Studies on the in vitro mechanism and in vivo therapeutic effect of the antimicrobial peptide ACP5 against Trichophyton mentagrophytes.

Trichophyton mentagrophytes is a zoophilic dermatophyte that can cause dermatophytosis in humans and animals. Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of T. mentagrophytes. Our findings suggest that cationic antimicrobial peptide (ACP5) not only possesses stronger activity against T. mentagrophytes than fluconazole, but also shows lower toxicity to L929 mouse fibroblast cells than terbinafine. Notably, its resistance development rate after resistance induction was lower than terbinafine. The present study aimed to evaluate the fungicidal mechanism of ACP5 in vitro and its potential to treat dermatophyte infections in vivo. ACP5 at 1 ×MIC completely inhibited T. mentagrophytes spore germination in vitro. ACP5 severely disrupts the mycelial morphology, leading to mycelial rupture. Mechanistically, ACP5 induces excessive ROS production, damaging the integrity of the cell membrane and decreasing the mitochondrial membrane potential, causing irreversible damage in T. mentagrophytes. Furthermore, 1% ACP5 showed similar efficacy to the commercially available drug 1% terbinafine in a guinea pig dermatophytosis model, and the complete eradication of T. mentagrophytes from the skin by ACP5 was verified by tissue section observation. These results indicate that ACP5 is a promising candidate for the development of new agent to combat dermatophyte resistance.

Interleukin-21 knockout reduces bone loss in ovariectomized mice by inhibiting osteoclastogenesis.

Estrogen deficiency accelerates osteoporosis in elderly women. However, the role of IL-21 in postmenopausal osteoporosis remains unclear. Female wild-type (WT) C57BL/6 and IL-21 knockout (KO) mice were used for ovariectomy (OVX). Here, IL-21 levels were significantly increased in the serum and bone tissues of WT-OVX mice. The trabecular bone space of the femur was significantly increased, and the bone mass was reduced in OVX mice, accompanied by a significant decrease in the maximum load, energy absorption, and elastic modulus indices. In contrast, IL-21 knockout effectively alleviated the effects of OVX on bone mass. Serum TRACP-5b and receptor activator of nuclear factor kappa B ligand (RANKL) levels and osteoclastogenesis were significantly higher in OVX mice than in sham mice, while serum TRACP-5b and RANKL levels and osteoclastogenesis were significantly decreased in IL-21 KO + OVX mice compared to WT + OVX mice. IL-21 knockdown reduces TRACP-5b, RANKL, and osteoclastogenesis, effectively preventing bone resorption and alleviating the progression of OVX-induced osteoporosis.

Leptin and melatonin's effects on OVX rodents' bone metabolism.

Purpose: This study aims to examine the effects of leptin and melatonin intervention on bone metabolism in ovariectomize (OVX) rodents, as well as their potential mechanisms of action. Methods: Prepare an OVX model of osteoporosis in rodents and validate the model by collecting bilateral tibia samples for Micro-CT scanning and histological analysis. A control group of normal size, the OVX group, the OVX+Sema4D (Semaphorin 4D) group, the OVX+Sema4D+Leptin group, the OVX+Sema4D+ Melatonin(MT) group and the OVX+Sema4D+Leptin+ MT group were the experimental groups. Adenovirus vector construction and tibial medullary injection validation were conducted in accordance with the aforementioned experimental groups. Four groups of rats were injected with the Sema4D overexpression adenovirus vector into the tibial medullary cavity, and two groups were injected with the Leptin overexpression adenovirus vector. The repair of osteoporosis was observed using micro-CT and histological analysis. Immunohistochemical detection of bone morphogenetic protein-2 (BMP-2) expression in bone tissue was employed to ascertain the amount of osteoclasts in the upper tibial metaphysis, utilizing TRAP(tartrate-resistant acid phosphatase) staining. Results: Increased levels of BV/TV, Tb.N, BMD, and BMC were seen in the OVX+ Sema4D+Leptin, OVX+ Sema4D+MT, and OVX+ Sema4D+Leptin+ MT groups compared to the OVX group, whereas Tb. Sp levels were lowered. When compared to the Sema4D overexpression group, the trabecular bone structure of the OVX + Sema4D + Leptin, OVX + Sema4D + MT, and OVX + Sema4D + Leptin + MT groups is largely intact, tends to be closer, and the amount of trabecular bone increases. The OVX + Sema4D + Leptin + MT group in particular.The expression of BMP-2 was dramatically upregulated (p<0.05), the number of TRAP-stained osteoclasts was significantly reduced (p<0.05), and BALP(bone-derived alkaline phosphatase) and TRAP-5b(tartrate-resistant acid phosphatase-5b) activities were significantly downregulated (p<0.05). Conclusion: In rats with osteoporosis, leptin and melatonin can be seen to augment the trabecular microstructure of the bone, augment bone growth, diminish trabecular harm, and mend the bone. The combined effect is more powerful.

An in vitro model for discovery of osteoclast specific biomarkers towards identification of racehorses at risk for catastrophic fractures.

