Ligustilide, a major bioactive component of Angelica sinensis, promotes bone formation via the GPR30/EGFR pathway.

Angelica sinensis (Oliv.) Diels is a widely-used traditional Chinese herbal medicine in treating osteoporosis. Ligustilide (LIG) is the main component of A. sinensis and is considered to be the most effective biologically active ingredient in this plant. LIG has been found to have multiple pharmacological activities, such as anti-atherosclerosis, neuroprotection, anticancer, anti-inflammatory and analgesic. However, little is known regarding its anti-osteoporotic effects. The aims of this study were to investigate any protective effect of LIG on bone formation. The results showed that LIG significantly ameliorated inhibition of bone formation in zebrafish caused by prednisolone. LIG promoted osteoblast differentiation, including that of the pre-osteoblastic cell line MC3T3-E1 and bone marrow mesenchymal stem cells. LIG greatly improved the viability of MC3T3-E1 cells exposed to H2O2, attenuated H2O2-induced apoptosis and increased the expression of Bcl-2. Furthermore, LIG treatment lead to marked activation of phosphorylated EGFR and ERK1/2. These effects could be obviously inhibited by blocking GPR30 signaling with the specific inhibitor G15. Collectively, the results reveal that GPR30 is a positive switch for LIG to increase bone formation via regulation of EGFR, and these results provide evidence for the potential of LIG to treat osteoporosis.

Vitamin D3 protects against prednisolone-induced liver injury associated with the impairment of the hepatic NF-κB/iNOS/NO pathway.

The study was carried out to define whether prednisolone-induced damage to hepatic cells is accompanied by excessive nitric oxide (NO) levels associated with nuclear factor kappa B (NF-κB)/inducible NO synthase (iNOS) activation and evaluate the efficacy of the treatment with vitamin D3. Histopathological examination, activities of liver transaminases (alanine aminotransferase and aspartate aminotransferase), and cell death assays consistently showed that prednisolone (5 mg/kg body weight, 30 days) induces chronic liver injury in female Wistar rats. Specifically, increased hepatocellular necrosis and caspase-3-dependent apoptosis were observed. Prednisolone enhanced iNOS protein expression, NO generation, and tyrosine nitration in liver cells. Despite unchanged hepatic level of the NF-κB/p65 protein, prednisolone increased inhibitory κB-α (IκB-α) degradation, nuclear translocation, and phosphorylation of NF-κB/p65 at Ser311, indicating that NF-κB activation can be involved in the induction of iNOS/NO. All changes were associated with a 2.9-fold decrease in the serum content of 25-hydroxyvitamin D3 and significant reduction of hepatic vitamin D3 receptor (VDR) expression that points reliably to vitamin D3 deficiency and failures in VDR signaling. Vitamin D3 co-administration (100 IU/rat, 30 days) prevented glucocorticoid-evoked abnormalities in hepatic tissue. In conclusion, prednisolone-induced liver disturbances were associated with the impairment of NF-κB/iNOS/NO responses that can be ameliorated by vitamin D3 treatment through VDR-mediated mechanisms.

Prednisolone and vitamin D(3) modulate oxidative metabolism and cell death pathways in blood and bone marrow mononuclear cells.

The study was designed to evaluate reactive oxygen species (ROS)/nitric oxide (NO) formation and apoptotic/necrotic cell death elicited by prednisolone in peripheral blood and bone marrow mononuclear cells and to define the efficacy of vitamin D3 to counter glucocorticoid (GC)-induced changes. It was shown that prednisolone (5 mg per kg of female Wistar rat's body weight for 30 days) evoked ROS and NO overproduction by blood mononuclear cells (monocytes and lymphocytes) that correlated with increased cell apoptosis and necrosis. In contrast, prednisolone did not affect ROS/NO levels in bone marrow mononuclear cells that corresponded to lower level of cell death than in the control. Alterations of prooxidant processes revealed in mononuclear cells and associated with GC action were accompanied by vitamin D3 deficiency in animals, which was assessed by the decreased level of blood serum 25-hydroxivitamin D3 (25OHD3). Vitamin D3 administration (100 IU per rat daily for 30 days, concurrently with prednisolone administration) completely restored 25OHD3 content to the control values and significantly reversed ROS and NO formation in blood mononuclear cells, thus leading to decreased apoptosis. In bone marrow, vitamin D3 activated ROS/NO production and protein nitration that may play a role in prevention of prednisolone-elicited increase in bone resorption. We conclude that vitamin D3 shows a profound protection against GC-associated cellular damage through regulating intracellular ROS/NO formation and cell death pathways.

[The ROS-generating and antioxidant systems in the liver of rats treated with prednisolone and vitamin D3].

