[Effect of Aralia echinocaulis containing serum on Wnt/ß-catenin signaling pathway of primary osteoblast].

This paper was aimed to investigate the effect of Aralia echinocaulis containing serum on expression of ß-catenin, Wnt-1, Frizzed-2, TCF and Axin in Wnt/ß-catenin signaling pathway of primary osteoblasts. SD healthy female rats (n=80) were used to make A. echinocaulis containing serum by gastric perfusion for seven days with distilled water, A. echinocaulis decoction high dosage, middle dosage, and low dosage. In vitro, primary osteoblasts were cultured and identified. The third generation primary osteoblasts were taken and cultured for 48 h, then cells were treated with the different drug serums for 10 days and calcified nodules were counted by alizarin red staining. The cells were collected after treatment for 48 h and the expression levels of ß-catenin, Wnt-1, Frizzled-2, TCF and Axin were detected by Real-time PCR and Western blot. The results suggested that the in vitro cells were primary osteoblasts; and after treatment, various doses groups could promote the mineralization ability of primary osteoblasts, up-regulate the mRNA and protein expression levels of ß-catenin, Wnt-1, Frizzled-2, and TCF, and down-regulate the mRNA and protein expression levels of Axin. These findings indicated that A. echinocaulis containing serum can enhance the differentiation and proliferation of osteoblasts by regulating the expression levels of ß-catenin, Wnt-1, Frizzled-2, TCF and Axin in Wnt/ß-catenin signaling pathway of primary osteoblasts.

Shared genetic architecture highlights the bidirectional association between major depressive disorder and fracture risk.

Background: There is limited evidence suggesting that osteoporosis might exacerbate depressive symptoms, while more studies demonstrate that depression negatively affects bone density and increases fracture risk. Aims: To explore the relationship between major depressive disorder (MDD) and fracture risk. Methods: We conducted a nested case-control analysis (32 670 patients with fracture and 397 017 individuals without fracture) and a matched cohort analysis (16 496 patients with MDD and 435 492 individuals without MDD) in the same prospective UK Biobank data set. Further, we investigated the shared genetic architecture between MDD and fracture with linkage disequilibrium score regression and the MiXeR statistical tools. We used the conditional/conjunctional false discovery rate approach to identify the specific shared loci. We calculated the weighted genetic risk score for individuals in the UK Biobank and logistic regression was used to confirm the association observed in the prospective study. Results: We found that MDD was associated with a 14% increase in fracture risk (hazard ratio (HR) 1.14, 95% CI 1.14 to 1.15, p<0.001) in the nested case-control analysis, while fracture was associated with a 72% increase in MDD risk (HR 1.72, 95% CI 1.64 to 1.79, p<0.001) in the matched cohort analysis, suggesting a longitudinal and bidirectional relationship. Further, genetic summary data suggested a genetic overlap between MDD and fracture. Specifically, we identified four shared genomic loci, with the top signal (rs7554101) near SGIP1. The protein encoded by SGIP1 is involved in cannabinoid receptor type 1 signalling. We found that genetically predicted MDD was associated with a higher risk of fracture and vice versa. In addition, we found that the higher expression level of SGIP1 in the spinal cord and muscle was associated with an increased risk of fracture and MDD. Conclusions: The genetic pleiotropy between MDD and fracture highlights the bidirectional association observed in the epidemiological analysis. The shared genetic components (such as SGIP1) between the diseases suggest that modulating the endocannabinoid system could be a potential therapeutic strategy for both MDD and bone loss.

TGFß1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice.

TGFß1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFß1+CCR5+) is the major source of TGFß1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFß1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFß1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFß1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFß1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFß1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFßRII specifically deleted in MPCs had lower numbers of TGFß1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFß1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Acellular nerve grafts supplemented with induced pluripotent stem cell-derived exosomes promote peripheral nerve reconstruction and motor function recovery.

