| Objective: Leonurine hydrochloride is a kind of extracted from Leonurus artemisia hydrochloride monohydrate,according to previous studies,the medicinal value of leonurine hydrochloride has many kinds,mainly concentrated in the treatment of gynecological diseases and the latest treatment of diseases of cardiovascular system.It has been preliminarily proved that herba leonuri alleviates primary osteoporosis,and the related mechanism is achieved by inhibiting the proliferation and differentiation of osteoclasts.However,the specific mechanism of osteoblasts is not clear.The purpose of this study is to investigate the effects of leonurus hydrochloride on osteoblasts through a variety of in vivo and in vitro experiments,and to explore the possible mechanisms.Methods:1.18 8 week old female C57BL/6 mice weighing 19-22 grams was raised in SPF laboratory and animal feeding room of Benxi experimental center of Shengjing Hospital affiliated to China Medical University.After one week of adaptive feeding,mice were randomly divided into three groups,6 in each group.They were sham operation group,osteoporosis model group,osteoporosis model plus Leonurine hydrochloride intervention group.There were no other operations to suture the peritoneum and the skin only after the sham operation group opened the abdominal cavity.The osteoporotic model group and the osteoporosis model + leonurin hydrochloride intervention group,after opening the abdominal cavity,the bilateral ovaries were ligated and removed by 6-0 lines,then the peritoneum and skin suture were performed.One week after operation,the osteoporosis model plus leonurine hydrochloride intervention group was given intraperitoneal injection of leonurine hydrochloride,the dose was 15mg/kg/d.The sham operation group and the osteoporosis model group were given the same amount of normal saline intraperitoneally.After 8weeks of intervention,bone mineral density was measured,and the distal femur and proximal tibia bone tissue and peripheral blood were detected.2.The pathological sections of the distal femur bone tissue,HE staining of distal femoral stem general structure of metaphyseal trabecular bone,and immunohistochemistry on tissue sections,were used to detect the expression of osteogenic differentiation protein Runx2 and Wnt/beta-catenin pathway downstream protein beta-catenin.3.The X-ray and micro-CT methods were used to detect the bone density and the changes of bone microstructures in mice.4.The ELISA method and the special kit were used to detect the osteocalcin and the detection of alkaline phosphatase in the peripheral blood of mice.5.The effect of leonurine hydrochloride on the cell viability of MC3T3-E1 in vitro of mouse pre-osteoblasts was measured by CCK-8 colorimetric assay.6.Using alkaline phosphatase staining and alizarin red staining,the effect of leonurus hydrochloride on MC3T3-E1 differentiation and mineralization of mouse osteoblast precursor cells was detected.7.The use of real-time PCR to detect the image effect of leonurine hydrochloride on MC3T3-E1 mice in vitro,the main indicators include some key protein Wnt/ beta-catenin pathway(LRP5,GSK-3 beta,beta-catenin)m RNA expression,and osteoblast differentiation(ALP,Runx2,Osterix,Col1-alpha 1)and bone mature protein related(OPG)expression of m RNA.8.We detected protein expression of osteoblast differentiation protein Runx2 and Wnt/ beta-catenin pathway downstream of beta-catenin and phosphorylated GSK-3beta by Western blotting(Western blot).Results:1.Bone mineral density of osteoporosis model group was significantly lower than that of normal control group(P<0.01),and bone mineral density of osteoporosis modeling group and leonurine hydrochloride group was significantly higher than that of modeling osteoporosis group(P<0.01).2.Compared with sham operation group,osteocalcin(OCN)in serum of osteoporotic mice was significantly decreased(P<0.01).Compared with osteoporosis modeling group,osteocalcin in serum of osteoporosis model+LH group increased(P<0.01).This is similar to the result that serum alkaline phosphatase(ALP)of osteoporosis modeling mice was lower than that of sham operation group and osteoporosis model plus leonurine hydrochloride group(P<0.01),the expression of leonurine hydrochloride can significantly alleviate osteoporosis in mice by alkaline phosphatase(P<0.01).3.HE