BACKGROUND: Focal bone microcracks with osteoclast recruitment and bone lysis, may reduce fracture resistance in racehorses. As current imaging does not detect all horses at risk for fracture, the discovery of novel serum biomarkers of bone resorption or osteoclast activity could potentially address this unmet clinical need. The biology of equine osteoclasts on their natural substrate, equine bone, has never been studied in vitro and may permit identification of specific biomarkers of their activity. OBJECTIVES: (1) Establish osteoclast cultures on equine bone, (2) Measure biomarkers (tartrate resistant acid phosphatase isoform 5b [TRACP-5b] and C-terminal telopeptide of type I collagen [CTX-I]) in vitro and (3) Study the effects of inflammation. STUDY DESIGN: In vitro experiments. METHODS: Haematopoietic stem cells, from five equine sternal bone marrow aspirates, were differentiated into osteoclasts and cultured either alone or on equine bone slices, with or without a pro-inflammatory stimulus (IL-1ß or LPS). CTX-I and TRACP-5b were immunoassayed in the media. Osteoclast numbers and bone resorption area were assessed. RESULTS: TRACP-5b increased over time in osteoclast cultures without bone (p < 0.0001) and correlated with osteoclast number (r = 0.63, p < 0.001). CTX-I and TRACP-5b increased with time for cultures with bone (p = 0.002; p = 0.02 respectively), correlated with each other (r = 0.64, p < 0.002) and correlated with bone resorption (r = 0.85, p < 0.001; r = 0.82, p < 0.001 respectively). Inflammation had no measurable effects. MAIN LIMITATIONS: Specimen numbers limited. CONCLUSIONS: Equine osteoclasts were successfully cultured on equine bone slices and their bone resorption quantified. TRACP-5b was shown to be a biomarker of equine osteoclast number and bone resorption for the first time; CTX-I was also confirmed to be a biomarker of equine bone resorption in vitro. This robust equine specific in vitro assay will help the study of osteoclast biology.

Isoliquiritigenin Decreases Bone Resorption and Osteoclast Differentiation.

SCOPE: A dose-ranging study is performed using young estrogen-depleted rats to determine whether dietary isoliquiritigenin (ILQ) alters bone metabolism and if the effects are associated with estrogen receptor signaling. METHODS AND RESULTS: Six-week-old rats (ovariectomized at 4 weeks of age) are fed diets containing 0, 100, 250, or 750 ppm ILQ (n = 5/treatment) for 7 days. Gene expression in femur and uterus, blood markers of bone turnover, body composition, and uterine weight and epithelial cell height are determined. Because ILQ lowers bone resorption, the effect of ILQ on in vitro differentiation of osteoclasts from bone marrow of mice is assessed. Treatment resulted in a dose-dependent increases in serum ILQ but no changes in serum osteocalcin, a marker of global bone formation. Contrastingly, ILQ administration results in reduced serum CTX-1, a marker of global bone resorption, and reduces tartrate resistant acid phosphatase expression in osteoclast culture. ILQ treatment and endogenous estrogen production had limited overlap on gene expression in femur and uterus. However, uterine epithelial cell hyperplasia is observed in two of five animals treated with 750 ppm. CONCLUSIONS: In conclusion, dietary ILQ reduces bone resorption in vivo and osteoclast differentiation in vitro, by mechanisms likely differing from actions of ovarian hormones.

Effect of Magnetic Nanoparticles on Hormone Level Changes During Perimenopausal Period and Regulation of Bone Metabolism.

This work aimed to explore the effect of nerve magnetic stimulation based on superparamagnetic Fe3O4 nanoparticle (NP) on bone metabolism during the perimenopausal period. First, the multifunctional water-soluble polymer PTMP-PMAA was utilized as the ligand. PTMP-MAA@ Fe3O4 NP with high magnetization was prepared by the co-precipitation method, and NPX diffraction pattern analysis and in vitro stability analysis were implemented. Then, NPs were co-cultured with 293T cells, and the cytotoxicity was detected by the CCK-8 method. Subsequently, 3-month-old female young SD rats and 11~15-month-old natural menopausal SD rats were taken as the research objects. According to the vaginal smear, the rats were randomly rolled into a young control, perimenopausal period model, estrogen treatment, and osteoporosis prevention groups. Rats in the estrogen treatment group were given Premarin suspension by gavage. Rats in the osteoporosis prevention group were injected stereotaxically with PTMP-MAA@ Fe3O4 NP suspension, and a rotating magnetic field was applied to the brain for nerve magnetic stimulation. The rats were sacrificed three days after treatment and brain tissues were taken for pathological analysis. Rat humerus was weighted and dual-energy X-ray was utilized to determine bone density and bone mineral content. Serum was collected and radioimmunoassay and ELISA were employed to detect estradiol (E2), osteocalcin (Boneglaprotein, BGP), oxytocin (OT), bone alkaline phosphatase (BALP), type I collagen carboxy-terminated cross-linked peptide (CTX-I), and tartrate-resistant acid phosphatase (TRACP-5b) in the serum of rats in each group. The results showed that PTMP-MAA@ Fe3O4 NP had good biocompatibility, and the CCK-8 test results showed that PTMP-MAA@ Fe3O4 NP had low cytotoxicity. Compared with the young control group, the humeral dry weight, wet weight, bone density, and bone mineral content, serum E2, OT, and BGP content in the perimenopausal period model group were reduced, while the serum BALP, CTX-I, and TRACP -5b content was increased (P<0.05). It was verified that nerve magnetic stimulation based on PTMP-MAA@ Fe3O4 NP increased the serum estrogen level of female rats during the perimenopausal period, increased the bone density of rats, promoted bone formation, and regulated bone metabolism.