The mechanisms of glucocorticoid-induced disturbances of liver function is currently not fully clarified. Vitamin D3 was previously shown to play an important role in the regulation of impaired oxidative metabolism and detoxification function of the liver associated with the effects of hepatotoxic compounds. The study was undertaken to define the intensity of oxidative metabolism in the rat liver and survival of hepatocytes after prolonged prednisolone administration and to assess whether vitamin D3 is capable to counter glucocorticoid-induced changes. It has been shown that prednisolone (0.5 mg per animal for 30 days) leads to 1.6-fold increase in the percentage of necrotic cells among isolated hepatocytes as compared with the control. The glucocorticoid-induced impairment of hepatocellular function was accompanied by enhanced generation of reactive oxygen species (ROS), accumulation of TBA-active products and carbonylated proteins but reduced levels of free SH-groups of low molecular weight compounds. It was demonstrated a decrease in the activities of key enzymes of antioxidant system (SOD, catalase, glutathione peroxidase), whereas the activities of pro-oxidant enzymes NAD(P)H-quinone oxidoreductase and semicarbazide-sensitive amine oxidase were shown to be increased. Vitamin D3 (and to greater extent in combination with α-tocopherol) administration (100 IU) on the background of glucocorticoid therapy caused normalizing effects on the level of ROS formation, oxidative modification of biomolecules and activity of antioxidant enzymes resulting in better survival of hepatocytes. These data suggest a potential role of vitamin D3 in the regulation of oxidative metabolism alterations related to hepatotoxic action of glucocorticoids.

Glucocorticosteroids increase cell entry by hepatitis C virus.

BACKGROUND & AIMS: Corticosteroids are used as immunosuppressants in patients with autoimmune disorders and transplant recipients. However, these drugs worsen hepatitis C virus (HCV) recurrence after liver transplantation, suggesting that they may directly exacerbate HCV infection. METHODS: The influence of immunosuppressive drugs on HCV replication, assembly, and entry was assessed in Huh-7.5 cells and primary human hepatocytes using cell culture- and patient-derived HCV. Replication was quantified by immunofluorescence, luciferase assays, quantitative reverse-transcriptase polymerase chain reaction, or core enzyme-linked immunosorbent assays. Expression of HCV entry factors was evaluated by cell sorting and immunoblot analyses. RESULTS: Glucocorticosteroids slightly reduced HCV RNA replication but increased efficiency of HCV entry by up to 10-fold. This was independent of HCV genotype but specific to HCV because vesicular stomatitis virus glycoprotein-dependent infection was not affected by these drugs. The increase in HCV entry was accompanied by up-regulation of messenger RNA and protein levels of occludin and the scavenger receptor class B type I-2 host cell proteins required for HCV infection; increase of entry by glucocorticosteroids was ablated by RU-486, an inhibitor of glucocorticosteroid signaling. Glucocorticosteroids increased propagation of cell culture-derived HCV approximately 5- to 10-fold in partially differentiated human hepatoma cells and increased infection of primary human hepatocytes by cell culture- and patient-derived HCV. CONCLUSIONS: Glucocorticosteroides specifically increase HCV entry by up-regulating the cell entry factors occludin and scavenger receptor class B type I. Our data suggest that the potential effects of high-dose glucocorticosteroids on HCV infection in vivo may be due to increased HCV dissemination.

A study of the immunology, virology, and safety of prednisone in HIV-1-infected subjects with CD4 cell counts of 200 to 700 mm(-3).

Adult Clinical Trials Group Study 349 examined the immunology, virology, and safety of 40 mg/d prednisone as an adjunct to antiretroviral therapy in 24 HIV-infected subjects with >200 CD4+ T cells/mm in a randomized placebo-controlled trial. After 8 weeks, median lymphocyte and CD4+ cell numbers increased >40% above baseline values (p =.08). No effect was observed on markers of cell activation or apoptosis, although the proportion of CD28+ CD8+ T cells increased (p =.006). Prednisone inhibited monocyte TNFalpha production without affecting T-cell responses to antigens or mitogens. Two subjects assigned to prednisone were subsequently found to have asymptomatic osteonecrosis of the hip. Many questions remain regarding the role of activation-induced sequestration and apoptosis as causes of progressive CD4+ T-cell loss in AIDS. The potential role of corticosteroids as tools to examine this question will be limited by concerns regarding their toxicity; however, further studies of other agents to limit cellular activation in AIDS are warranted.

Extracellular superoxide dismutase and glomerular mesangial cells: its production and regulation.

