Modulation Of Dehydroepiandrosterone And Bushen Ningxin Decoction On Osteoblasts-Immune Network

Postmenopausal osteoporosis (PMO) is a metabolic bone disease characterized by a decrease of bone mass after menopause. Normal bone remodeling depends upon a balance between the osteoblast (OB)-mediated bone formation and osteoclast-mediated bone resorption. After surgical and natural menopause, the bone resorption exceeds the bone formation, which leads to a decrease in bone mass. Skeletal system homeostasis is greatly influenced by endocrine and immune factors. Recent investigations describe an association between these pathogenetic changes and disturbances of the endocrine system, especially sex steroid hormones, and immune system. Up to now, approaches to managing PMO have been directed towards prevention of bone resorption. Estrogen replacement therapy, which appears to act primarily through inhibition of resorption, has been widely used in postmenopausal women for several years, although recent observational studies have called into question the wisdom of the long-term estrogen therapy owing to safety concerns. The OB is bone-forming cells that regulate bone metabolism, is regulated by the local microenvironment, and play a central role in the pathogenesis of PMO. A number of investigations have demonstrated that regulation of the OB metabolism is critical for the maintenance of the skeleton, so the promising approach is to administer compounds capable of stimulating the anabolic activity of OB in PMO.Dehydroepiandrosterone, DHEA, is among the most plentiful circulating adrenal hormones, and is one of the most abundant adrenal sex steroid hormone precursors in adult women. It exerts biological actions that cannot be attributed to its metabolites. These findings coupled to the fact that an increased evidence indicates that DHEA as additional factors modulating bone mineral density (BMD)in Postmenopausal women emphasize the need for more information concerning potential clinical benefit of DHEA for treatment of PMO. However, both the molecule and biochemistry mechanisms of whereby DHEA exerts its effect on bone metabolism remain shrouded in mystery. While PMO has been extensively studied in recent years, the utilization of Traditional Chinese Herbal Medicine for the prevention and treatment has seldom been examined. We had showed previously that BSNXD could prevent and treat PMO efficiently, but the precise mechanism, however, remains to be elucidated.The present study is to elucidate the mechanisms underlying DHEA and BSNXD -mediated OB lifespan and immunity in vitro and in vivo.Part I Modulation of DHEA on Osteoblast-Immune NetworkObjective To investigate modulation of DHEA on morphometry of bone tissue, proliferation and apoptosis of OB, expression of Th1 /Th2 cytokines and costimulatory molecule of CD4⁺T lymphocytes, and to study the effect of blockade of ER, AR and MAPK signaling pathway on the OB and CD4⁺T in response to DHEA.Methods BALB/c mice were ovariectomized for foundation of osteoporosis, and then were afforded with DHEA (OVX+DHEA group), 17β-estradiol (0VX+E2 group) and Saline (OVX group), respectively. The femur, vertebra and spleen of each group were collected in 12 weeks of the administration. Femur and vertebra were measured for morphometry of bone tissue. The OB was isolated from vertebra and cultured. The proliferative cell nuclear antigen (PCNA) of OB was analyzed by flow cytometry (FCM). The expression of Th1-type (IL-2 and IFN-γ) and Th2-type cytokines (IL-4 and IL-10) in splenic CD4⁺ T cells were analyzed respectively by FCM. The OB was isolated by the enzyme-digested assay from calvaria of neonatal BALB/c mice, and then the effect of DHEA on OB ultrastructure was evaluated by TEM. After the primary OB was pretreated withICI 182,780, an estrogen receptor antagonist, Flutamide, an androgen receptor antagonist, or U0126, a specific inhibitor of MAPK pathway, they were treated with a series of concentrations of DHEA or E2. The DNA content of OB was analyzed by FCM; the early apoptotic cells were analyzed by FCM with the Annexin-V-FITC/PI dual labeling technique; the phosphorylation of ERK1/2 in OB was analyzed by Western blot. The OB was treated with series concentration E2 or DHEA in vitro, real time RT-PCR analysis and western blotting analysis for aromatase, ERct, ERp and AR mRNA and protein expression in OB. The OB was isolated from calvaria of neonatal mice, and cultured by the enzyme-digested method. The CD4⁺T cells are isolated from spleen with MACS separation. The OB or CD4⁺T cell was pretreated with dissolvent, ICI 182,780, Flutamide or CsA, they were treated with DHEA or E2 in vitro, respectively, and then stimulated by LPS. The expression of costimulatory molecules on the OB and CD4⁺T were analyzed by FCM.Results The area of bone trabecula in OVX group decreased significantly as compared to that of the Sham