Effects of Cynanchum wilfordii on osteoporosis with inhibition of bone resorption and induction of bone formation.

Cynanchum wilfordii Hemsley has been used for the treatment of musculoskeletal diseases in traditional Republic of Korean medicine. The present study investigated the effects of C. wilfordii water extract (CW) on postmenopausal osteoporosis. Female mice were used and randomly assigned into a normal group and three ovariectomized (OVX) groups: OVX with vehicle (OVX + vehicle); OVX with 17ß­estradiol (E2; 10 µg/kg/day); and OVX with CW (1 mg/kg/day). Oral administration of CW or E2 intraperitoneal injection began 9 weeks after OVX and continued for 3 weeks. Following sacrifice, bone histology, bone mineral density (BMD) and bone mineral content (BMC) of the femur were observed. Serum osteocalcin concentration was analyzed. In addition, the expression levels of osteoprotegerin (OPG) and osterix were evaluated in human osteoblast­like Saos­2 cells. In the lateral and medial epicondyles of the CW­administrated group, dense and well­formed bone marrow cells with reduced bone marrow pores were observed. CW decreased the number of tartrate resistant acid phosphatase­positive multinucleated osteoclasts. BMD and BMC were increased following increased serum osteocalcin levels by CW treatment. The expression levels of OPG and osterix were upregulated by CW treatment in vitro. The results suggested that C. wilfordii has an advantageous effect on osteoporosis and possesses the potential to be used in osteoporosis treatment.

Effects of calcitonin on orthodontic tooth movement and associated root resorption in rats.

OBJECTIVES: Our main aim was to evaluate the effects of calcitonin (CT) on orthodontic tooth movement (OTM) and orthodontic root resorption in a rat model. MATERIAL AND METHODS: Eighty male Wistar rats were randomly divided into five groups. Rats in the negative control group were not given any appliances or injections. All the remaining rats were used to establish a model of OTM. The positive control group were then injected with normal saline, while rats in the three experimental groups were injected with 0.2 IU, 1 IU or 5 IU/kg/day CT. Nickel-titanium closed-coil springs were used to deliver an initial 50 g mesial force to the left maxillary first molar for 14 days in rats in the positive control group and the experimental groups. Each group was randomly subdivided into two groups, one for analysis of tooth movement, tissue changes and tartrate-resistant acid phosphatase (TRAP)-positive cells in alveolar bone, the other to examine root resorption by scanning electron microscopy. RESULTS: The OTM distance, the number of force-induced osteoclasts and root resorption areas were significantly decreased in CT-injected rats in a dose-dependent manner. CONCLUSIONS: Administration of CT reduces the root resorption area and may therefore be effective as a novel adjunctive orthodontic approach to diminish undesired tooth movement via enhancing anchorage or preventing relapse after OTM.

Effects of antiplatelet therapy on platelet extracellular vesicle release and procoagulant activity in health and in cardiovascular disease.

Dual antiplatelet therapy with aspirin and clopidogrel is commonly used to prevent recurrent ischemic events in patients with cardiovascular disease. Whilst their effects on platelet reactivity are well documented, it is unclear, however, whether antiplatelet therapy inhibits platelet extracellular vesicle (EV) release. The aim of this study was to investigate the effects of antiplatelet therapy on platelet EV formation and procoagulant activity. Blood samples from 10 healthy controls not receiving antiplatelet therapy were incubated in vitro with aspirin or a P2Y12 inhibitor (MeSAMP). Blood samples from 50 patients receiving long-term dual antiplatelet therapy and undergoing coronary angiography were also studied. Platelet reactivity was assessed by Multiplate™ impedance aggregometry. Platelet EV formation and procoagulant activity of pretreated and untreated blood samples in response to arachidonic acid (AA), adenosine diphosphate (ADP), ADP+PGE1, and thrombin receptor-activating peptide (TRAP) stimulation were assessed by flow cytometry and Procoag-PL assays, respectively. Incubation of normal platelets with aspirin significantly inhibited AA-induced platelet reactivity, EV formation, and procoagulant activity, whilst MeSAMP significantly inhibited platelet reactivity and EV formation in response to AA, ADP, and TRAP, but had minimal effect on procoagulant activity. Most patients receiving dual antiplatelet therapy showed an appropriate reduction in platelet reactivity in response to their treatment; however there was not complete inhibition of increased platelet and EV procoagulant activity in response to ADP, AA, or TRAP. In addition, we could not find any correlation between platelet reactivity and procoagulant activity in patients receiving dual antiplatelet therapy.