Investigation on the physical properties and biocompatibility of zirconia-alumina-silicate@diopside composite materials and its in vivo toxicity study in embryonic zebrafish.

Bioceramic materials have a wide range of applications in the biomedical field, such as in the repair of bone defects and dental surgery. Silicate-based bioceramics have attracted biomedical researchers' interest due to their bioactivity and biodegradability. In this study, extended the scope of ZAS utilization in bone tissue engineering by introducing calcium-magnesium-silicate (diopside, CMS) as an interface material aim to develop a machinable bioceramic composite (ZASCMS) by the sol-gel method. The physicochemical characterization, in vitro biological properties and in vivo zebrafish cytotoxicity study of ZAS-based composites as a function of CMS contents, 0, 25, 50, 75 and 100 wt%, were performed. Results showed that the as-prepared ZASCMS possessed porous architecture with well-interconnected pore structure. Results also revealed that the mechanical properties of ZASCMS composite materials were gradually improved with increasing CMS contents. The ZASCMS composites with more than 50 wt% CMS had the highest compressive strength and modulus of 6.78 ± 0.62 MPa and 340.10 ± 16.81 MPa, respectively. Regarding in vitro bioactivities, the composite scaffolds were found to stimulate osteoblast-like UMR-106 cell adhesion, growth, and proliferation. The antibacterial activity of the ZASCMS composite scaffolds was tested against Staphylococcus epidermidis (S. epidermidis) and Escherichia coli (E. coli) also exhibited an antibacterial property. Furthermore, the in vivo studies using embryonic zebrafish were exposed to as-prepared particles (0-500 µg mL-1) and showed that the synthesized ZAS, CMS and ZASCMS composite particles were non-toxic based on the evaluation of survivability, hatching rate and embryonic morphology. In conclusions, our results indicated that the synthesized composite exhibited their biological properties and antibacterial activity, which could well be a promising material with high potential to be applied in orthopaedic and dental tissue engineering.

CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance.

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl- and HCO3 - across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO3 - efflux through CFTR often required the intracellular H+/HCO3 - production and/or the Na+-dependent basolateral HCO3 - uptake, the intracellular pH (pHi) balance might be disturbed, especially as a consequence of increased endogenous H+ and HCO3 - production. However, measurement of pHi by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO3 - transport. In addition, although the plasma membrane Na+/K+-ATPase (NKA) is normally essential for basolateral HCO3 - uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pHi or NKA abundance on cell membrane.

Anomalous bone changes in ovariectomized type 2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition.

Estrogen deprivation accelerates bone resorption, leading to imbalance of bone remodeling and osteoporosis in postmenopausal women. In the elderly, type 2 diabetes mellitus (T2DM) frequently coexists as an independent factor of bone loss. However, little is known about the skeletal changes in a combined condition of estrogen deficiency and T2DM. Herein, we performed ovariectomy (OVX) in nonobese Goto-Kakizaki (GK) T2DM rats to examine changes associated with calcium and phosphate metabolism and bone microstructures and strength. As expected, wild-type (WT) rats subjected to ovariectomy (OVX-WT) had low trabecular bone volume and serum calcium with increased dynamic histomorphometric and serum bone markers, consistent with the high turnover state. T2DM in GK rats also led to low trabecular volume and serum calcium. However, the dynamic histomorphometric markers of bone remodeling were unaffected in these GK rats, indicating the distinct mechanism of T2DM-induced bone loss. Interestingly, OVX-GK rats were found to have anomalous and unique changes in bone turnover-related parameters, i.e., decreased osteoblast and osteoclast surfaces with lower COOH-terminal telopeptide of type I collagen levels compared with OVX-WT rats. Furthermore, the levels of calciotropic hormones, i.e., parathyroid hormone and 1,25(OH)2D3, were significantly decreased in OVX-GK rats. Although the OVX-induced bone loss did not further worsen in GK rats, a three-point bending test indicated that OVX-GK bones exhibited a decrease in bone elasticity. In conclusion, T2DM and estrogen deficiency both led to microstructural bone loss, the appearance of which did not differ from each factor alone. Nevertheless, the combination worsened the integrity and suppressed the turnover, which might eventually result in adynamic bone disease.

