There has been a strong interest in searching for natural therapies for osteoporosis. Genistein, an isoflavone abundant in soy, and icariin, a prenylated flavonol glycoside isolated from Epimedium Herb, have both been identified to exert beneficial effects in preventing postmenopausal bone loss. However, the relative potency in osteogenesis between the individual phytoestrogen flavonoids remains unknown. The present study compared ability of genistein and icariin in enhancing differentiation and mineralization of cultured rat calvarial osteoblasts in vitro. Dose-dependent studies in osteoblast differentiation measuring alkaline phosphatase (ALP) activity revealed optimal concentrations of genistein and icarrin for stimulating osteogenesis to be both at 10(-5) M. Time course studies comparing the two compounds both at 10(-5) M demonstrated that icariin treatment always produced higher ALP activity, more and larger areas of CFU-F(ALP) colonies and mineralized nodules, more osteocalcin secretion, and calcium deposition, and a higher level of mRNA expression of osteogenesis-related genes COL1α2, BMP-2, OSX, and RUNX-2. However, they inhibited the proliferation of osteoblasts to a similar degree. In conclusion, although future in vivo studies are required to investigate whether icariin is more efficient in improving bone mass and/or preventing bone loss, our in vitro studies have demonstrated that icariin has a stronger osteogenic activity than genistein. In addition, while the prenyl group on C-8 of icariin could be the active group that takes part in osteoblastic differentiation and explains its greater potency in osteogenesis, mechanisms of action, and reasons for the relative potency of icariin versus genistein need to be further studied.

Icariin, a prenylated flavonol glycoside isolated from Epimedii herba, has been found to be a potent stimulator of osteogenic differentiation and has potential application in preventing bone loss. However, the signaling pathway underlying its osteogenic effect remains unclear. We hypothesized that the osteogenic activity of icariin is related to the nitric oxide (NO) signal pathway and PI3K/AKT pathway in its upstream. Rat bone marrow stromal cells (rBMSCs) were cultured in osteogenic medium and treated with icariin or together with L-NAME, ODQ, PDE5, and/or LY294002 (the inhibitor of NOS, sGC, cGMP, and PI3K respectively), and effects were examined on the expression of signal messengers (NOS, NO, sGC, cGMP, PKG and PI3K) and the levels of osteogenic markers (alkaline phosphatase or ALP, osteocalcin and calcified nodules). It was found that icariin dose-dependently increased ALP activity, and treatment at the optimal concentration (10(-5)M) increased NOS activity, iNOS and eNOS expression, NO production, sGC and cGMP contents and PKG expression besides the phosphorylation of AKT. The addition of L-NAME, ODQ and PDE5 significantly inhibited the icariin effects on above markers respectively. The addition of LY294002 decreased the p-AKT level, NOS activity, eNOS expression and NO production significantly, but had no significant effect on iNOS expression. The addition of any of the four inhibitors also abolished the osteogenic effect of icariin on rBMSCs as indicated by ALP activity, osteocalcin synthesis, calcium deposition and the number and areas of calcified nodules. These results suggest that the osteogenic effect of icariin involves the PI3K-AKT-eNOS-NO-cGMP-PKG signal pathway. Furthermore, dosage response studies showed that icariin at 10(-6)M (a physiologically achievable concentration in vivo) also activated this signal pathway.

Adrenalectomy has been shown to reverse most facets of the syndrome of the genetically obese fa/fa rat. However, a detailed analysis of the hypothalamo-pituitary-adrenal (HPA) axis in these animals is lacking. In the present study, morning corticosteronemia was higher in obese rats of both sexes than in lean ones, whereas evening corticosteronemia was higher only in obese male rats. The HPA axis was further investigated using stressful stimuli. Immobilization, ether, and cold stresses resulted in greater corticosterone levels in obese than in lean animals. These abnormalities consisted in upward shifts of the corticosterone response in obese females and absolute increases in that of obese males, indicating that such alterations were more pronounced in obese male than obese female rats. Due to this, the putative origin of the increased corticosterone output of obese rats was studied in males. Greater levels of ACTH were reached in obese than in lean rats when submitted to a cold stress (6 C). Dexamethasone produced a complete suppression of corticosterone output in both lean and obese rats. During the recovery from such suppression, corticosterone levels rose to higher values in obese than in lean rats. This observation together with the greater cold-induced ACTH output in obese rats suggest that the increased activity of the HPA axis of these animals is of central origin. Whatever its precise etiology within the central nervous system, it is proposed that the increased HPA axis activity in obese rats and its resultant hypercorticism play a role in the establishment and maintenance of their syndrome.

