Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

Osteocyte RANKL Drives Bone Resorption in Mouse Ligature-Induced Periodontitis.

Mouse ligature-induced periodontitis (LIP) has been used to study bone loss in periodontitis. However, the role of osteocytes in LIP remains unclear. Furthermore, there is no consensus on the choice of alveolar bone parameters and time points to evaluate LIP. Here, we investigated the dynamics of changes in osteoclastogenesis and bone volume (BV) loss in LIP over 14 days. Time-course analysis revealed that osteoclast induction peaked on days 3 and 5, followed by the peak of BV loss on day 7. Notably, BV was restored by day 14. The bone formation phase after the bone resorption phase was suggested to be responsible for the recovery of bone loss. Electron microscopy identified bacteria in the osteocyte lacunar space beyond the periodontal ligament (PDL) tissue. We investigated how osteocytes affect bone resorption of LIP and found that mice lacking receptor activator of NF-κB ligand (RANKL), predominantly in osteocytes, protected against bone loss in LIP, whereas recombination activating 1 (RAG1)-deficient mice failed to resist it. These results indicate that T/B cells are dispensable for osteoclast induction in LIP and that RANKL from osteocytes and mature osteoblasts regulates bone resorption by LIP. Remarkably, mice lacking the myeloid differentiation primary response gene 88 (MYD88) did not show protection against LIP-induced bone loss. Instead, osteocytic cells expressed nucleotide-binding oligomerization domain containing 1 (NOD1), and primary osteocytes induced significantly higher Rankl than primary osteoblasts when stimulated with a NOD1 agonist. Taken together, LIP induced both bone resorption and bone formation in a stage-dependent manner, suggesting that the selection of time points is critical for quantifying bone loss in mouse LIP. Pathogenetically, the current study suggests that bacterial activation of osteocytes via NOD1 is involved in the mechanism of osteoclastogenesis in LIP. The NOD1-RANKL axis in osteocytes may be a therapeutic target for bone resorption in periodontitis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Perspective: The current state of Cre driver mouse lines in skeletal research: Challenges and opportunities.

The Cre/Lox system has revolutionized the ability of biomedical researchers to ask very specific questions about the function of individual genes in specific cell types at specific times during development and/or disease progression in a variety of animal models. This is true in the skeletal biology field, and numerous Cre driver lines have been created to foster conditional gene manipulation in specific subpopulations of bone cells. However, as our ability to scrutinize these models increases, an increasing number of issues have been identified with most driver lines. All existing skeletal Cre mouse models exhibit problems in one or more of the following three areas: (1) cell type specificity-avoiding Cre expression in unintended cell types; (2) Cre inducibility-improving the dynamic range for Cre in inducible models (negligible Cre activity before induction and high Cre activity after induction); and (3) Cre toxicity-reducing the unwanted biological effects of Cre (beyond loxP recombination) on cellular processes and tissue health. These issues are hampering progress in understanding the biology of skeletal disease and aging, and consequently, identification of reliable therapeutic opportunities. Skeletal Cre models have not advanced technologically in decades despite the availability of improved tools, including multi-promoter-driven expression of permissive or fragmented recombinases, new dimerization systems, and alternative forms of recombinases and DNA sequence targets. We review the current state of skeletal Cre driver lines, and highlight some of the successes, failures, and opportunities to improve fidelity in the skeleton, based on successes pioneered in other areas of biomedical science.

Sostdc1 Suppression in the Absence of Sclerostin Potentiates Anabolic Action of Cortical Bone in Mice.