Factors inhibiting intestinal calcium absorption: hormones and luminal factors that prevent excessive calcium uptake.

Besides the two canonical calciotropic hormones, namely parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH)2D3], there are several other endocrine and paracrine factors, such as prolactin, estrogen, and insulin-like growth factor that have been known to directly stimulate intestinal calcium absorption. Generally, to maintain an optimal plasma calcium level, these positive regulators enhance calcium absorption, which is indirectly counterbalanced by a long-loop negative feedback mechanism, i.e., through calcium-sensing receptor in the parathyroid chief cells. However, several lines of recent evidence have revealed the presence of calcium absorption inhibitors present in the intestinal lumen and extracellular fluid in close vicinity to enterocytes, which could also directly compromise calcium absorption. For example, luminal iron, circulating fibroblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive calcium uptake under certain conditions. Interestingly, the intestinal epithelial cells themselves could lower their rate of calcium uptake after exposure to high luminal calcium concentration, suggesting a presence of an ultra-short negative feedback loop independent of systemic hormones. The existence of neural regulation is also plausible but this requires more supporting evidence. In the present review, we elaborate on the physiological significance of these negative feedback regulators of calcium absorption, and provide evidence to show how our body can efficiently restrict a flood of calcium influx in order to maintain calcium homeostasis.

Fabrication of biocomposite scaffolds made with modified hydroxyapatite inclusion of chitosan-grafted-poly(methyl methacrylate) for bone tissue engineering.

In the present study, composite scaffolds of chitosan-graft-poly(methyl methacrylate) (Chi-g-PMMA) and mineral ions-loaded hydroxyapatite (mHA) (obtained by the hydrothermal treatment of hydroxyapatite (HA) in a simulated body fluid (SBF) solution (mHA@Chi-g-PMMA)) were prepared by the blending method. The physical properties, bioactivity, biological properties and their capabilities for sustained drug and protein release were studied. Physicochemical analysis showed a successful incorporation of the mineral ions in the HA particles and a good distribution of the mHA within the Chi-g-PMMA polymer matrix. The compressive strength and the Young's modulus were 15.760 ± 0.718 and 658.452 ± 17.020 MPa, respectively. In bioactivity studies, more apatite formation on the surface were seen after immersion in the SBF solution. In vitro growth experiments using UMR-106 osteoblast-like cells on the mHA@Chi-g-PMMA scaffold case showed that the attachment, viability and proliferation of the cells on the scaffolds had improved after 7 d of immersion. The in vitro release of two compounds (the cancer drug, doxorubicin (DOX)) and bovine serum albumin (BSA)), which had been attached to separate mHA@Chi-g-PMMA scaffolds, were studied to determine their suitability as drug delivery vehicles. It was found that the sustained release of DOX was 73.95% and of BSA was 57.27% after 25 h of incubation. These experimental results demonstrated that the mHA@Chi-g-PMMA composite can be utilized as a scaffold for bone cells ingrowth and also be used for drug delivery during the bone repairing.

Estrogen deprivation aggravates cardiac hypertrophy in nonobese Type 2 diabetic Goto-Kakizaki (GK) rats.

