| ObjectivesIn this study,we studied the effects of CSA on the prevention and treatment of glucocorticoid-induced osteoporosis in rats,and the cellular and molecular mechanisms of CSA on the prevention and treatment of glucocorticoid-induced osteoporosis in osteoblasts and osteoclasts.Methods1.Forty-five six-month-old female SPF SD rats were divided into three groups: CON group,GIOP group and CSA group.The rats in GIOP group and CSA group were injected with dexamethasone(1.0mg / kg)once every 3 days.The CON group was intervened with equal volume of normal saline.After 12 weeks of intervention,rats in the three groups were given saline and CSA intraperitoneal injection respectively.The rats of GIOP group were given 200 ?l saline intraperitoneally every day,and the rats of CSA group were given 10 mg / kg 200 ?l intraperitoneally every day.The treatment lasted for 12 weeks.After the treatment,the rats in each group were given intraperitoneal blood samples to detect ALP,lumbar bone samples were taken for ?CT scanning,dual energy X-ray measurement of bone density,tissue sections and biomechanical experiments.2.We culture osteoblast and osteoclast,and observe the effect of dexamethasone on the cell activity,differentiation,mineralization and apoptosis of osteoblast.To observe the protective effect of CSA on osteoblast under glucocorticoid intervention,and to observe the effect of CSA on the bone resorption and differentiation of osteoclast,as well as the expression of related genes and proteins.Results1.Bone mass of rats: compared with CON group,BMD and BMC of rats in GIOP group were significantly lower(P < 0.05),and BMD and BMC in treatment group were significantly higher than those in GIOP group(P < 0.05),but there was no statistical difference in AREA between the groups.2.Bone microstructure of rats: compared with CON group,BV / TV,TB.N Tb.Th,v BMD decreased significantly(P < 0.05),SMI and Tb.Sp compared with GIOP group,BV / TV,TB.N,TB Tb.Th And v BMD were significantly higher than those of GIOP group(P <0.05),SMI was significantly lower than GIOP group(P < 0.05).In addition,?CT two-dimensional images showed that the trabeculae of GIOP group were significantly sparse and discontinuous compared with CON group.Compared with CON group,the bone of rats in GIOP group was significantly loose and porous,and CSA group significantly improved the number and shape of trabecula.3.Bone strength of rats: compared with CON group,the compression strength of bones in GIOP group decreased significantly(P < 0.05).Compared with GIOP group,the compression strength,compression stiffness and energy absorption of CSA group were significantly higher than those of GIOP group(P < 0.05).4.Morphology of lumbar vertebrae in rats: under 4X and 10 X microscope,with the increase of rats' age,the bone damage in GIOP group was gradually worsened;compared with CON group,the number of bone trabeculae in GIOP group was significantly reduced at different time points,and the arrangement of bone trabeculae was disordered,sparse,broken and the gap widened.Under 40 X microscope,compared with CON group,GIOP group significantly reduced the number of bone cells at different time points,and there were few osteoblast,osteoclast and marrow stem cells on the surface of trabecula,and the number of fat bubbles in marrow cavity increased significantly.Compared with GIOP group,CSA can significantly reverse the bone morphological damage caused by DEX at different time points under low and high fold microscope.5.ALP activity in serum of rats: compared with CON group,ALP activity in GIOP group was significantly lower(P < 0.05),and that in CSA group was significantly higher than that in GIOP group(P < 0.05).6.Activity of osteoblast: DEX inhibited the activity of osteoblast in a dose-dependent manner.Above 50 ?mol / L,DEX significantly inhibited the activity of osteoblast(P <0.05).CSA had no significant toxicity to osteoblast,and CSA within 10 ?mol / L did not significantly affect the activity of osteoblast(P > 0.05).In the presence of DEX,CSA was added to coculture,and it was found that the increase of CSA concentration had a positive effect on the activity of osteoblast.CSA above 2.5 ?mol / l could significantly increase the activity of osteoblast(P < 0.05).7.Osteoblast apoptosis: compared with CON group,DEX can induce osteoblast apoptosis(P < 0.05).After adding CSA,it was found that 5 ?mol / L and 10 ?mol / L CSA could significantly reduce the apoptosis rate of osteoblast(P < 0.05).8.Mineralization ability of osteoblast: the results of ALP staining of osteoblast showed that CON group,DEX group and CSA group had no significant effect on ALP staining results(P > 0.05).After mineralization induction of osteoblast and alizarin red staining,the bone mineralization ability of DEX composition was significantly lower than that of CON group(P < 0.05),and that of CSA group was significantly higher than that of DEX group(P <0.05).9.Osteoblast differentiation related genes: compared with the CON group,the ALP gene expression in DEX group decreased,and the ALP gene expression level increased significantly after adding CSA(P < 0.05).Compared with CON group,the expression of OCN gene in DEX group decreased,and the expression level of OCN gene increased significantly after adding CSA(P < 0.05).Compared with the blank control group,the expression of OPN gene in DEX group decreased,and the expression level of OPN gene increased after adding CSA,but not significantly(P > 0.05).Compared with the blank control group,the expression of Runx2 gene in DEX group decreased,and the expression level of Runx2 gene increased significantly after adding CSA(P < 0.05).10.Apoptosis related genes of osteoblast: caspase-3 expression increased significantly after adding DEX(P < 0.05),and decreased significantly after adding 5 ?mol / L and 10 ?mol/ L CSA(P < 0.05).The expression of caspase-6 was significantly increased after adding DEX(P < 0.05),but there