Peripheral nerve injury is a great challenge in clinical work due to the restricted repair gap and weak regrowth ability. Herein, we selected induced pluripotent stem cells (iPSCs) derived exosomes to supplement acellular nerve grafts (ANGs) with the aim of restoring long-distance peripheral nerve defects. Human fibroblasts were reprogrammed into iPSCs through non-integrating transduction of Oct3/4, Sox2, Klf4, and c-Myc. The obtained iPSCs had highly active alkaline phosphatase expression and expressed Oct4, SSEA4, Nanog, Sox2, which also differentiated into all three germ layers in vivo and differentiated into mature peripheral neurons and Schwann cells (SCs) in vitro. After isolation and biological characteristics of iPSCs-derived exosomes, we found that numerous PKH26-labeled exosomes were internalized inside SCs through endocytotic pathway and exhibited a proliferative effect on SCs that were involved in the process of axonal regeneration and remyelination. After that, we prepared ANGs via optimized chemical extracted process to bridge 15 mm long-distance peripheral nerve gaps in rats. Owing to the promotion of iPSCs-derived exosomes, satisfactory regenerative outcomes were achieved including gait behavior analysis, electrophysiological assessment, and morphological analysis of regenerated nerves. Especially, motor function was restored with comparable to those achieved with nerve autografts and there were no significant differences in the fiber diameter and area of reinnervated muscle fibers. Taken together, our combined use of iPSCs-derived exosomes with ANGs demonstrates good promise to restore long-distance peripheral nerve defects, and thus represents a cell-free strategy for future clinical applications.

Morroniside ameliorates inflammatory skeletal muscle atrophy via inhibiting canonical and non-canonical NF-κB and regulating protein synthesis/degradation.

No drug options exist for skeletal muscle atrophy in clinical, which poses a huge socio-economic burden, making development on drug interventions a general wellbeing need. Patients with a variety of pathologic conditions associated with skeletal muscle atrophy have systemically elevated inflammatory factors. Morroniside, derived from medicinal herb Cornus officinalis, possesses anti-inflammatory effect. However, whether and how morroniside combat muscle atrophy remain unknown. Here, we identified crucial genetic associations between TNFα/NF-κB pathway and grip strength based on population using 377,807 European participants from the United Kingdom Biobank dataset. Denervation increased TNFα in atrophying skeletal muscles, which inhibited myotube formation in vitro. Notably, morroniside treatment rescued TNFα-induced myotube atrophy in vitro and impeded skeletal muscle atrophy in vivo, resulting in increased body/muscles weights, No. of satellite cells, size of type IIA, IIX and IIB myofibers, and percentage of type IIA myofibers in denervated mice. Mechanistically, in vitro and/or in vivo studies demonstrated that morroniside could not only inhibit canonical and non-canonical NF-κB, inflammatory mediators (IL6, IL-1b, CRP, NIRP3, PTGS2, TNFα), but also down-regulate protein degradation signals (Follistatin, Myostatin, ALK4/5/7, Smad7/3), ubiquitin-proteasome molecules (FoxO3, Atrogin-1, MuRF1), autophagy-lysosomal molecules (Bnip3, LC3A, and LC3B), while promoting protein synthesis signals (IGF-1/IGF-1R/IRS-1/PI3K/Akt, and BMP14/BMPR2/ALK2/3/Smad5/9). Moreover, morroniside had no obvious liver and kidney toxicity. This human genetic, cells and mice pathological evidence indicates that morroniside is an efficacious and safe inflammatory muscle atrophy treatment and suggests its translational potential on muscle wasting.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).

TNF-Polarized Macrophages Produce Insulin-like 6 Peptide to Stimulate Bone Formation in Rheumatoid Arthritis in Mice.

