| BackgroundWith the GCs (glucocorticoids) widely used in clinical applications, its side-effects have become increasingly prominent, including GIOP (glucocorticoid-induced osteoporosis) which has been accounted for the first one of the second osteoporosis. SLE (Systemic lupus erythematosus) is one of the high incidence disease of Rheumatology department, and about 86 percent of SLE patients are less than 40 years. Most of these patients must take GCs for a long-term or even a lifetime. Because of these reasons,we do some research in the field of GIOP. The mechanisms of GIOP is not clear yet,and now researchers focus on the inhibition of osteogenesis differentiation caused by GCs. Osteoblast,osteocyte and even the bone tissue are differentiated from MSC (human bone marrow mesenchymal stem cells).Since BMSCs is one type of MSC which can be induced to osteoblasts under certain condition, these cells have become the effective tool cells in the field of osteogenesis research. The Hedgehog signaling pathway is the inherent genes which can promote the mammalian’s growth and development. Resently, its role in bone growth and development was under concern. Studies have shown that the Hedgehog pathway can promote the osteogenic differentiation of stem cells and maintain bone strength. But whether it can promote the osteogenic differentiation of hBMSC (human Bone Mesenchymal Stem Cell) remains controversial. Bisphosphonates are the FDA approved first-line drug treatment of GIOP. The mechanism of bisphosphonate is the inhibition effect on osteoclasts and the effects on osteogenic pathway is not clear. Bisphosphonates was not explicitly recommended in the group of pre-menopausal women and age<50 years old male with no history of fragility fractures in the GIOP guideline in 2010 ACR, for there’s no adequate data. Now, we focus on the controversial questions to do more research in the field of the clinical and basic mechanism of GIOP.Objective1) To find the effect of Hedgehog signaling pathway on the Hbmsc osteogenesis differention.2) To explore the the GIOP mechanism:may Hedgehog pathway partly blocked by glucocorticoids lead to the reduction of osteoblast osteogenic differentiation of hBMSC?3) To observe the effects of alendronate on hBMSC osteogenic differentiation through Hedgehog signaling pathway.4) To observe the effect of alendronate on the treatment of GIOP in the group of pre-menopausal women with no history of fragility fractures.Methods1. The effect of Hedgehog pathway on hBMSC osteogenesis differentiation1.1 hBMSC culture, identification:the primary hBMSC was cultured directly adherent to the flask. Identification: ① cell morphology, ②The multi-differentiation character: using osteogenic medium when hBMSCs cultured and after 3 weeks, the calcium nodules can be detected by alizarin red staining. ③urface markers (CD29, CD34, CD44, CD45)detected by flow cytometry expression.1.2 the effects of Hedgehog signaling pathway on hBMSC osteogenesis:hBMSCs were cultured after successfully identificated. After adding rh-SHHN cytokines, quantitative PCR was used to detect the expressions of Glil-mRNA. The final calcium nodules was detected by Alizarin red staining after 21 days treatment.2 The effects of high and moderate concentrations GCs on hBMSC osteogenesis differentiation through Hedgehog signaling pathway and downstream osteogenesis pathway.2.1 Cell experiments:hBMSCs were cultured after successfully identificated. After adding rh-SHHN cytokines and high and moderate concentrations GCs alone or in combination, quantitative PCR was used to detect the expressions of Glil, BMP2, RunX2, β-catenine-mRNA for 7 days,14days,21 days, seperately. Immunofluorescence was used to detect the nucleus and cytoplasm expressions of Glil. The final calcium nodules was detected by Alizarin red staining.2.2 Immunohistochemistry:the expression of Glil in bone tissue of long-term glucocorticoid treated (=6 months) SLE patients compared with those of newly diagnosed SLE patients was detected in the way of immunohistochemistry. Each group contain 20 cases.3. The effects of bisphosphonate on Hedgehog signaling pathway:3.1 The effect of bisphosphonate on hBMSC osteogenesis differentiation: hBMSCs were cultured in osteogenic medium alone or in combination with different concentrations of bisphosphonate. The mRNA expressions of Glil was detected by quantitative PCR and the final calcium nodules was detected by Alizarin red staining.3.2 The effects of bisphosphonate on relieve the inhibition of GCs on hBMSCs osteogenesis differentiation. hBMSC were cultured in osteogenic medium in combination with high concentration of GCs. Then adding low concentration of bisphosphonate. The mRNA expressions of Glil was detected by quantitative PCR and the final calcium nodules was detected by Alizarin red staining.3.3 Immunohistochemistry:the expression of Glil in bone tissue of long-term bisphosphonate treated (6 months-12months) SLE patients compared with those SLE patients without receiving bisphosphonate treatment was detected in the way of immunohistochemistry. Each group contains 10 cases.4 