Osteocyte mitochondria inhibit tumor development via STING-dependent antitumor immunity.

Bone is one of the most common sites of tumor metastases. During the last step of bone metastasis, cancer cells colonize and disrupt the bone matrix, which is maintained mainly by osteocytes, the most abundant cells in the bone microenvironment. However, the role of osteocytes in bone metastasis is still unclear. Here, we demonstrated that osteocytes transfer mitochondria to metastatic cancer cells and trigger the cGAS/STING-mediated antitumor response. Blocking the transfer of mitochondria by specifically knocking out mitochondrial Rho GTPase 1 (Rhot1) or mitochondrial mitofusin 2 (Mfn2) in osteocytes impaired tumor immunogenicity and consequently resulted in the progression of metastatic cancer toward the bone matrix. These findings reveal the protective role of osteocytes against cancer metastasis by transferring mitochondria to cancer cells and potentially offer a valuable therapeutic strategy for preventing bone metastasis.

KERS-Inspired Nanostructured Mineral Coatings Boost IFN-γ mRNA Therapeutic Index for Antitumor Immunotherapy.

Tumor-associated macrophage (TAM) reprogramming is a promising therapeutic approach for cancer immunotherapy; however, its efficacy remains modest due to the low bioactivity of the recombinant cytokines used for TAM reprogramming. mRNA therapeutics are capable of generating fully functional proteins for various therapeutic purposes but accused for its poor sustainability. Inspired by kinetic energy recovery systems (KERS) in hybrid vehicles, a cytokine efficacy recovery system (CERS) is designed to substantially augment the therapeutic index of mRNA-based tumor immunotherapy via a "capture and stabilize" mechanism exerted by a nanostructured mineral coating carrying therapeutic cytokine mRNA. CERS remarkably recycles nearly 40% expressed cytokines by capturing them onto the mineral coating to extend its therapeutic timeframe, further polarizing the macrophages to strengthen their tumoricidal activity and activate adaptive immunity against tumors. Notably, interferon-γ (IFN-γ) produced by CERS exhibits ≈42-fold higher biological activity than recombinant IFN-γ, remarkably decreasing the required IFN-γ dosage for TAM reprogramming. In tumor-bearing mice, IFN-γ cmRNA@CERS effectively polarizes TAMs to inhibit osteosarcoma progression. When combined with the PD-L1 monoclonal antibody, IFN-γ cmRNA@CERS significantly boosts antitumor immune responses, and substantially prevents malignant lung metastases. Thus, CERS-mediated mRNA delivery represents a promising strategy to boost antitumor immunity for tumor treatment.

An On-Demand Collaborative Innate-Adaptive Immune Response to Infection Treatment.

Deep tissue infection is a common clinical issue and therapeutic difficulty caused by the disruption of the host antibacterial immune function, resulting in treatment failure and infection relapse. Intracellular pathogens are refractory to elimination and can manipulate host cell biology even after appropriate treatment, resulting in a locoregional immunosuppressive state that leads to an inadequate response to conventional anti-infective therapies. Here, a novel antibacterial strategy involving autogenous immunity using a biomimetic nanoparticle (NP)-based regulating system is reported to induce in situ collaborative innate-adaptive immune responses. It is observed that a macrophage membrane coating facilitates NP enrichment at the infection site, followed by active NP accumulation in macrophages in a mannose-dependent manner. These NP-armed macrophages exhibit considerably improved innate capabilities, including more efficient intracellular ROS generation and pro-inflammatory factor secretion, M1 phenotype promotion, and effective eradication of invasive bacteria. Furthermore, the reprogrammed macrophages direct T cell activation at infectious sites, resulting in a robust adaptive antimicrobial immune response to ultimately achieve bacterial clearance and prevent infection relapse. Overall, these results provide a conceptual framework for a novel macrophage-based strategy for infection treatment via the regulation of autogenous immunity.

3D-printed hydrogel particles containing PRP laden with TDSCs promote tendon repair in a rat model of tendinopathy.