Both Type 2 diabetes mellitus (T2DM) and estrogen deprivation have been shown to be associated with the development of cardiovascular disease and adverse cardiac remodeling. However, the role of estrogen deprivation on adverse cardiac remodeling in nonobese T2DM rats has not been clearly elucidated. We hypothesized that estrogen-deprivation aggravates adverse cardiac remodeling in Goto-Kakizaki (GK) rats. Wild-type (WT) and GK rats at the age of 9 months old were divided into two subgroups to have either a sham operation (WTS, GKS) or a bilateral ovariectomy (WTO, GKO) (n = 6/subgroup). Four months after the operation, the rats were killed, and the heart was excised rapidly. Metabolic parameters, cardiomyocytes hypertrophy, cardiac fibrosis, and biochemical parameters were determined. GK rats had hyperglycemia with hypoinsulinemia, and estrogen deprivation did not increase the severity of T2DM. Cardiac hypertrophy, cardiac oxidative stress, and phosphor-antinuclear factor κB were higher in WTO and GKS rats than WTS rats, and they markedly increased in GKO rats compared with GKS rats. Furthermore, cardiac fibrosis, transforming growth factor-ß, Bax, phosphor-p38, and peroxisome proliferator- activated receptor γ coactivator-1α expression were increased in GKS and GKO rats compared with the lean rats. However, mitochondrial dynamics proteins including dynamin-related protein 1 and mitofusin-2 were not altered by T2DM and estrogen deprivation. Although estrogen deprivation did not aggravate T2DM in GK rats, it increased the severity of cardiac hypertrophy by provoking cardiac inflammation and oxidative stress in nonobese GK rats.

CFTR-mediated anion secretion across intestinal epithelium-like Caco-2 monolayer under PTH stimulation is dependent on intermediate conductance K+ channels.

Parathyroid hormone (PTH), a pleiotropic hormone that maintains mineral homeostasis, is also essential for controlling pH balance and ion transport across renal and intestinal epithelia. Optimization of luminal pH is important for absorption of trace elements, e.g., calcium and phosphorus. We have previously demonstrated that PTH rapidly stimulated electrogenic [Formula: see text] secretion in intestinal epithelial-like Caco-2 monolayers, but the underlying cellular mechanism, contributions of other ions, particularly Cl- and K+, and long-lasting responses are not completely understood. Herein, PTH and forskolin were confirmed to induce anion secretion, which peaked within 1-3 min (early phase), followed by an abrupt decay and plateau that lasted for 60 min (late phase). In both early and late phases, apical membrane capacitance was increased with a decrease in basolateral capacitance after PTH or forskolin exposure. PTH also induced a transient increase in apical conductance with a long-lasting decrease in basolateral conductance. Anion secretion in both phases was reduced under [Formula: see text]-free and/or Cl--free conditions or after exposure to carbonic anhydrase inhibitor (acetazolamide), CFTR inhibitor (CFTRinh-172), Na+/H+ exchanger (NHE)-3 inhibitor (tenapanor), or K+ channel inhibitors (BaCl2, clotrimazole, and TRAM-34; basolateral side), the latter of which suggested that PTH action was dependent on basolateral K+ recycling. Furthermore, early- and late-phase responses to PTH were diminished by inhibitors of PI3K (wortmannin and LY-294002) and PKA (PKI 14-22). In conclusion, PTH requires NHE3 and basolateral K+ channels to induce [Formula: see text] and Cl- secretion, thus explaining how PTH regulated luminal pH balance and pH-dependent absorption of trace minerals.

Evaluation of bioactive glass incorporated poly(caprolactone)-poly(vinyl alcohol) matrix and the effect of BMP-2 modification.

Composite materials having mechanical and biological properties similar to those of human bones are needed for bone regeneration and repair. In the present study, composites were made by incorporating bioactive glass (BG) into polycaprolactone (PCL)-polyvinyl alcohol (PVA) (PCLPVA) matrix. Composites with different BG contents of 10, 25 and 50wt% were fabricated by an in-situ blending method. Physicochemical properties measurements found that the composite with 50wt% BG in the PCLPVA organic matrix exhibited the best mechanical properties (compressive strength and compressive young's modulus up to 32.26MPa and 530.91MPa, respectively). We investigated the effects of the BG content on cell adhesion, proliferation and osteogenic activity of UMR-106 cells grown on the scaffolds using in vitro cell culture assay. The composite scaffolds having 25wt% BG showed a significant increase in their cell adhesion capability and a faster cell proliferation. They also exhibited cell adhesion and spreading morphology after only 5days of culturing. For these reasons, we chose to attach the bone morphogenetic protein (BMP)-2 to this composite. The resulting composite (labeled BMP-2-loaded PCLPVABG25) showed significant improvement in the UMR-106 cells adhesion, in the enhancement in osteogenic differentiation and osteoinductivity of this composite.