The development of Wnt-based osteoanabolic agents has progressed rapidly in recent years, given the potent effects of Wnt modulation on bone homeostasis. Simultaneous pharmacologic inhibition of the Wnt antagonists sclerostin and Dkk1 can be optimized to create potentiated effects in the cancellous bone compartment. We looked for other candidates that might be co-inhibited along with sclerostin to potentiate the effects in the cortical compartment. Sostdc1 (Wise), like sclerostin and Dkk1, also binds and inhibits Lrp5/6 coreceptors to impair canonical Wnt signaling, but Sostdc1 has greater effects in the cortical bone. To test this concept, we deleted Sostdc1 and Sost from mice and measured the skeletal effects in cortical and cancellous compartments individually. Sost deletion alone produced high bone mass in all compartments, whereas Sostdc1 deletion alone had no measurable effects on either envelope. Mice with codeletion of Sostdc1 and Sost had high bone mass and increased cortical properties (bone mass, formation rates, mechanical properties), but only among males. Combined administration of sclerostin antibody and Sostdc1 antibody in wild-type female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. In conclusion, Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Targeting Sclerostin and Dkk1 at Optimized Proportions of Low-Dose Antibody Achieves Similar Skeletal Benefits to Higher-Dose Sclerostin Targeting in the Mature Adult and Aged Skeleton.

Age-associated low bone mass disease is a growing problem in the US. Development of osteoanabolic therapies for treating skeletal fragility has lagged behind anti-catabolic therapies, but several bone-building molecules are clinically available. We reported previously that antibody-based neutralization of the Lrp5/Lrp6 inhibitor Dkk1 has minimal effects on bone gain, but can potentiate the already potent osteoanabolic effects of sclerostin inhibition (another Lrp5/Lrp6 inhibitor highly expressed by osteocytes). In this communication, we test whether an optimized ratio of sclerostin and Dkk1 antibodies (Scl-mAb and Dkk1-mAb, respectively), administered at low doses, can maintain the same bone-building effects as higher dose Scl-mAb, in adult (6 months of age) and aged (20 months of age) wild-type mice. A 3:1 dose of Scl-mAb:Dkk1-mAb at 12.5 mg/kg was equally efficacious as 25 mg/kg of Scl-mAb in both age groups, using radiographic (DXA, µCT), biomechanical, (3-point bending tests), and histological (fluorochrome-based bone formation parameters) outcome measures. For some bone properties, including trabecular thickness and bone mineral density in the spine, and endocortical bone formation rates in the femur, the 3:1 treatment was associated with significantly improved skeletal properties compared to twice the dose of Scl-mAb. Cortical porosity in aged mice was also reduced by both Scl-mAb and low-dose 3:1 treatment. Overall, both treatments were efficacious in the mature adult (6 mo.) and aged (20 mo.) skeletons, suggesting Wnt targeting is a viable strategy for improving skeletal fragility in the very old. Further, the data suggest that low dose of combination therapy can be at least equally efficacious as higher doses of Scl-mAb monotherapy.

Suppression of Sost/Sclerostin and Dickkopf-1 Augment Intervertebral Disc Structure in Mice.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss, but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dickkopf-1 (dkk1). Anti-sclerostin antibody (scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We aimed to (i) determine if pharmacological neutralization of sclerostin, dkk1, or their combination would stimulate Wnt signaling and augment IVD structure and (ii) determine the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n = 6-7/group) were subcutaneously injected 2×/week for 5.5 weeks with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg), or vehicle (veh). Separately, IVD of sost KO and wild-type (WT) mice (n = 8/group) were harvested at 16 weeks of age. First, compared with vehicle, injection of scl-Ab, dkk1-Ab, and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and ß-catenin gene expression. Despite these similarities, only injection of scl-Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared with WT, sost KO enlarged IVD height, increased proteoglycan staining, and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co-transcription factor ß-catenin in the IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress-related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl-Ab outperformed dkk1-Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Notum Deletion From Late-Stage Skeletal Cells Increases Cortical Bone Formation and Potentiates Skeletal Effects of Sclerostin Inhibition.