Long-term chronic inflammation after Achilles tendon injury is critical for tendinopathy. Platelet-rich plasma (PRP) injection, which is a common method for treating tendinopathy, has positive effects on tendon repair. In addition, tendon-derived stem cells (TDSCs), which are stem cells located in tendons, play a major role in maintaining tissue homeostasis and postinjury repair. In this study, injectable gelatine methacryloyl (GelMA) microparticles containing PRP laden with TDSCs (PRP-TDSC-GM) were prepared by a projection-based 3D bioprinting technique. Our results showed that PRP-TDSC-GM could promote tendon differentiation in TDSCs and reduce the inflammatory response by downregulating the PI3K-AKT pathway, thus promoting the structural and functional repair of tendons in vivo.

GSTP1-mediated S-glutathionylation of Pik3r1 is a redox hub that inhibits osteoclastogenesis through regulating autophagic flux.

Glutathione S-transferase P1(GSTP1) is known for its transferase and detoxification activity. Based on disease-phenotype genetic associations, we found that GSTP1 might be associated with bone mineral density through Mendelian randomization analysis. Therefore, this study was performed both in vitro cellular and in vivo mouse model to determine how GSTP1 affects bone homeostasis. In our research, GSTP1 was revealed to upregulate the S-glutathionylation level of Pik3r1 through Cys498 and Cys670, thereby decreasing its phosphorylation, further controlling the alteration of autophagic flux via the Pik3r1-AKT-mTOR axis, and lastly altering osteoclast formation in vitro. In addition, knockdown and overexpression of GSTP1 in vivo also altered bone loss outcomes in the OVX mice model. In general, this study identified a new mechanism by which GSTP1 regulates osteoclastogenesis, and it is evident that the cell fate of osteoclasts is controlled by GSTP1-mediated S-glutathionylation via a redox-autophagy cascade.

Synergistic Effect of Reverse Drilling and Bone Dust on Femoral Tendon-Bone Healing After Anterior Cruciate Ligament Reconstruction in a Rabbit Model.

BACKGROUND: Anterior cruciate ligament (ACL) injuries and bone tunnel enlargement (BTE) after ACL reconstruction (ACLR) remain frequent issues. Bone dust (BD) produced by tunnel preparation with osteogenic ability and reverse drilling (RD), an easy compaction technique, make it accessible to enhance tendon-bone healing in the clinic. HYPOTHESIS: RD and BD synergistically promote tendon-bone healing by improving peritunnel bone and preventing BTE in femurs. STUDY DESIGN: Controlled laboratory study. METHODS: In total, 96 New Zealand White rabbits underwent ACLR. The semitendinosus tendon was freed before medial parapatellar arthrotomy. After the native ACL was transected, bone tunnels were prepared through the footprint of the native ACL. All animals were randomly assigned to 1 of 4 groups according to different tunnel preparation methods: group 1 (irrigation after extraction drilling [ED]; control group), group 2 (irrigation after RD), group 3 (no irrigation after ED), and group 4 (no irrigation after RD). BD was harvested by irrigating tunnels and was characterized by morphology and size. The specimens underwent microarchitectural, histological, and biomechanical evaluations at 4, 8, and 12 weeks postoperatively. RESULTS: Micro-computed tomography demonstrated more peritunnel bone and less BTE in the femurs of group 4 compared with the other groups. Histologically, BD possessed osteogenic activity in bone tunnels postoperatively. Meanwhile, group 4 regenerated a higher amount of the tendon-bone interface and more peritunnel bone than group 1. Biomechanically, group 4 showed higher failure loads and stiffness than group 1. However, peritunnel bone loss, active osteoclasts, and significant BTE were found in the femurs of group 1 and group 3 at 12 weeks postoperatively, while no strong correlation was found between BTE and inflammatory cytokines. Scanning electron microscopy and particle size analysis suggested that BD produced by ED and RD had no difference in size. CONCLUSION: Tendon-bone healing was facilitated by the synergistic effect of RD and BD in femurs. CLINICAL RELEVANCE: This study provides a more accessible and effective surgical strategy to promote tendon-bone healing after ACLR by increasing peritunnel bone and preventing BTE in femurs.

Norcantharidin ameliorates estrogen deficient-mediated bone loss by attenuating the activation of extracellular signal-regulated kinase/ROS/NLRP3 inflammasome signaling.