The clinical research of bisphosphonate:4.1 The effect of alendronate on the bone metabolism markers and BMD in the premenopausal SLE women taking long-term GCs.47 pre-menopausal female SLE patients taking long-term use of glucocorticoids with no history of fragility fractures, were divided into three groups:low doses of GCs therapy+calcium group; low doses of GCs treatment+calcium+calcitriol group; small doses of GCs treatment+ calcium+alendronate group. The concentration of serum calcium and serum phosphorus, ALP (alkaline phosphatase), PTH(parathyroid hormone), N-MID (N-terminal osteocalcin), β-CT (beta collagen level of decomposition fragments) and BMD (bone mineral density) of lumbar spine, total hip and femoral neck were measured on the baseline, the third month and the 6th month to explore the effects of alendronate.4.2 The effect of alendronate on the bone metabolism markers and BMD in the premenopausal newly diagnosis SLE women taking large doses of GCs.45 newly diagnosed SLE patients, taking large doses of GCs treatment at the beginning of diagnosis, were divided into three groups:high doses of GCs therapy+calcium group; high doses of GCs treatment+calcium+calcitriol group; high doses of GCs treatment+calcium+alendronate group. The concentration of serum calcium and serum phosphorus, ALP, PTH, N-MID, β-CT and BMD of lumbar spine, total hip and femoral neck were measured on the baseline, the third month and the 6th month to explore the effects of alendronate.5. Statistical analysis:All data is shown as s±S.SPSS17.0 statistical software was used for analysis. Clinical data using a repeated measures variance, while basic experiments using one-way onova, factorial analysis and t test, P<0.05 was considered significant.Results1 The effect of rh-SHHN on the differentiation of hBMSC1.1 The expression of Glil-mRNA:After dealing with rh-SHHN cytokines for 7 days the expression of hBMSC Glil-mRNA was in the highest expression (P= 0.006) then decreased gradually.1.2 Alizarin red staining------the calcium nodules can be prominently observed: after the treatment for 21 days, there’s a large number of calcium nodules in the rh-SHHN use only group.2 The effects of high and moderate GCs on hBMSC osteogenesis differentiation through Hedgehog pathway.2.1 hBMSCs were cultured in non-osteogenesic medium, the expression of Glil-mRNA increased:when co-cultured with different concentrations of MP (10-5M/L,10-3M/L), compared with the group which rh-SHHN use only for 7 days, the expression of hBMSC Glil-mRNA and the downstream osteogenic gene------RunX2-mRNA significantly decreased (P<0.001, P=0.035) separately and the Glil of the nucleus staining intensity weakened in the way of immunofluorescence. The expressions of hBMSC osteogenic gene------RunX2, BMP2,β-catenine-mRNA have no statistically meaning among groups for 14 and 21 days.2.2 Alizarin red staining:after the treatment for 21 days, there’s a large number of calcium nodules in the rh-SHHN use only group than in the high concentration of MP group.2.4 The expression of Glil declined after using long-term of GCs in the way of immunohistochemistry:the Glil average integral optical density of bone significantly decreased (P<0.001) in the long-term glucocorticoid treated SLE patients compared with newly diagnosed SLE patients,3. The effects of bisphosphonate on Hedgehog signaling pathway:3.1 The effect of bisphosphonate on hBMSC Glil-mRNA:hBMSCs were cultured in osteogenesic medium, when adding low concentration of bisphosphonate, the expressions Glil-mRNA increased significantly (P=0.007), while high concentration of bisphosphonate significantly decreases the Glil-mRNA> expressions (P=0.041); compared with hBMSC cultured with osteogenesis medium and high concentration of GCs, when adding low concentration of bisphosphonate, the expressions of Glil-mRNA increased significantly (P=0.037).3.2 hBMSCs were cultured in osteogenesic medium, after the intervention of 21 days, when taking alizarin red staining, low concentration of bisphosphonate group can be observed a large number of calcium nodules compared with control group; the group adding low concentration of bisphosphonate plus high concentration of GCs can be observed a large number of calcium nodules compared with those containing high cocentration of GCs.3.3 The effects of bisphosphonate on relieve the inhibition of GCs on bone Glil immunohistochemistry:the Glil average integral optical density of bone significantly decreased (P=0.002) in the long-term alendronate treated SLE patients compared with those untreated with alendronate.4 The clinical research of bisphosphonate4.1 Alendronate treatment of patients with small doses of GCs4.1.1 Biochemical indicators:Among the three groups, there’s no significant difference of serum concentrations of Ca, PO4, ALP, PTH before and after treatment.4.1.2 Bone turnover markers:①serum N-MID:the serum N-MID level in the calcium group significantly increased after treatment on the third month (P<0.01); the serum N-MID level significantly increased on the sixth month (P<0.01) in patients given calcium and calcitriol treatment; the serum N-MID level significantly decreased after treatment (P<0.01) in the calcium plus alendronat group on the third month (P<0.01) and continued to decline up to 6 months (P<0.01).