Wnt signaling plays a vital role in the cell biology of skeletal patterning, differentiation, and maintenance. Notum is a secreted member of the α/ß-hydrolase superfamily that hydrolyzes the palmitoleoylate modification on Wnt proteins, thereby disrupting Wnt signaling. As a secreted inhibitor of Wnt, Notum presents an attractive molecular target for improving skeletal health. To determine the cell type of action for Notum's effect on the skeleton, we generated mice with Notum deficiency globally (Notum-/- ) and selectively (Notumf/f ) in limb bud mesenchyme (Prx1-Cre) and late osteoblasts/osteocytes (Dmp1-Cre). Late-stage deletion induced increased cortical bone properties, similar to global mutants. Notum expression was enhanced in response to sclerostin inhibition, so dual inhibition (Notum/sclerostin) was also investigated using a combined genetic and pharmacologic approach. Co-suppression increased cortical properties beyond either factor alone. Notum suppressed Wnt signaling in cell reporter assays, but surprisingly also enhanced Shh signaling independent of effects on Wnt. Notum is an osteocyte-active suppressor of cortical bone formation that is likely involved in multiple signaling pathways important for bone homeostasis © 2021 American Society for Bone and Mineral Research (ASBMR).

The Wnt pathway: An important control mechanism in bone's response to mechanical loading.

The conversion of mechanical energy into biochemical changes within living cells is process known as mechanotransduction. Bone is a quintessential tissue for studying the molecular mechanisms of mechanotransduction, as the skeleton's mechanical competence is crucial for vertebrate movement. Bone cell mechanotransduction is facilitated by a number of cell biological pathways, one of the most prominent of which is the Wnt signaling cascade. The Wnt co-receptor Lrp5 has been identified as a crucial protein for mechanical signaling in bone, and modifiers of Lrp5 activity play important roles in mediating signaling efficiency through Lrp5, including sclerostin, Dkk1, and the co-receptor Lrp4. Mechanical regulation of sclerostin is mediated by certain members of the Hdac family. Other mechanisms that influence Wnt signaling-some of which are mechanoresponsive-are coming to light, including R-spondins and their role in organizing the Rnf43/Znrf3 and Lgr4/5/6 complex that liberates Lrp5. While the identity of the key Wnt proteins involved in bone cell mechanical signaling are elusive, the likely pool of key players is narrowing. Identification of Wnt-based molecular targets that can be modulated pharmacologically to make mechanical stimulation (e.g., exercise) more beneficial is an emerging approach to improving skeletal integrity and reducing fracture risk.

Improving Bone Health by Optimizing the Anabolic Action of Wnt Inhibitor Multitargeting.

Sclerostin antibody (romosozumab) was recently approved for clinical use in the United States to treat osteoporosis. We and others have explored Wnt-based combination therapy to disproportionately improve the anabolic effects of sclerostin inhibition, including cotreatment with sclerostin antibody (Scl-mAb) and Dkk1 antibody (Dkk1-mAb). To determine the optimal ratio of Scl-mAb and Dkk1-mAb for producing maximal anabolic action, the proportion of Scl-mAb and Dkk1-mAb were systematically varied while holding the total antibody dose constant. A 3:1 mixture of Scl-mAb to Dkk1-mAb produced two to three times as much cancellous bone mass as an equivalent dose of Scl-mAb alone. Further, a 75% reduction in the dose of the 3:1 mixture was equally efficacious to a full dose of Scl-mAb in the distal femur metaphysis. The Scl-mAb/Dkk1-mAb combination approach was highly efficacious in the cancellous bone mass, but the cortical compartment was much more subtly affected. The osteoanabolic effects of Wnt pathway targeting can be made more efficient if multiple antagonists are simultaneously targeted. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Skeletal Protection and Promotion of Microbiome Diversity by Dietary Boosting of the Endogenous Antioxidant Response.

There is an unmet need for interventions with better compliance that prevent the adverse effects of sex steroid deficiency on the musculoskeletal system. We identified a blueberry cultivar (Montgomerym [Mont]) that added to the diet protects female mice from musculoskeletal loss and body weight changes induced by ovariectomy. Mont, but not other blueberries, increased the endogenous antioxidant response by bypassing the traditional antioxidant transcription factor Nrf2 and without activating estrogen receptor canonical signaling. Remarkably, Mont did not protect the male skeleton from androgen-induced bone loss. Moreover, Mont increased the variety of bacterial communities in the gut microbiome (α-diversity) more in female than in male mice; shifted the phylogenetic relatedness of bacterial communities (ß-diversity) further in females than males; and increased the prevalence of the taxon Ruminococcus1 in females but not males. Therefore, this nonpharmacologic intervention (i) protects from estrogen but not androgen deficiency; (ii) preserves bone, skeletal muscle, and body composition; (iii) elicits antioxidant defense responses independently of classical antioxidant/estrogenic signaling; and (iv) increases gut microbiome diversity toward a healthier signature. These findings highlight the impact of nutrition on musculoskeletal and gut microbiome homeostasis and support the precision medicine principle of tailoring dietary interventions to patient individualities, like sex. © 2020 American Society for Bone and Mineral Research (ASBMR).