staining showed that the number and width of the distal femur trabecula in the primary osteoporosis mice decreased obviously and the interval was obviously increased.The prognosis of leonurus hydrochloric acid can improve the sparsity of trabecular bone obviously.4.Immunohistochemistry showed that compared with the sham operation group,the expression of beta-catenin and Runx2 in the distal femur tissue of osteoporosis group decreased significantly(P<0.01).However,the expression of beta-catenin and Runx2 in the osteoporotic group and the group of leonurus hydrochloride was significantly increased(P<0.01).5.Micro-CT detection of proximal tibia bone microstructure showed that compared with sham operation group,width and number of osteoporosis model group in trabecular bone and total volume,percentage of trabecular area and trabecular bone volume,percentage of trabecular bone were significantly decreased(P<0.01),trabecular bone spacing width increased significantly(P<0.01);and the model of osteoporosis + leonurine hydrochloride group,compared with the osteoporosis model group,leonurine hydrochloride increased number of trabecular bone volume and percentage of trabecular bone(P<0.01),decreased trabecular spacing(P<0.05).6.Through the CCK-8 detection of Leonurine hydrochloride MC3T3-E1 on osteoblast precursor cells of mouse in vitro activity,proliferation of100ummol/L showed lower concentrations of Leonurine hydrochloride had no significant effects on MC3T3-E1 cell,concentrations of 100μmmol/L,resulting in the proliferation rate of MC3T3-E1 decreased significantly(P<0.01).7.Alkaline phosphatase and alizarin red staining showed that Leonurine hydrochloride(10μmmol/L,1μmmol/L)could significantly promote the secretion of alkaline phosphatase and calcification nodules in MC3T3-E1 cells.At the same time to detect the concentration of alkaline phosphatase in MC3T3-E1 cells of intervention of Leonurine hydrochloride 1 days and 5 days,1 days between each intervention showed no obvious differences in the concentration of alkaline phosphatase,after 5 days of intervention,the intervention group 10μmmol/L and1μmmol/L of Leonurine hydrochloride alkaline phosphatase expression significantly,statistically significant difference respectively(P<0.01,P<0.05).8.RT-PCR showed that MC3T3-E1 cells in vitro exposed to Leonurine hydrochloride of 10ummol/L,the Wnt pathway related gene LRP5,an important node of beta-catenin m RNA expression increased significantly,and different concentrations of Leonurine intervention(10μmmol/L,1μmmol/L,0.1μmmol/L)have prompted the osteogenic differentiation genes(ALP,unx2,Osterix,Col1-alpha 1)and OPG gene expression increased.9.Compared with the control group,after the intervention of Leonurine hydrochloride,the expression of Wnt-catenin and p-GSK-3 β increased significantly,and the total GSK-3 beta expression was not significantly different.The expression of important protein Runx2 in osteogenic differentiation was significantly increased.Conclusion:1.Leonurine hydrochloride can significantly increase the bone mineral density of mice with primary osteoporosis.2.Leonurine hydrochloride can significantly increase the number of trabecular bone and reduce the distance between the average trabecular bone,improve the bone microstructure and increase bone mineral density in primary osteoporosis mice.3.Leonurine hydrochloride can significantly improve the serum levels of osteocalcin and the expression of alkaline phosphatase in the serum of mice with primary osteoporosis.4.Leonurine hydrochloride can increase the expression of Wnt/ β-catenin pathway downstream protein beta-catenin and the expression of osteoblast transcription factor Runx2 in bone of mice,and increase the activity of osteogenic differentiation.5.Leonurine hydrochloride can activate the Wnt /β-catenin signaling pathway in vitro,increase the expression level of LRP5,beta-catenin m RNA,and increase the phosphorylation level and proportion of GSK-3 β in osteoblasts.6.Leonurine hydrochloride can improve the alkaline phosphatase activity and expression of MC3T3-E1 in vitro,and promote the mineralization process of osteoblast.7.Leonurine hydrochloride can increase the expression of related genes of osteoblast differentiation(ALP,Runx2,osterix,Col1-alpha 1)and the m RNA expression of mature related gene OPG. |