The risk of osteoporosis is increased in rheumatoid arthritis (RA). Anti-tumor necrosis factor (TNF) therapy has markedly improved the outcomes of RA patients but does not improve osteoporosis in some reports. This could be a combined result of disease severity and other therapeutic agents, such as glucocorticoids that accelerate osteoporosis progression. We evaluated the effects of anti-TNF therapy on osteoporosis in an animal model of RA and explored the possible mechanisms involved. Six-week-old TNF transgenic (TNF-Tg) mice with early stage erosive arthritis were treated with TNF antibody (Ab) or control immunoglobulin (IgG) weekly for 4 weeks. We found that TNF Ab completely blocked the development of erosive arthritis in TNF-Tg mice, but only slightly increased vertebral bone mass, associated with reduction in parameters of both bone resorption and formation. Similarly, TNF Ab slightly increased trabecular bone mass in tibias of 8-month-old TNF-Tg mice with advanced erosive arthritis. Interestingly, TNFα increased osteoblast differentiation from mouse bone marrow stromal cells (BMSCs) containing large number of macrophages but not from pure mesenchymal progenitor cells (MPCs). TNFα-polarized macrophages (TPMs) did not express iNos and Arginase 1, typical markers of inflammatory and resident macrophages. Interestingly, TPMs stimulated osteoblast differentiation, unlike resident and inflammatory macrophages polarized by IL-4 and interferon-λ, respectively. RNA-seq analysis indicated that TPMs produced several anabolic factors, including Jagged1 and insulin like 6 (INSL6). Importantly, inhibition of either Jagged1 or INSL6 blocked TNFα-induced osteoblast differentiation. Furthermore, INSL6 Ab significantly decreased the expansion of TNF-induced MPCs in BMSCs, and anti-TNF Ab reduced INSL6 expression by macrophages in vitro and in TNF-Tg mice in vivo. We conclude that TPMs produce INSL6 to stimulate bone formation and anti-TNF Ab blocks not only enhanced bone resorption but also the anabolic effect of TPMs on bone, limiting its effect to increase bone mass in this model of RA. © 2021 American Society for Bone and Mineral Research (ASBMR).

TNF Receptor-Associated Factor 6 Mediates TNFα-Induced Skeletal Muscle Atrophy in Mice During Aging.

During aging, muscle mass decreases, leading to sarcopenia, associated with low-level chronic inflammation (inflammaging), which induces sarcopenia by promoting proteolysis of muscle fibers and inhibiting their regeneration. Patients with a variety of pathologic conditions associated with sarcopenia, including rheumatoid arthritis (RA), have systemically elevated TNFα serum levels, and transgenic mice with TNFα overexpression (TNF-Tg mice, a model of RA) develop sarcopenia between adolescence and adulthood before they age. However, if and how TNFα contributes to the pathogenesis of sarcopenia during the normal aging process and in RA remains largely unknown. We report that TNFα levels are increased in skeletal muscles of aged WT mice, associated with muscle atrophy and decreased numbers of satellite cells and Type IIA myofibers, a phenotype that we also observed in adult TNF-Tg mice. Aged WT mice also have increased numbers of myeloid lineage cells in their skeletal muscles, including macrophages and granulocytes. These cells have increased TNFα expression, which impairs myogenic cell differentiation. Expression levels of TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase, which mediates signaling by some TNF receptor (TNFR) family members, are elevated in skeletal muscles of both aged WT mice and adult TNF-Tg mice. TRAF6 binds to TNFR2 in C2C12 myoblasts and mediates TNFα-induced muscle atrophy through NF-κB-induced transcription of the muscle-specific E3 ligases, Atrogen1 and Murf1, which promote myosin heavy-chain degradation. Haplo-deficiency of TRAF6 prevents muscle atrophy and the decrease in numbers of satellite cells, Type IIA myofibers, and myogenic regeneration in TRAF6+/- ;TNF-Tg mice. Our findings suggest that pharmacologic inhibition of TRAF6 signaling in skeletal muscles during aging could treat/prevent age- and RA-related sarcopenia by preventing TNFα-induced proteolysis and inhibition of muscle fiber regeneration. © 2020 American Society for Bone and Mineral Research.