Osteoporosis, characterized by reduced bone mass, aberrant bone architecture, and elevated bone fragility, is driven by a disruption of bone homeostasis between bone resorption and bone formation. However, up to now, no drugs are perfect for osteoporosis treatment due to different defects. In this study, we demonstrated that norcantharidin (NCTD) could inhibit osteoclast formation and bone resorption by attenuating the ERK, ROS and NLRP3 inflammasomes pathways in vitro. Moreover, our in vivo study further confirms its preventive effects on estrogen-deficiency bone loss by inhibiting osteoclast formation and functions. Therefore, we could conclude that NCTD might be a potential candidates for the prevention and treatment of osteoporosis.

Cepharanthine Ameliorates Chondrocytic Inflammation and Osteoarthritis via Regulating the MAPK/NF-κB-Autophagy Pathway.

Osteoarthritis is a worldwide joint disease caused by abnormal chondrocytic metabolism. However, traditional therapeutic methods aimed at anti-inflammation for early-stage disease are palliative. In the present study, we demonstrated that cepharanthine (CEP), extracted from the plant Stephania cepharantha, exerted protective medicinal efficacy on osteoarthritis for the first time. In our in vitro study, CEP suppressed the elevated expression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and inducible nitric oxide synthase (iNOS) stimulated by IL-1ß or TNF-α by inhibiting the activation of MAPK and NF-κB signaling pathways, and upregulated the protein expression of aggrecan, collagen II, and Sox9. Also, CEP could reverse the reduced level of cellular autophagy in IL-1ß or TNF-α-induced chondrocytes, indicating that the protective effect of CEP on osteoarthritis was achieved by restoring MAPK/NF-κB-mediated autophagy. Furthermore, in a murine OA model, CEP mitigated cartilage degradation and prevented osteoarthritis in the CEP-treated groups versus the OA group. Hence, our results revealed the therapeutic prospect of CEP for anti-osteoarthritic treatment.

Additive Effect of Parathyroid Hormone and Zoledronate Acid on Prevention Particle Wears-Induced Implant Loosening by Promoting Periprosthetic Bone Architecture and Strength in an Ovariectomized Rat Model.

Implant-generated particle wears are considered as the major cause for the induction of implant loosening, which is more susceptible to patients with osteoporosis. Monotherapy with parathyroid hormone (PTH) or zoledronate acid (ZOL) has been proven efficient for preventing early-stage periprosthetic osteolysis, while the combination therapy with PTH and ZOL has exerted beneficial effects on the treatment of posterior lumbar vertebral fusion and disuse osteopenia. However, PTH and ZOL still have not been licensed for the treatment of implant loosening to date clinically. In this study, we have explored the effect of single or combined administration with PTH and ZOL on implant loosening in a rat model of osteoporosis. After 12 weeks of ovariectomized surgery, a femoral particle-induced periprosthetic osteolysis model was established. Vehicle, PTH (5 days per week), ZOL (100 mg/kg per week), or combination therapy was utilized for another 6 weeks before sacrifice, followed by micro-CT, histology, mechanical testing, and bone turnover examination. PTH monotherapy or combined PTH with ZOL exerted a protective effect on maintaining implant stability by elevating periprosthetic bone mass and inhibiting pseudomembrane formation. Moreover, an additive effect was observed when combining PTH with ZOL, resulting in better fixation strength, higher periprosthetic bone mass, and less pseudomembrane than PTH monotherapy. Taken together, our results suggested that a combination therapy of PTH and ZOL might be a promising approach for the intervention of early-stage implant loosening in patients with osteoporosis.

Renoprotective mechanisms of morin in cisplatin-induced kidney injury.

In this study, we investigated the renoprotective effects of morin on cisplatin-induced kidney injury in mice. Serum creatinine and blood urea nitrogen (BUN) levels, glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) activities were determined according to the corresponding kits. The mRNA levels of TNF-α and IL-1ß in kidney tissues were measured by quantitative real-time PCR (qRT-PCR). The activities of cytochrome P450 2E1 (CYP2E1), nuclear factor kappa B (NF-κB) p65, P38 mitogen-activated protein kinase (MAPK), Bax, p53 and cleaved caspase 3 were evaluated by western blotting. The results showed that the model of cisplatin-induced kidney injury was successfully replicated, and morin significantly attenuated histopathological changes and decreased the levels of TNF-α and IL-1ß in the kidneys. In addition, morin attenuated the activation of CYP2E1, phospho-NF-κB p65, phospho-P38 MAPK, Bax, phospho-p53 and cleaved caspase 3 in CP-induced kidney injury. In conclusion, these results indicated that the renoprotective mechanisms of morin may be attributed to the suppression of oxidative stress, inflammation and apoptosis in CP-induced kidney injury.