group (P<0.01); the area of bone trabecula in OVX+DHEA group and OVX+E2 group is higher significantly than that of OVX group (P＜0.01). Moreover, the OB treated by the DHEA showed more organelles, expanded endoplasmic reticulum(ER) and abundant mitochondria by TEM. The expression of PCNA in the OB of OVX group increased significantly compared to the Sham group (P＜0.05). Compared to the OVX group, the average fluorescence intensity and positive cell percentage of PCNA in the OB of OVX+DHEA group increased significantly (P<0.05 or P<0.01). DNA content analysis showed DHEA increased the S-phase (P＜0.01) and G₂/M phase (P＜0.01), decreased the G₀/G₁ phase (P＜0.01) of OB cell cycle, improved the proliferation index (PI) significantly (P＜0.01), while E2 appeared no effect on PI of OB compared to control; DHEA and E2 were all capable of inhibiting the serum deprivation-induced early apoptosis of OB (P＜0.05, P＜0.01, respectively), and induced ERKs phosphorylation in OB (P＜0.01). Those effects of DHEA were neither blocked by ICI 182,780 nor by Flutamide, but could be blocked by thespecific inhibitor of MAPK pathway, U0126. However, the effects of E2 on OB apoptosis and ERKs phosphorylation were not blocked by Flutamide, but blocked by ICI 182,780 and U0126. The aromatase, ERα, ERβ and AR mRNA and protein were steadily expressed in murine OB. Compared to control, DHEA and E2 increased significantly the expression of aromatase mRNA, ERα and ERβ mRNA and protein respectively (P＜0.01, P＜0.05), and DHEA increased significantly, but E2 apparently decreased the ratio of ERβ/ERα mRNA (P＜0.05); moreover, DHEA but not E2 could increase the expression of aromatase protein, AR mRNA and protein significantly (P＜0.01). The average fluorescence intensity and positive cell percentage of the costimulatory molecule CD80, CD86 on the OB (LPS stimulated) increased significantly after treated with DHEA or E2 (P＜0.05 or P＜0.01); moreover, the expression of CD80, CD86 on the LPS stimulated OB in the DHEA treating group and the control was suppressed significantly by CsA (PO.01) but not by ICI 182,780 or Flutamide in vitro; the effect of E2, however, was suppressed significantly by CsA or ICI 182,780 but not by Flutamide. The Th1 cytokine expression in OVX+DHEA group and OVX+E2 group increased significantly as compared to that of the OVX group (P＜0.01, P<0.05). The IL-4 and IL-10 expression in splenic CD4⁺ T cells of OVX+DHEA group reduced significantly as compared to that of the OVX group (P<0.01). The ratio of IFN-γ/IL-4 was significantly decreased in the OVX group compared to the Sham group (P<0.05). Compared to the OVX group, the ratio of IFN-γ/IL-4 was significantly increased in OVX+DHEA group and OVX+E2 group (P<0.01). In OVX+DHEA group, the expression of PCNA in the OB was positively related to that of IFN-γ in the CD4+T cells (r=0.931, P＜0.05), and negatively related to that of IL-4 (r= -0.815, P＜0.05). The average fluorescence intensity of the CD28, CTLA-4 and positive cell percentage of CTLA-4 on the LPS-stimulated CD4⁺T cells in the DHEA treating group did not show a significant change, but DHEA increased the expansion of the CD4⁺CD28⁺ T cells (LPS-stimulated) significantly (P＜0.05).Conclusions DHEA administration could increase the proliferation of OB in vivo, improve the bone tissue morphometry of the PMO model. DHEA promotesproliferation and inhibits apoptosis of OB by a mechanism independent of either AR or ER, suggesting it exerts effects presumably via a DHEA-specific receptor directly, not by way of conversion to androgens or estrogens, which involves phospho-pERK1/2. DHEA could increase the expression of aromatase significantly, it can contribute to increase estrogen concentration in bone tissue; DHEA could increase the expression of ERα, ERβ and AR, to cause augmenting on the OB in response to the ligand such as estrogen. DHEA up-regulates the expression of CD80 and CD86 on the LPS-stimulated OB in vitro through a mechanism independent of either AR or ER, which can be blocked by CsA. Moreover, DHEA can up-regulate expression of Th1-type cytokines, promote a shift in Thl/Th2 ratio balance toward the Th1-bias, thus improve the immune state of the PMO model. DHEA can also increase expansion of the CD4⁺CD28⁺ cells, which appears to improve the osteo-immune network.Part II Modulation of BSNXD on Osteoblast-Immune NetworkObjective To investigate modulation of BSNXD on morphometry of bone tissue, proliferation and apoptosis of OB, expression of Th1 /Th2 cytokines and costimulatory molecules of CD4⁺T lymphocytes, and to study effect of blockade of ER, AR and MAPK signaling pathway on the OB and CD4⁺T in response to pharmacological serum of BSNXD.Methods BALB/c mice were ovariectomized for foundation of osteoporosis, and then were afforded with BSNXD (OVX+ BSNXD group), DHEA (OVX+ DHEA group), 17p-estradiol (0VX+E2 group) and Saline (OVX group), respectively. The