Evaluation of pharmacist-led strategy to reduce outpatient fluoroquinolone prescribing from the emergency department.

OBJECTIVE: The purpose of this study was to evaluate the effectiveness of a pharmacist-led initiative to reduce outpatient fluoroquinolone prescribing in the emergency department (ED). DESIGN: Eight common indications for fluoroquinolone prescribing were selected for intervention. The intervention consisted of a multimodal prescriber education program providing empirical recommendations for antibacterial agents on the basis of guidelines, the local ED antibiogram, and clinical trials. The electronic medical record was reviewed to identify all prescriptions for moxifloxacin, ciprofloxacin, and levofloxacin oral tablets written at the time of discharge by ED providers. SETTING AND PARTICIPANTS: This study was conducted in a 62-bed emergency department at an academic community medical center with approximately 90,000 ED visits per year. Adult patients who were prescribed an oral fluoroquinolone at discharge from the ED were included. OUTCOME MEASURES: The primary outcome of the study was fluoroquinolone days of therapy (DOT) prescribed per 100 ED visits. RESULTS: In the preintervention group, the primary outcome of fluoroquinolone DOT per 100 ED visits was 18.4, 17.8, 16.5, 19.8, and 16.8 for the months of December 2017 through April 2018, respectively. The fluoroquinolone DOT per 100 ED visits in the postintervention group was 8.7, 7.9, 8.0, 6.3, and 6.0 for the months of December 2018 through April 2019, respectively. The fluoroquinolone DOT per 100 ED visits was found to be significantly shorter in the postintervention group with P = 0.009. CONCLUSION: The results of this study reveal that pharmacist-led interventions, focused on multimodal provider education, were effective at reducing ED discharge prescriptions for fluoroquinolones.

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism-the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1-a gene know to regulate skeletal development-is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.

Yu Ping Feng San, an ancient Chinese herbal decoction, regulates the expression of inducible nitric oxide synthase and cyclooxygenase-2 and the activity of intestinal alkaline phosphatase in cultures.

Yu Ping Feng San (YPFS), a Chinese herbal decoction comprising Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu), and Saposhnikoviae Radix (SR; Fangfeng), has been used clinically to treat inflammatory bowel diseases (IBD). Previously, we demonstrated a dual role of YPFS in regulating cytokine release in cultured macrophages. In this study, we elucidated the anti-inflammatory effect of YPFS that is mediated through modulating the expression of three key enzymes involved in IBD: inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intestinal alkaline phosphatase (IALP). In a lipopolysaccharide (LPS)-induced chronic-inflammation model of cultured murine macrophages, YPFS treatment suppressed the activation of iNOS and COX-2 expression in a dose-dependent manner. Conversely, application of YPFS in cultured small intestinal enterocytes markedly induced the expression of IALP in a time-dependent manner, which might strengthen the intestinal detoxification system. A duality of YPFS in modulating the expression of iNOS and COX-2 was determined here. The expression of iNOS and COX-2 in macrophages was induced by YPFS, and this activation was partially blocked by the NF-κB-specific inhibitor BAY 11-7082, indicating a role of NF-κB signaling. These YPFS-induced changes in gene regulation strongly suggest that the anti-inflammatory effects of YPFS are mediated through the regulation of inflammatory enzymes.

Ferulic acid enhances the chemical and biological properties of astragali radix: a stimulator for danggui buxue tang, an ancient Chinese herbal decoction.