Inhibition of Lipopolysaccharide (LPS)-induced inflammatory responses by selenium in bovine mammary epithelial cells in primary culture.

Selenium, in the form of selenoproteins, plays a pivotal role in anti-inflammatory processes and antioxidant defense system. The aim of this study was to examine the effects of selenium on lipopolysaccharide (LPS)-induced inflammatory responses in bovine mammary epithelial cells (bMEC) and to investigate the potential mechanism. bMEC viability was measured by MTT assay. TNF-α, IL-1ß, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) expressions were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The activation of nuclear factor-kappa B (NF-κB) was determined by Western blotting. The results showed that the mRNA expressions of these inflammatory factors were significantly inhibited by selenium in a dose-dependent manner. At protein levels, Western blot analysis demonstrated that selenium dose-dependently decreased NF-κB p65 translocating from the cytoplasm to the nucleus. Taken together, these results suggest that the anti-inflammatory property of selenium in LPS-stimulated primary bMEC may be attributed to the downregulation of NF-κB activation.

Niacin attenuates the production of pro-inflammatory cytokines in LPS-induced mouse alveolar macrophages by HCA2 dependent mechanisms.

Niacin has been reported to have potent anti-inflammatory effects in LPS-induced acute lung injury. However, the molecular mechanism of niacin has not been fully understood. The aim of the present study was to investigate the effects of niacin on the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß in LPS-induced mouse alveolar macrophages and explore its underlying mechanism. Mouse alveolar macrophages were incubated in the presence or absence of various concentrations of niacin (1, 10, 100 µmol/l) 1h before LPS (1 µg/ml) challenge. The results showed that niacin reduced the levels of TNF-α, IL-6 and IL-1ß in LPS-challenged alveolar macrophages. Furthermore, NF-κB activation was inhibited by niacin through blocking the phosphorylation of NF-κB p65 and IκBα. In addition, silencing HCA2 abrogated the effect of niacin on the production of pro-inflammatory cytokines. These findings suggested that niacin attenuated the LPS-induced pro-inflammatory cytokines possibly mediated by HCA2 in LPS-challenged alveolar macrophages.

Dietary selenium deficiency exacerbates lipopolysaccharide-induced inflammatory response in mouse mastitis models.

Selenium (Se) is an essential micronutrient that plays a critical role in anti-inflammatory processes and antioxidant defense system. In this study, we investigated the effects of dietary selenium deficiency on lipopolysaccharide (LPS)-induced mastitis in mouse models. Se content in the liver was assessed by fluorescent atomic absorption spectrometry. Glutathione peroxidase (GPx) activity in the blood, myeloperoxidase (MPO) activity, tumor necrosis actor alpha (TNF-α), and interleukin (IL)-1ß in the supernatant of the mammary tissue were determined according to the corresponding kits. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions were evaluated by Western blotting. The results showed that the Se-deficient mouse model was successfully replicated, and selenium deficiency exacerbated mammary gland histopathology, increased the expressions of TNF-α and IL-1ß, and facilitated the activation of iNOS and COX-2 in LPS-induced mouse mastitis. In conclusion, our studies demonstrated that selenium deficiency resulted in more severe inflammatory response in LPS-induced mouse mastitis.

Effects of niacin on Staphylococcus aureus internalization into bovine mammary epithelial cells by modulating NF-κB activation.