was no significant change after adding CSA(P > 0.05).The expression of caspase-7 in osteoblast increased significantly after adding DEX(P < 0.05),and decreased after adding CSA,but there was no significant difference(P > 0.05).The expression of caspase-9 in osteoblast increased significantly after adding DEX(P < 0.05),and decreased significantly after adding 5 ?mol / L and 10 ?mol / L CSA(P < 0.05).11.Cytotoxic experiment of osteoclast: We use 0-10 ?mol / L gradient concentration of CSA to carry out cytotoxic experiment on osteoclast.Compared with the control group,there was no significant change in absorbance,indicating that CSA had no significant toxicity on osteoclast(P > 0.05).12.Osteoclast differentiation experiment: when using RANKL to induce osteoclast differentiation,CSA with concentration of 1 ?mol / L,2.5 ?mol / L,5 ?mol / L and 10 ?mol/ L was added.It was found that the number of mature osteoclast in CSA group with concentration of 2.5 ?mol / L,5 ?mol / L and 10 ?mol / L decreased significantly(P <0.05).When using RANKL to induce osteoclast differentiation,10 ?mol / L CSA was added in different time periods.Compared with the control group,the number of osteoclast differentiation in 1-3 days group and 1-6 days group decreased significantly(P < 0.05).13.Osteoclast actin ring: CSA can inhibit the formation of osteoclast actin ring and the accumulation of osteoclast nucleus(P < 0.05).14.Bone resorption test of osteoclast: mature osteoclast were implanted into a hydroxyapatite coated culture dish,the number of cells and the absorption area of hydroxyapatite coating were measured,and it was found that CSA could inhibit the bone resorption area of a single osteoclast(P < 0.05).15.The expression of NFATc1,c-fos,ctsk and acp5 in osteoclast increased significantly after adding DEX,and the expression of ctsk and acp5 decreased significantly after adding5 ?mol / L and 10 ?mol / L CSA(P < 0.05).After adding 10 ?mol / L CSA,the expression of NFATc1 and c-fos decreased significantly(P < 0.05).16.Osteoclast differentiation related protein: NFATc1 protein had no significant difference between the two groups on day 0 and day 1 of culture,but significantly increased on day 3and day 5 of culture.Compared with the control group,CSA group had a lower increase,with a significant difference.The expression of c-fos protein in CSA group was slightly higher at 0 d,but there was no significant difference.There was no significant difference between the two groups at 1 d.the expression of c-fos protein in CSA group was significantly higher at 3 d and 5 d.compared with the control group,the increase of CSA group was lower,and the difference was significant.There was no significant difference in v-atpase-d2 protein between the two groups on day 0 and day 1 of culture,but it was significantly higher in the two groups on day 3 and day 5 of culture.Compared with the control group,the increase of v-atpase-d2 protein in the CSA group was lower on day 5,and the difference was significant.There was no significant difference in ctsk protein between the two groups on day 0 and day 1 of culture,but it increased significantly on day3 and day 5 of culture.Compared with the control group,the increase of ctsk protein in CSA group was lower and the difference was significant.There was no significant difference in intergrin ? 3 protein between the two groups on day 0 and day 1 of culture,but it was significantly higher in the two groups on day 3 and day 5 of culture.Compared with the control group,the increase of intergrin ? 3 protein in the CSA group was lower and the difference was significant.ConclusionAfter glucocorticoid intervention in rats,there were serious bone loss,bone micro structure damage,bone strength reduction and pathological structure damage,serum ALP activity decreased significantly,which was consistent with previous research results,indicating that GIOP animal model was successfully constructed.Cs A treatment significantly improved BMD,BMC,BS / TV,TB.N Tb.Th,v BMD,reduce SMI Tb.Sp It can enhance the bone strength,improve the condition of bone trabecula(quantity,structure,osteoblast,OC,stem cells in bone microenvironment),and reverse the change of ALP activity caused by DEX.Under the intervention of dexamethasone,the activity of osteoblast decreased and the apoptosis rate of osteoblast increased significantly,accompanied by the decrease of mineralization ability of osteoblast,the expression of ALP,OCN,OPN and Runx2 genes related to osteoblast differentiation decreased significantly,and the expression of caspase family genes increased,indicating that dexamethasone inhibited the cell viability,differentiation process of osteoblast and mineralization and apoptosis of osteoblast may be one of the mechanisms of osteoporosis.After the treatment with CSA,the activity and mineralization of osteoblast increased,the expression of ALP,OCN,OPN and Runx2 genes related to osteoblast differentiation also increased,and the apoptosis decreased,which represented the promotion of osteoblast function and differentiation.It is suggested that the mechanism of the prevention and treatment of glucocorticoid induced osteoporosis may come from the protection of osteoblast from the inhibition of hormones,the maintenance of the original cell viability,differentiation and mineralization ability,and the avoidance of cell apoptosis.There was no direct cytotoxic effect on osteoblast,and the number of osteoblast was not significantly affected.However,in the experiment of osteoblast bone absorption,the ability of osteoblast to absorb bone decreased under the intervention of CSA.It can also inhibit the formation of actin ring in osteoblast,and then affect the maintenance of osteoblast normal morphology.Coumarin can also inhibit the expression of osteoblast differentiation related genes and proteins,and hinder the normal differentiation process of osteoblast. |
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