Background The capacity for bone healing reportedly is limited in osteoporosis with a less than ideal environment for healing of bone grafts. We therefore developed a composite bone substitute with rhBMP-2 loaded gelatin microsphere (GM) and calcium phosphate cement (CPC) to use in osteoporosis. Questions/purposes We asked whether (1) controlled release of rhBMP-2 could be improved in this composite bone substitute and (2) increasing factors released from the bone substitute could accelerate osteoporotic bone healing. Methods We soaked rhBMP-2/GM/CPC and rhBMP-2/ CPC composites in simulated body fluid for 28 days and then determined the amount of rhBMP-2 released. Both composites were implanted in bone defects of osteoporotic goats and left in place for 45 and 140 days; the specimens then were evaluated mechanically (pushout test) and morphologically (CT scanning, histology). Results The in vitro study showed the new composite released more rhBMP-2 compared with rhBMP-2/CPC. CT showed the defects healed more quickly with new grafts. The bone mineralization rate was greater in rhBMP-2/GM/ CPC than in rhBMP-2/CPC after 45 days of implantation and the pushout test was stronger after 45 and 140 days of implantation. Conclusions The new graft composite released more loaded factors and appeared to repair osteoporotic bone defects. Clinical Relevance These preliminary data suggest the new composite can be used as a bone substitute to accelerate healing of fractures and bone defects in osteoporosis.

No abstract

SOD activity was quantified by the use of a cytochrome c-modified gold electrode. The electrode responded rapidly to superoxide radicals in solution. Steady-state superoxide concentrations were established by control of the calibration conditions. On this basis very low SOD activities were detected (10-200 munits/mL). This method showed good correlation with the standard photometric test and was applied for the determination of SOD activity entrapped into liposomes. Interference by hydrogen peroxide and uric acid was characterized and minimized using long-chain thiols for the first electrode modification step. The complete modification proved to be stable for several days.

LHRH (median eminence) and LH (pituitary and plasma) from male and female Sprague-Dawley rats were assayed 1 month after streptozotocin injection and compared with values in controls either fed ad libitum or offered a restricted diet. Plasma LH was also assayed after stimulation with exogenous LHRH or naloxone. In diabetic males, the median eminence LHRH content and the plasma LH response to exogenous LHRH were unaltered, pituitary LH was increased, and plasma LH was decreased under basal conditions and after naloxone treatment. In diabetic females, while the median eminence LHRH content and the plasma LH response to exogenous LHRH or naloxone were reduced, pituitary and plasma LH levels were not different. Measurements made in undernourished rats excluded the possibility that the alterations found in diabetic animals were nutrition dependent. In parallel experiments, hypothalami and pituitaries were examined morphologically. In diabetic animals, degenerate axons, mainly of the LHRH type, were found in the arcuate nucleus and median eminence, and LH gonadotrophs were altered and more numerous. Strong differences between control males and females were revealed by morphometry; moreover, diabetic females had higher brain weights and fewer LH gonadotroph changes than diabetic males. These studies indicate that 1) the hypothalamo-pituitary changes that occur early in our streptozotocin-treated rats are unrelated to undernourishment and are possibly caused by insulin deficiency; 2) the LHRH axonal lesions might play a primary pathogenic role in the hypothalamo-pituitary disorder; 3) some anatomical data indicate that the brain and pituitary are less severely affected by diabetes in female than in male animals; and 4) differences between control males and females may account for some of the dissimilarities between the sexes observed under diabetic conditions.

The absorption of recombinant human insulin-like growth factor I (rhIGF-I), [¹²⁵I]rhIGF-I, xylose and [³H]lysine, administered into a clamped jejunal segment in anesthetized neonatal calves, was studied by measuring their appearance in the mesenteric vein draining the gut segment. Only trace amounts of IGF-I and of [¹²⁵I]rhIGF-I and/or ¹²⁵I were absorbed following dissolution in saline, buffer or colostrum. The absorption rate of both [³H]lysine and/or 3H and of xylose was much greater than the absorption of rhIGF-I, [¹²⁵I]rhIGF-I and/or ¹²⁵I but was also transient. Biological effects of ingested IGF-I in neonatal calves should barely occur as a consequence of intestinal absorption even in 1-day-old calves, known to absorb various peptides and proteins.