②serum β-CT:the serum β-CT level increased after treatment in the calcium and calcium plus calcitriol group but the difference was not significant; the serum β-CT level significantly decreased after treatment (P<0.01) in the calcium plus alendronat group on the third month (P<0.01) and continued to decline up to 6 months (P<0.01).4.1.3 BMD:① Lumbar spine BMD:the lumbar spine BMD in calcium group significantly decreased after treatment on the third month (P<0.05) and continued to decline up to 6 months (P<0.01); the lumbar spine BMD significantly increased (P<0.01) after 3 months of treatment in calcium plus alendronat group and continued to rise to 6 months (P<0.01); there was no significant difference of lumbar spine BMD before and after treatment in calcium plus calcitriol group.②Left femur BMD:the left femur BMD in calcium group significantly decreased after treatment on the third month (P<0.01) and continued to decline up to 6 months (P<0.01); the left femur BMD significantly increased after 6 months of treatment in calcium plus alendronat group (P<0.01); there was no significant difference of the left femur BMD before and after treatment in calcium plus calcitriol group.ゝemoral neck BMD:the femoral neck BMD in calcium group significantly decreased after treatment on the third month (P<0.01) and continued to decline up to 6 months (P<0.01); the left femur BMD significantly increased after 6 months of treatment in calcium plus alendronat group (P<0.05); there was no significant difference of the left femur BMD before and after treatment in calcium plus calcitriol group.4.2 Alendronate treatment of SLE patients with large doses of GCs4.2.1 Biochemical indicators:There’s no significant difference of serum concentrations of Ca, PO4, ALP before and after treatment. The serum level of PTH significantly increased after the treatment of 3 months (P<0.01) and continues to increase until the 6th month (P<0.01) in calcium group.;the serum level of PTH significantly increased after the treatment of 6 months in the calcium plus alendronate group (P<0.01); The serum level of PTH significantly decreased after the treatment of 3 months (P<0.05) and continues to decrease until the 6th month(P <0.01) in calcium plus alendronate group.4.2.2 Bone turnover markers:①The serum N-MID level was significantly increased on the sixth month (P<0.05, P<0.01) in calcium group and calcium plus calcitriol group separately; the serum N-MID level significantly decreased in the calcium plus alendronat group on the sixth month (P<0.01)②the serum β-CT:the serum β-CT level significantly increased after treatment in the calcium and calcium plus calcitriol group on the third month(P<0.05, P<0.05) and on the sixth month (P<0.05) separately; the serum P-CT level significantly decreased after treatment (P<0.01) in the calcium plus alendronat group on the third month (P<0.01) and continued to decline up to 6 months (P<0.01).4.2.3 BMD:① Lumbar spine BMD:the lumbar spine BMD in calcium group significantly decreased after treatment on the third month (P<0.01) and continued to decline up to 6 months (P<0.01); the lumbar spine BMD in calcium plus calcitriol group significantly decreased after treatment of six months(P0.01) the lumbar spine BMD significantly increased (P<0.01) after 6 months treatment in calcium plus alendronat group.②Left femur BMD:the left femur BMD in calcium group and calcium plus calcitriol group significantly decreased after treatment on the third month (P <0.01,P<0.05) and continued to decline up to 6 months (P<0.01,P<0.01) separately; there was no significant difference of the left femur BMD before and after treatment in calcium plus alendronat group.③Femoral neck BMD:the femoral neck BMD in calcium group and calcium plus calcitriol group significantly decreased after treatment on the third month (P <0.01,P<0.05) and continued to decline up to 6 months (P<0.01,P<0.01) separately; there was no significant difference of the femoral neck BMD before and after treatment in calcium plus alendronat group. Conclusionl.rh-SHHN can improve the exprssion of Glil----the target gene of Hedgehog pathway and finally lead to osteogenesis differentiation.2.High and moderate doses of glucocorticoids inhibit hBMSC osteogenesis by partly inhibiting Glil------the target gene of Hedgehog Pathway and the dowmstream osteogenic gene------RunX2, which can be detected in vivo and vitro experiment.3. Low concentration of alendronate can not only stimulate hBMSC osteogenesis differentiation but also can remit the inhibition effects of GCs through the way of Hedgehog.4. We can observed after using alendronate, the BMD of SLE patients treated with small doses of GCs therapy and high-dose GCs therapy can be increased or maintained at the same level before the use of GCs. Finally, the incidence of femural head necrosis can be reduced in the alendronate group. |
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