Danggui buxue tang, an ancient formula composed of astragali radix and Angelicae sinensis radix, has been used for treating menopausal irregularity in women for more than 800 years in China. In danggui buxue tang, the complete functions of astragali radix require the assistance of Angelicae sinensis radix, and both herbs have to work harmoniously in order to achieve the maximal therapeutic purposes. In order to analyze the relationship of the two herbs, the role of ferulic acid, a major chemical within Angelicae sinensis radix, in chemical and biological properties of astragali radix was determined. Using ferulic acid in the extraction of astragali radix, the amounts of astragaloside IV, calycosin, and formononetin were increased in the final extract; however, the astragali radix polysaccharide showed a minor increase. The chemical-enriched astragali radix extract showed robust induction in osteogenic and estrogenic activities in cultured osteosarcoma MG-63 and breast MCF-7 cells. However, ferulic acid itself did not show such biological responses. The current results strongly suggest that Angelicae sinensis radix-derived ferulic acid is a positive regulator for danggui buxue tang, which enhanced the solubilities of active ingredients derived from astragali radix, and which therefore increased the biological efficacies of danggui buxue tang.

Quantification of the transcripts encoding different forms of AChE in various cell types: real-time PCR coupled with standards in revealing the copy number.

Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7 × 10(4) in PC12 (rat neuronal cell), 1.3 × 10(4) in Caco-2 (human intestinal cell), 0.67 × 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3 × 10(4), 3.3 × 10(4), 2.7 × 10(4), 133.3, and 46.7, respectively, and AChER were 0.07 × 10(4), 0.13 × 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.

Characterizations of cholinesterases in golden apple snail (Pomacea canaliculata).

Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.

Song bu li decoction, a traditional uyghur medicine, protects cell death by regulation of oxidative stress and differentiation in cultured PC12 cells.

Song Bu Li decoction (SBL) is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia) and heart disorders (arrhythmia and palpitation). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS) formation. The transcriptional activity of antioxidant response element (ARE), as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF-) induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress.

Kai-xin-san, a chinese herbal decoction containing ginseng radix et rhizoma, polygalae radix, acori tatarinowii rhizoma, and poria, stimulates the expression and secretion of neurotrophic factors in cultured astrocytes.

Kai-xin-san (KXS), a Chinese herbal decoction prescribed by Sun Simiao in Beiji Qianjin Yaofang about 1400 years ago, contains Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma, and Poria. In China, KXS has been used to treat stress-related psychiatric diseases with the symptoms of depression and forgetfulness. Although animal study has supported the antidepression function of KXS, the mechanism in cellular level is still unknown. Here, a chemically standardized water extract of KXS was applied onto cultured astrocytes in exploring the action mechanisms of KXS treatment, which significantly stimulated the expression and secretion of neurotrophic factors, including NGF, BDNF, and GDNF, in a dose-dependent manner: the stimulation was both in mRNA and protein levels. In addition, the water extracts of four individual herbs did not significantly stimulate the expression of neurotrophic factors, which could explain the optimized effect of KXS in a herbal decoction. The KXS-induced expression of neurotrophic factors did not depend on signaling mediated by estrogen receptor or protein kinase. The results suggested that the antidepressant-like action of KXS might be mediated by an increase of expression of neurotrophic factors in astrocytes, which fully supported the clinical usage of this decoction.

Flavonoids induce the expression of synaptic proteins, synaptotagmin, and postsynaptic density protein-95 in cultured rat cortical neuron.

Flavonoids, a family of phenolic compounds, are widely present in our daily diet and exist in traditional Chinese medicines, in which they act as the major active functional ingredients. Different lines of evidence indicate that flavonoids have positive impacts on human health. Here, different subclasses of flavonoids were analyzed for their inductive roles in promoting the expression of synaptic proteins, synaptotagmin, and post-synaptic density protein-95 in cultured rat cortical neurons. Among the screened 65 flavonoids, (-)-catechin, luteolin, and isorhamnetin, in micromolar concentration, were found to induce the expression of synaptic proteins in a dose-dependent manner: the induction values were from 2- to 8-fold that of the control. Similar results were revealed in the flavonoid-treated hippocampal neurons. The identification of these synapse-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

Yu Ping Feng San, an ancient Chinese herbal decoction containing Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix, regulates the release of cytokines in murine macrophages.

Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.