Extracellular superoxide dismutase (EC-SOD) is synthesized in mesenchymally derived cells and prevents the oxygen radical-induced injury. We studied whether kidney mesangial cells (MCs) produce EC-SOD and how its production is associated with chemokine secretion. Under unstimulated condition, MCs produced EC-SOD, and its production was correlated positively with cyclic adenosine monophosphate (cAMP), but negatively with interleukin (IL)-6 or IL-8 production. By prednisolone or phorbol myristate acetate treatment, EC-SOD levels were correlated negatively with levels of IL-6 and IL-8. The presence of adenylate cyclase inhibitor 2',3'-dideoxyadenosine lost the prednisolone effect. The stimulation of EC-SOD production might be one of the important effects of prednisolone via cAMP pathway in MCs.

Effect of prednisolone on increased expression of laminin by human peritoneal mesothelial cells cultured with high glucose.

This study aimed to clarify the role of laminin (a component of the extracellular matrix) in the mechanism of peritoneal fibrosis in continuous ambulatory peritoneal dialysis (CAPD) patients, and the effect of prednisolone on such fibrosis. We used semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and ELISA to study laminin mRNA expression and laminin protein production by human peritoneal mesothelial cells (HPMCs) cultured under various conditions. After 6 hours, medium 199 with a high glucose content (4.0%) increased laminin mRNA expression by 1.38-fold relative to control medium (0.1% glucose). Prednisolone (1 mumol/L) suppressed this increase by 92.9%. The laminin protein level in culture supernatant was increased 1.83-fold after incubation for 24 hours in high-glucose medium. Prednisolone (1 mumol/L) suppressed this increase by 58.3%. The effects of prednisolone were prevented by a glucocorticoid receptor antagonist (RU486) at 100 mumol/L. We conclude that culture of HPMCs in high-glucose medium increases laminin mRNA and protein expression, while prednisolone suppresses these changes via the glucocorticoid receptor, suggesting that prednisolone may prevent peritoneal fibrosis in CAPD patients.

Role of IL-2 and interferon in the generation of natural cytotoxic activity in influenza virus-stimulated PBL cultures: analysis with the use of prednisolone.

We have examined the role of interleukin 2, interferon-gamma and interferon-alpha in the generation of natural cytotoxic (NC) activity and cytotoxic T-lymphocyte (CTL) activity in peripheral blood lymphocyte cultures stimulated with influenza virus, using the immunosuppressive effects of prednisolone. In addition to an inhibitory effect on the generation of CTL activity, prednisolone also inhibited the generation of NC activity in a similar dose-and time-dependent manner. Prednisolone suppressed the production of interferon-gamma when it was added on the first day of culture of PBL with influenza virus. Levels of interferon-alpha were not affected. The effects of prednisolone on the generation of NC activity and CTL activity in kinetic terms were not paralleled by the effects on interferon-alpha and interferon-gamma production. The diminished generation of NC activity could be reversed by the addition of interleukin 2 (IL-2), but interferon-gamma had little if any restorative effects. Interferon-alpha had no effect. These findings support the hypothesis that IL-2 is the major inducer of NC activity in CTL generation cultures. The inhibitory effect on CTL generation could only be reversed by IL-2 and not by interferon-alpha- and interferon-gamma. Thus, in the absence of IL-2, interferon-alpha and interferon-gamma cannot support the generation of CTL activity or the concomitantly induced NC activity.

Steroid antagonism of the hypertensinogenic activity of adrenocortical steroids.

Previous studies in sheep have provided evidence for a separate "hypertensinogenic" class of adrenocortical steroid activity which is not simply related to their classical mineralocorticoid (MC) and/or glucocorticoid (GC) actions. This study investigated the structure-activity relationships of the effects of structural analogues of prednisolone on mean arterial pressure (MAP), and MC and GC actions in sheep. Infusions of these synthetic GC at 0.6 and 24 mg/day produced variable pressor effects which were dissociated from their MC and GC actions. In other experiments, the minimum adrenocortical steroid requirement to reproduce the onset of ACTH-dependent hypertension was determined. Infusion of cortisol, aldosterone, 17 alpha-hydroxy progesterone and 17 alpha,20 alpha-dihydroxy-4-pregnene-3-one was found to be sufficient to reproduce the hypertensive response to ACTH administration in sheep. A subsequent experiment showed that substitution of cortisol by the more potent synthetic GC, prednisolone had no effect on MAP. Therefore, cortisol appears to exert an essential action in ACTH hypertension which is not dependent on its GC activity. Other studies have found that prednisolone (100 mg/day) antagonized 9 alpha-fluoro-prednisolone (0.6 mg/day) induced hypertension but not its MC effects. The effect of progesterone (500 mg/day) and the progesterone analogues, norethisterone, medroxy-progesterone and 16 alpha-methyl progesterone on ACTH (5 micrograms/kg per day) hypertension was investigated. Progesterone completely blocked the hypertension and MC effects of ACTH infusion, while medroxy-progesterone partially blocked the increase in MAP. These data support our concept of a "hypertensinogenic" class of steroid activity.