Na+/H+ exchanger 3 inhibitor diminishes hepcidin-enhanced duodenal calcium transport in hemizygous ß-globin knockout thalassemic mice.

Recent investigation has shown that the liver-derived iron-regulating hormone, hepcidin, can potentiate intestinal calcium absorption in hemizygous ß-globin knockout thalassemic (BKO) mice. Since the upregulation of Fe2+ and H+ cotransporter, divalent metal transporter (DMT)-1, has been shown to correlate with thalassemia-induced intestinal calcium absorption impairment, the inhibition of the apical Na+/H+ exchanger (NHE)-3 that is essential for cytoplasmic pH regulation and transepithelial sodium absorption was hypothesized to negatively affect hepcidin action. Herein, the positive effect of hepcidin on the duodenal calcium transport was evaluated using Ussing chamber technique. The results showed that BKO mice had lower absorptive surface area and duodenal calcium transport than wild-type mice. Besides, paracellular transport of zinc in BKO mice was compromised. Hepcidin administration completely restored calcium transport. Since this hepcidin action was totally abolished by inhibitors of the basolateral calcium transporters, Na+/Ca2+ exchanger (NCX1) and plasma membrane Ca2+-ATPase (PMCA1b), the enhanced calcium flux potentially occurred through the transcellular pathway rather than paracellular pathway. Interestingly, the selective NHE3 inhibitor, 100 nM tenapanor, markedly inhibited hepcidin-enhanced calcium transport. Accordingly, hepcidin is one of the promising therapeutic agents for calcium malabsorption in ß-thalassemia. It mainly stimulates the transcellular calcium transport across the duodenal epithelium in an NHE3-dependent manner.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

Hepcidin and 1,25(OH)2D3 effectively restore Ca2+ transport in ß-thalassemic mice: reciprocal phenomenon of Fe2+ and Ca2+ absorption.

Previously, ß-thalassemia, an inherited anemic disorder with iron overload caused by loss-of-function mutation of ß-globin gene, has been reported to induce osteopenia and impaired whole body calcium metabolism, but the pathogenesis of aberrant calcium homeostasis remains elusive. Herein, we investigated how ß-thalassemia impaired intestinal calcium absorption and whether it could be restored by administration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or hepcidin, the latter of which was the liver-derived antagonist of intestinal iron absorption. The results showed that, in hemizygous ß-globin knockout (BKO) mice, the duodenal calcium transport was lower than that in wild-type littermates, and severity was especially pronounced in female mice. Both active and passive duodenal calcium fluxes in BKO mice were found to be less than those in normal mice. This impaired calcium transport could be restored by 7-day 1,25(OH)2D3 treatment. The 1,25(OH)2D3-induced calcium transport was diminished by inhibitors of calcium transporters, e.g., L-type calcium channel, NCX1, and PMCA1b, as well as vesicular transport inhibitors. Interestingly, the duodenal calcium transport exhibited an inverse correlation with transepithelial iron transport, which was markedly enhanced in thalassemic mice. Thus, 3-day subcutaneous hepcidin injection and acute direct hepcidin exposure in the Ussing chamber were capable of restoring the thalassemia-associated impairment of calcium transport; however, the positive effect of hepcidin on calcium transport was completely blocked by proteasome inhibitors MG132 and bortezomib. In conclusion, both 1,25(OH)2D3 and hepcidin could be used to alleviate the ß-thalassemia-associated impairment of calcium absorption. Therefore, our study has shed light on the development of a treatment strategy to rescue calcium dysregulation in ß-thalassemia.

Prolactin receptor and osteogenic induction of prolactin in human periodontal ligament fibroblasts.