femur, vertebra and spleen of each group were collected in 12 weeks of the administration. Femur and vertebra were measured for morphometry of bone tissue. The OB was isolated from vertebra and cultured. The PCNA of OB was analyzed by FCM. The expression of Th1-type and Th2-type cytokines in splenic CD4⁺ T cell were analyzed respectively by FCM. The OB was isolated bythe enzyme-digested assay from calvaria of neonatal BALB/c mice and then the effect of pharmacological serum of BSNXD on OB ultrastructure was evaluated by TEM. After the primary OB was pretreated with ICI 182,780, Flutamide or U0126, they were treated with a series of concentrations of pharmacological serum of BSNXD or control serum. The DNA content of OB was analyzed by FCM; the early apoptotic cells were analyzed by FCM with the Annexin-V-FITC/PI dual labeling technique. The OB was treated with series concentration of pharmacological serum of BSNXD or control serum in vitro. Real time RT-PCR was used to analyze for aromatase, ERα, ERβ and AR mRNA expression in OB. The OB was isolated from calvaria of neonatal mice; the CD4⁺T cells are isolated from spleen with MACS separation. The OB or CD4⁺T cell was pretreated with dissolvent, ICI 182,780, Flutamide or CsA, they were treated with series concentrations of pharmacological serum of BSNXD or control serum in vitro, respectively, and then stimulated by LPS. The expression of costimulatory molecules on the OB and CD4⁺T were analyzed by FCM.Results The area of bone trabecula in OVX+ BSNXD group is higher than that of OVX group (P＜0.05). Moreover, the OB treated by the BSNXD pharmacological serum in 20% concentration showed more organelles, expanded endoplasmic reticulum, and abundant mitochondria by TEM. Compared to the OVX group, the expression of PCNA in OB of OVX+ BSNXD group increased significantly (P＜0.05). DNA content analysis showed that the pharmacological serum at the concentration of 10-20% increased the S-phase and G₂/M phase, decreased the G₀/G₁ phase of OB cell cycle, improved the PI significantly (P＜0.01, P＜0.05). The BSNXD pharmacological serum in 10-20% concentration was capable of inhibiting the serum deprivation-induced early apoptosis of OB (P＜0.05, P＜0.01, respectively). Those effects of BSNXD pharmacological serum could be blocked partly by ICI 182,780, Flutamide or U0126. Compared to control, the BSNXD pharmacological serum increased the expression of aromatase, ERα, ERβ and AR mRNA in OB in 10-20% concentration (P＜0.01, P＜0.05), and increased the ratio of ERβ/ERα mRNA apparently in 20% concentration (P＜0.01). Theexpression of CD80, CD86 on the OB (LPS-stimulated) increased significantly after treated with the BSNXD pharmacological serum (P＜0.01); moreover, the expression of the CD80, CD86 on the LPS-stimulated OB in the BSNXD pharmacological serum and the control was suppressed significantly by CsA (P＜0.01), but not by ICI 182,780 or Flutamide in vitro. The Th1 cytokine expression in OVX+ BSNXD group increased significantly as compared to that of the OVX group (P＜0.05). The IL-4 and IL-10 expression in splenic CD4⁺ T cells of OVX+ BSNXD group reduced significantly as compared to that of the OVX group (P＜0.05). Compared to the OVX group, the ratio of IFN-γ/IL-4 was significantly increased in OVX+ BSNXD group (P＜0.01). In the BSNXD group, the expression of PCNA in the OB was positively related to that of IFN-γ in the CD4+T cells (r=0.943, P＜0.05), and negatively related to that of IL-4 (r= -0.934, P＜0.05). The average fluorescence intensity and positive cell percentage of the CD28 and CTLA-4 on CD4⁺T cells (LPS-stimulated or not) in the BSNXD pharmacological serum treating group did not show a significant change compared to control.Conclusions BSNXD treatment could increase the proliferation of OB in vivo, and improve the bone tissue morphometry of the PMO model. The BSNXD pharmacological serum can promote proliferation and inhibit apoptosis of OB in vitro through multiple pathways such as ER, AR, etc. The effects of the BSNXD pharmacological serum involves multiple signal pathways includind MAPK signal pathway, phospho-pERK1/2. The BSNXD pharmacological serum could increase the transcription of aromatase significantly, and in turn contribute to increase estrogen concentration in bone tissue; the BSNXD pharmacological serum could increase the transcription of ERα, ERβ and AR, to cause augmenting on the OB in response to the ligand such as estrogen. The BSNXD pharmacological serum up-regulates the expression of CD80 and CD86 on the LPS-stimulated OB in vitro through a mechanism independent of either AR or ER, which can be blocked by CsA. Moreover, the BSNXD pharmacological serum can up-regulate expression of Th1-type cytokines, promote a shift in Th1/Th2 ratio balance toward the Th1-bias,and thus improves the immune state of the PMO model. Therefore, BSNXD combined DHEA will be a promising approach to managing PMO.