group was highly decreased.(2)BMD of OVX group was significantly decreased than Sham group.After administration of PSP for 8 weeks,BMD ofH-PSP group was significantly increased than OVX group.(3)Serum ALP,OC,TRAP,Ca and P of OVX group were highly increased than Sham group.After giving different doses of PSP,the levels of ALP,TRAP and P in H-PSP group were significantly decreased,OC in H,M-PSP group was highly decreased,Ca in H,M,L-PSP group was highly decreased than OVX group.(4)The levels of BV/TV,Tb.N and Tb.Th in each dose of PSP were increased to different degrees than OVX group and Tb.Sp decreased.(5)MicroRNA microarray analysis showed 15 differential expression mi RNAs,6 of which were up-regulated and 9 were down-regulated.2.(1)rBMSCs were long spindle and polarity arranged in shape,and short fusiform and larger in shape after osteogenic differentiation.(2)There was no significant difference in OD values between each group when detecting cell proliferation with MTT method.(3)Cell membrane and cytoplasm were dark blue and lumpy with ALP staining.(4)Mineralized nodes were crimson and significantly increased in 25mg/L PSP group than in control group.(5)mRNA expression of osteogenesis related genes of ALP,Runx2 and OCN was highly increased in different doses of PSP group.(6)MicroRNA microarray analysis showed 3 down-regulated differential expression miRNAs.3.(1)Osteoclast looked like fried eggs with cytoplasm red,vacuoles and cell multinucleated with TRAP staining.(2)MicroRNA microarray analysis showed27 differential expression miRNAs,3 of which were up-regulated and 24 were down-regulated.4.(1)Limd1 was Predicted as the target gene regulated by rno-miR-1224.(2)Protein expression of Limd1 was highly decreased at the point of 30,60min;Mst1 and Lats1 decreased at 30,60min;TAZ decreased at 60min;TEAD3 decreased at 30,60 min;CTGF decreased at 15 min.Conclusion:(1)PSP has the effect of anti-osteoporosis with the resistance of bone loss in postmenopausal osteoporosis.(2)The expression of miRNA plays an important role in regulation of bone mass in rats,rBMSCs' induction into osteoblast and BMMs' differentiation into osteoclast.(3)mi R-1224 affects the process of osteoclast proliferation by regulating downstream factors of Hippo signaling pathway. |
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