Prolactin is an important hormone involved in the interaction between maternal, extraembryonic, and fetal tissues that remains in high levels during the entire duration of pregnancy. Although many systemic alterations occur during pregnancy, such as hormonal changes, that are known to be associated with periodontitis and tooth loss, PRL function in human periodontal ligament fibroblasts (HPDLF) had never been studied. Herein, we investigated the role of PRL in the regulation of HPDLF proliferation and differentiation. HPDLF were cultured in differentiating medium with various concentrations of PRL. The present study demonstrated that HPDLF and primary human PDL cells that were extracted for orthodontic purpose expressed both short and long isoforms of PRLR mRNA and its proteins. An incubation with of high concentration of PRL (600 and 1,000 ng/mL) modestly decreased the HPDLF number. In contrast, PRL at a non-reproductive level (10 ng/mL) and pregnant level (100 ng/mL) significantly upregulated the markers of osteogenesis, such as RUNX2, BMP2, and POSTN, but not SOX9. Mineral nodule formation was induced, whereas proteoglycan accumulation was reduced by PRL suggesting that HPDLF were undergoing differentiation into preosteoblastic cells. In conclusion, the presence of hPRLR in human PDL together with PRL-induced upregulation of osteogenic markers strongly suggested a direct regulatory role of PRL in PDL and periodontal tissue development.

Premature chondrocyte apoptosis and compensatory upregulation of chondroregulatory protein expression in the growth plate of Goto-Kakizaki diabetic rats.

Type 2 diabetes mellitus (T2DM) is much more detrimental to bone than previously thought. Specifically, it is associated with aberrant bone remodeling, defective bone microstructure, poor bone quality, and growth retardation. The T2DM-associated impairment of bone elongation may result from a decrease in growth plate function, but the detailed mechanism has been unknown. The present study, therefore, aimed to test hypothesis that T2DM led to premature apoptosis of growth plate chondrocytes in Goto-Kakizaki (GK) type 2 diabetic rats, and thus triggered the compensatory responses to overcome this premature apoptosis, such as overexpression of Runt-related transcription factor (Runx)-2 and vascular endothelial growth factor (VEGF), the essential mediators for bone elongation. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of epiphyseal sections successfully revealed increases in chondrocyte apoptosis in the hypertrophic zone (HZ) and chondro-osseous junction of GK rats. Quantitative immunohistochemical analysis further confirmed the overexpression of parathyroid hormone-related protein (PTHrP), Runx2 and VEGF, but not Indian hedgehog (Ihh) in the HZ. Analysis of blood chemistry indicated suppression of bone remodeling with a marked decrease in parathyroid hormone level. In conclusion, GK rats manifested a premature increase in chondrocyte apoptosis in the HZ of growth plate, and a compensatory overexpression of chondroregulatory proteins, such as PTHrP, Runx2, and VEGF. Our results, therefore, help explain how T2DM leads to impaired bone elongation and growth retardation.

Bromocriptine modulates the expression of PTHrP receptor, Indian hedgehog, and Runx2 proteins in the growth plate of lactating rats.

In lactating rats, the endochondral bone growth is markedly enhanced, leading to the lengthening of long bone. This lactation-induced bone elongation could be abolished by a dopaminergic D2 receptor agonist bromocriptine, but how bromocriptine altered the expression of major chondroregulatory proteins in the growth plate cartilage was elusive. Here, we performed a quantitative immunohistochemical analysis to determine the expression of various peptides and transcription factors known to control the growth plate chondrocyte proliferation and differentiation [i.e., parathyroid hormone-related protein (PTHrP), PTHrP receptor, Indian hedgehog (Ihh), and runt-related transcription factor 2 (Runx2)], in bromocriptine-treated lactating rats. The results showed that bromocriptine markedly increased Ihh expression in hypertrophic chondrocytes during early and mid-lactation, while the expression of PTHrP receptor, but not its ligand PTHrP, was upregulated in the proliferative and hypertrophic zones during mid and late lactation. In contrast, the expression of Runx2, an important transcription factor for chondrocyte differentiation, was suppressed in the hypertrophic chondrocytes of bromocriptine-treated rats. In conclusion, bromocriptine increased Ihh and PTHrP receptor expressions and decreased Runx2 expression, which might, in turn, enhance chondrocyte proliferation and delay chondrocyte hypertrophy, thereby slowing down endochondral bone growth. This finding could explain how bromocriptine compromised the lactation-induced bone elongation.