Niacin is a precursor of coenzymes NAD and NADP and plays a critical role in electron transfer during the metabolic process. In addition to its nutrimental function, niacin has long been used for the treatment of lipid disorders and cardiovascular disease. However, the effect of niacin on Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMEC) remains unclear. Here we sought to examine the effect of niacin on S. aureus internalization into bovine mammary epithelial cells (bMEC) and to investigate the potential mechanism. In this study, the growth of S. aureus supplemented with niacin (0.5-2 mM) was monitored turbidimetrically at 600 nm for 24 h and cell viability was measured by MTT assay. Gentamicin protection assay was carried out to determine the effect of niacin on S. aureus internalization into bMEC. To determine the potential mechanism, tracheal antimicrobial peptide (TAP) and ß-defensin (BNBD5) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The activation of nuclear factor-kappa B (NF-κB) was determined by Western blotting. The results showed that niacin (0.5-2 mM) did not affect S. aureus growth and bMEC viability, whereas it inhibits S. aureus internalization ranging from 13% to 42% and down-regulated the mRNA expression of TAP and BNBD5 compared to the control group. No exactly relationship was discovered between S. aureus internalization into bMEC and antimicrobial peptide expression, while niacin inhibited S. aureus-induced NF-κB activation in a dose manner. These dates suggest that inhibiting NF-κB activation may be the potential mechanism of niacin on modulating S. aureus internalization into bMEC.

Thymol inhibits Staphylococcus aureus internalization into bovine mammary epithelial cells by inhibiting NF-κB activation.

Bovine mastitis is one of the most costly and prevalent diseases in the dairy industry and is characterised by inflammatory and infectious processes. Staphylococcus aureus (S. aureus), a Gram-positive organism, is a frequent cause of subclinical, chronic mastitis. Thymol, a monocyclic monoterpene compound isolated from Thymus vulgaris, has been reported to have antibacterial properties. However, the effect of thymol on S. aureus internalization into bovine mammary epithelial cells (bMEC) has not been investigated. In this study, we evaluated the effect of thymol on S. aureus internalization into bMEC, the expression of tracheal antimicrobial peptide (TAP) and ß-defensin (BNBD5), and the inhibition of NF-κB activation in bMEC infected with S. aureus. Our results showed that thymol (16-64 µg/ml) could reduce the internalization of S. aureus into bMEC and down-regulate the mRNA expression of TAP and BNBD5 in bMEC infected with S. aureus. In addition, thymol was found to inhibit S. aureus-induced nitric oxide (NO) production in bMEC and suppress S. aureus-induced NF-κB activation in a dose-dependent manner. In conclusion, these results indicated that thymol inhibits S. aureus internalization into bMEC by inhibiting NF-κB activation.

Curcumin attenuates inflammatory responses by suppressing TLR4-mediated NF-κB signaling pathway in lipopolysaccharide-induced mastitis in mice.

Curcumin, the main constituent of the spice turmeric, has been reported to have potent anti-inflammatory properties. However, the effect of curcumin on lipopolysaccharide (LPS)-induced mice mastitis has not been investigated. The aim of this study was to investigate whether curcumin could ameliorate the inflammation response in LPS-induced mice mastitis and to clarify the possible mechanism. The mouse model of mastitis was induced by injection of LPS through the duct of the mammary gland. Curcumin was applied 1h before and 12h after LPS treatment. The results showed that curcumin attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in a dose-dependent manner. Additionally, Western blotting results showed that curcumin inhibited the phosphorylation of IκB-α and NF-κB p65 and the expression of TLR4. These results indicated that curcumin has protective effect on mice mastitis and the anti-inflammatory mechanism of curcumin on LPS-induced mastitis in mice may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathways. Curcumin may be a potential therapeutic agent against mastitis.

Protective effect of taraxasterol on acute lung injury induced by lipopolysaccharide in mice.

Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have potent anti-inflammatory properties. However, the effect of taraxasterol on lipopolysaccharide (LPS)-induced mice acute lung injury has not been investigated. The aims of this study were to investigate whether taraxasterol could ameliorate the inflammation response in LPS-induced acute lung injury and to clarify the possible mechanism. Male BALB/c mice were pretreated with taraxasterol 1h before intranasal instillation of LPS. 7h after LPS administration, the myeloperoxidase (MPO) in lung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NF-κB, p46-p54 JNK, p42-p44 ERK, and p38 were determined by western blotting. The results showed that taraxasterol attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), lung wet/dry ratio, and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in a dose-dependent manner. Additionally, western blotting results showed that taraxasterol inhibited the phosphorylation of IκB-α, p65 NF-κB, p46-p54 JNK, p42-p44 ERK, and p38 caused by LPS. Our data suggest that anti-inflammatory effects of taraxasterol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPK signaling pathways.