[Restorative effect of traxanox on the suppression of antibody production in BALB/c mice].

The number of spleen-rosette forming cells (RFC) and thymus-RFC was assayed on day 4 after immunization with 5 X 10(8) sheep red blood cells (SRBC) in BALB/c mice. The number was decreased significantly by oral administration of cyclophosphamide (20 mg/kg) on days--1 and 0. The decrease in spleen-RFC and thymus-RFC by the pretreatment with cyclophosphamide was significantly restored or had a tendency to be restored by traxanox at doses of 3 and 30 mg/kg (p.o.). In addition, traxanox (30 mg/kg, p.o.) protected the reduced number of RFC in mice treated with cyclophosphamide (3 mg/kg, p.o.). Traxanox (30 mg/kg, p.o.) restored the decreased number of spleen-RFC and thymus-RFC in mice pretreated with dexamethasone (0.1 mg/kg, p.o.), and protected the decrease in the RFC in mice treated with prednisolone (10 mg/kg, p.o.). Also, phagocytosis of SRBC by the spleen adherent cells obtained from mice treated with dexamethasone (0.1 mg/kg, p.o.) was inhibited markedly in comparison with that by the spleen adherent cells of untreated mice. The transfer of the spleen adherent cells treated with dexamethasone into syngeneic recipient mice had no effect on the number of spleen hemolytic plaque forming cells (HPFC). On the other hand, the inhibition of phagocytosis of SRBC by spleen adherent cells of mice pretreated with dexamethasone was restored by the addition of traxanox (10 and 30 microM). The transfer of the spleen adherent cells treated with traxanox resulted in an increase in HPFC number. Traxanox (30 mg/kg, p.o.) protected the decrease in spleen-RFC and thymus-RFC in mice treated with indomethacin 1 mg/kg, p.o.) or acetylsalicylic acid (300 mg/kg, p.o.). Traxanox (3-30 mg/kg, p.o.) restored the decreased number of HPFC, spleen-RFC and thymus-RFC in mice pretreated with carrageenan (0.03 mg). The suppression of phagocytosis of SRBC by spleen adherent cells was restored by the treatment with traxanox (3 and 30 microM). The transfer of the spleen adherent cells treated with traxanox also resulted in an increase in HPFC count. These results strongly suggest that traxanox restores the artificially suppressed immune responses via macrophages.

[Modification of the leukocyte adherence inhibition test by prednisolone and aristolochic acid]. / Beeinflussung des Leukozyten-Adhärenz-Inhibitionstestes durch Prednisolon und Aristolochiasäure.

Drugs influence the leukocyte adherence inhibition test. Glucocorticoids reduce adherence more then aristolochic acid. When given together the glucocorticoid related adherence inhibition is abolished. In this way the known antagonistic behaviour of aristolochic acid towards glucocorticoids is confirmed.

Oral 1,25(OH)2D3: an effective prophylactic treatment for glucocorticoid osteopenia in rats.

Eighty-eight adult male Sprague-Dawley rats were given a diet with either (a) 0.5% Ca and 0.6% P or (b) 0.01% Ca and 0.6% P. Osteopenia was created by adding prednisolone to the diet. The prophylactic effect of oral 1,25(OH)2D3 on the osteopenia was studied. It was found that prednisolone osteopenia in the rat was associated with defective Ca absorption. By giving an oral dose of 1,25(OH)2D3, it was possible to maintain normal Ca absorption during prednisolone treatment and to prevent the bone loss. No significant hypercalcemia or any kidney calcifications were seen. These results are in contrast to earlier findings, in which subcutaneous administration of 1,25(OH)2D3 failed to prevent prednisolone osteopenia because of its tendency to increase bone resorption.

[Effect of alpha-mercaptopropionylglycine (alpha-MPG) and sodium dipropylacetate (DPA) on antibody formation (II). Immunosuppression induced by carcinostatic agents and glucocorticoid (author's transl)].

Previously, we reported that alpha-MPG and DPA stimulated humoral immuno-responses. In the present work, we investigated the effect of alpha-MPG and DPA on immunosuppression induced by carcinostatic agents including cyclophosphamide (CP), azathioprine (AP), methotrexate (MTX), actinomycin D (AcD) and mitomycin C(MMC) as well as prednisolone (Pred). The formation of hemolytic plaque forming cell (HPFC) in spleen of mice was clearly inhibited by treatment with immunosuppressive agents given s.c. for 5 days from the immunization. When alpha-MPG and DPA were given i.p. concomitantly with immunosuppressive agents, HPFC formation was not inhibited except when the combination of MTX and alpha-MPG was injected. With alpha-MPG, there was a tendency toward disappearance of the leucopenia induced by AP, MMC and Pred.