Prolactin stimulates the L-type calcium channel-mediated transepithelial calcium transport in the duodenum of male rats.

Elevated plasma levels of prolactin (PRL) have been reported in several physiological and pathological conditions, such as lactation, prolactinoma, and dopaminergic antipsychotic drug uses. Although PRL is a calcium-regulating hormone that stimulates intestinal calcium absorption in lactating rats, whether PRL is capable of stimulating calcium absorption in male rats has been elusive. Herein, the transepithelial calcium transport and electrical characteristics were determined in ex vivo duodenal tissues of male rats by Ussing chamber technique. We found that PRL receptors were abundantly present in the basolateral membrane of the duodenal epithelial cells. PRL (200-800 ng/mL) markedly increased the active duodenal calcium transport in a dose-dependent fashion without effect on the transepithelial resistance. The PRL-enhanced active duodenal calcium transport was completely abolished by L-type calcium channel blocker (nifedipine) as well as inhibitors of the major basolateral calcium transporters, namely plasma membrane Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger. Several intracellular mediators, such as JAK2, MEK, PI3K and Src kinase, were involved in the PRL-enhanced transcellular calcium transport. Moreover, PRL also stimulated the paracellular calcium transport in the duodenum of male rats in a PI3K-dependent manner. In conclusion, PRL appeared to be a calcium-regulating hormone in male rats by enhancing the L-type calcium channel-mediated transcellular and the paracellular passive duodenal calcium transport. This phenomenon could help restrict or alleviate negative calcium balance and osteoporosis that often accompany hyperprolactinemia in male patients.

Upregulated mRNA levels of SERT, NET, MAOB, and BDNF in various brain regions of ovariectomized rats exposed to chronic aversive stimuli.

Estrogen deficiency increases the risk of anxiety and mood disorders, presumably by deranging metabolism of the monoamine neurotransmitters and the expression of their reuptake transporters in the brain. Although estrogen-deficient individuals were also susceptible to stress, little was known regarding the effect of stress on the levels of transcripts related to brain monoamine metabolism. Herein, we used quantitative real-time PCR to quantify the mRNA levels of serotonin reuptake transporter (SERT), norepinephrine transporter (NET), monoamine oxidase-B (MAOB), tryptophan hydroxylase (TPH), and tyrosine hydroxylase (TH) in various brain regions of ovariectomized (OVX) rats which had been exposed for 4 weeks to chronic aversive stimuli (CAS), such as water deprivation, cage tilt, and illumination. We found that CAS induced stress responses in OVX rats as indicated by increases in the adrenal gland weight and sucrose intake. After CAS exposure, mRNA levels of SERT and NET were upregulated in the frontal cortex, hippocampus, amygdala, and periaqueductal gray. In addition, CAS also increased the mRNA levels of MAOB, an enzyme for dopamine degradation, in the same brain regions. However, CAS did not alter the mRNA levels of TPH or TH, both of which are rate-limiting enzymes for the synthesis of serotonin and norepinephrine in the dorsal raphé and locus coeruleus, respectively. Interestingly, mRNA expression of brain-derived neurotrophic factor precursor was upregulated in the hippocampus of CAS-exposed OVX rats, suggesting a compensatory mechanism which might counteract the stress-induced depression. Therefore, the present data have provided evidence to explain how stress affected brain monoamine metabolism in estrogen-deficient stressed patients.

Upregulation of osteoblastic differentiation marker mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens from Pueraria mirifica.

Phytoestrogens have attracted attention for their potential in the prevention of postmenopausal osteoporosis. Recently, phytoestrogen-rich herb Pueraria mirifica has been demonstrated to possess an osteogenic effect on bone in ovariectomized rats, but its underlying cellular mechanism was not known. Here, we investigated the effects of P. mirifica extract and its major isoflavone compound, puerarin, on cell viability, cell proliferation and the expression of differentiation markers in rat osteoblast-like UMR106 cells. After exposure to 17ß-estradiol (E2), genistein, P. mirifica extract and puerarin, proliferation but not viability of UMR106 cells was markedly decreased. Quantitative real-time PCR revealed that P. mirifica extract and puerarin significantly increased the mRNA expression of alkaline phosphatase (ALP) and osteoprotegerin, but not Runx2, osterix or osteocalcin. Puerarin also decreased the mRNA expression of receptor activator of nuclear factor-κB ligand, an osteoclastogenic factor, suggesting that it could induce bone gain by enhancing osteoblast differentiation and suppressing osteoclast function. Furthermore, after an exposure to high affinity estrogen receptor (ER) antagonist (ICI182780), the E2-, genistein-, P. mirifica extract- and puerarin-induced upregulation of ALP expressions were completely abolished. It could be concluded that P. mirifica extract and puerarin induced osteoblast differentiation rather than osteoblast proliferation in an ER-dependent manner. The present findings, therefore, corroborated the potential benefit of P. mirifica extract and puerarin in the prevention and treatment of postmenopausal osteoporosis.

Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells.

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells.

Electrogenic Na⁺/HCO3⁻ co-transporter-1 is essential for the parathyroid hormone-stimulated intestinal HCO3⁻ secretion.

Parathyroid hormone (PTH) was recently demonstrated to enhance the HCO(3)(-) secretion through the apical anion channel, cystic fibrosis transmembrane conductance regulator (CFTR), but how the HCO(3)(-) entered the epithelial cells was not well understood, in part, due to the lack of specific inhibitors of the basolateral HCO(3)(-) transporters. Moreover, the function of the PTH-stimulated HCO(3)(-) secretion has never been investigated in vivo. Here, we designed three specific pairs of small interfering RNA sequences to simultaneously knockdown three variants of the electrogenic Na(+)/HCO(3)(-) co-transporter (NBCe)-1 in the intestinal epithelium-like Caco-2 monolayer. The results showed that NBCe1 mRNA levels were markedly reduced, and the PTH-induced transepithelial current and voltage changes were diminished after triple knockdown as determined by quantitative real-time PCR and Ussing chamber technique, respectively. An in vivo ligated intestinal loop study further showed that there was an increased fluid secretion, presumably driven by HCO(3)(-) transport, in the ileum, but not in jejunum or colon, of rats administered intravenously with 2 µg/kg body weight of rat PTH 1-34. Therefore, the present results suggested that PTH stimulated intestinal HCO(3)(-) secretion, particularly in the ileum, by inducing the basolateral HCO(3)(-) uptake via NBCe1.

Anxiety-like behaviors and expression of SERT and TPH in the dorsal raphé of estrogen- and fluoxetine-treated ovariectomized rats.

The anxiolytic effect of fluoxetine (Flx) was often ineffective in postmenopausal and estrogen-deficient patients, but such effect had not been experimentally demonstrated, particularly in the female rat model of estrogen deficiency. Here we determined the anxiety-like behaviors in ovariectomized (Ovx) rats treated for 4weeks with 10µg/kg 17ß-estradiol s.c. (Ovx+E2), 10mg/kg Flx p.o. (Ovx+Flx) or a combination of both (Ovx+E2+Flx). Since Flx is known to induce anxiolysis in males, we first evaluated the Flx regimen in male rats. The results showed that anxiety-like behaviors were reduced in Flx-treated male rats. In contrast, Ovx+Flx rats still exhibited the same anxiety-like behaviors as in Ovx rats. Both Ovx+E2 and Ovx+E2+Flx rats, however, showed comparable reductions in anxiety-like behaviors, suggesting that Flx had no anxiolytic-like effect. Furthermore, E2 and E2+Flx similarly upregulated the mRNA expression of serotonin reuptake transporter (SERT) and tryptophan hydroxylase-2 in the dorsal raphé of Ovx rats, while having no effect on SERT expression in the frontal cortex, hippocampus, septum, amygdala and periaqueductal gray. In conclusion, Flx induced anxiolytic-like action in male rats. In Ovx rats, it was E2 and not Flx that exerted the anxiolytic-like action, which was mediated, in part, by altering serotonin metabolism in the dorsal raphé.