Hypolipidemic Effect of Rice Bran Oil Extract Tocotrienol in High-Fat Diet-Induced Hyperlipidemia Zebrafish (Danio Rerio) Induced by High-Fat Diet.

In recent years, the potent influence of tocotrienol (T3) on diminishing blood glucose and lipid concentrations in both Mus musculus (rats) and Homo sapiens (humans) has been established. However, the comprehensive exploration of tocotrienol's hypolipidemic impact and the corresponding mechanisms in aquatic species remains inadequate. In this study, we established a zebrafish model of a type 2 diabetes mellitus (T2DM) model through high-fat diet administration to zebrafish. In the T2DM zebrafish, the thickness of ocular vascular walls significantly increased compared to the control group, which was mitigated after treatment with T3. Additionally, our findings demonstrate the regulatory effect of T3 on lipid metabolism, leading to the reduced synthesis and storage of adipose tissue in zebrafish. We validated the expression patterns of genes relevant to these processes using RT-qPCR. In the T2DM model, there was an almost two-fold upregulation in pparγ and cyp7a1 mRNA levels, coupled with a significant downregulation in cpt1a mRNA (p < 0.01) compared to the control group. The ELISA revealed that the protein expression levels of Pparγ and Rxrα exhibited a two-fold elevation in the T2DM group relative to the control. In the T3-treated group, Pparγ and Rxrα protein expression levels consistently exhibited a two-fold decrease compared to the model group. Lipid metabolomics showed that T3 could affect the metabolic pathways of zebrafish lipid regulation, including lipid synthesis and decomposition. We provided experimental evidence that T3 could mitigate lipid accumulation in our zebrafish T2DM model. Elucidating the lipid-lowering effects of T3 could help to minimize the detrimental impacts of overfeeding in aquaculture.

Betulinic Acid Attenuates Osteoarthritis via Limiting NLRP3 Inflammasome Activation to Decrease Interleukin-1ß Maturation and Secretion.

Introduction: Osteoarthritis (OA) is the most common degenerative joint disorder. Prior studies revealed that activation of NLRP3 inflammasome could promote the activation and secretion of interleukin-1ß (IL-1ß), which has an adverse effect on the progression of OA. Betulinic acid (BA) is a compound extract of birch, whether it can protect against OA and the mechanisms involved are still unknown. Materials and Methods: In vivo experiments, using gait analysis, ELISA, micro-CT, and scanning electron microscopy (SEM), histological staining, immunohistological (IHC) and immunofluorescence (IF) staining, and atomic force microscopy (AFM) to assess OA progression after intraperitoneal injection of 5 and 15 mg/kg BA in an OA mouse model. In vitro experiments, caspase-1, IL-1ß, and the N-terminal fragment of gasdermin D (GSDMD-NT) were measured in bone marrow-derived macrophages (BMDMs) by using ELISA, western blot, and immunofluorescence staining. Results: We demonstrated that OA progression can be postponed with intraperitoneal injection of 5 and 15 mg/kg BA in an OA mouse model. Specifically, BA postponed DMM-induced cartilage deterioration, alleviated subchondral bone sclerosis, and relieved synovial inflammation. In vitro studies, the activated NLRP3 inflammasome produces mature IL-1ß by facilitating the cleavage of pro-IL-1ß, and BA could inhibit the activation of NLRP3 inflammasome in BMDMs. Conclusions: Taken together, our analyses revealed that BA attenuates OA via limiting NLRP3 inflammasome activation to decrease the IL-1ß maturation and secretion.

Variations in the Intestinal Microbiota of the Chinese Soft-Shelled Turtle (Trionyx sinensis) between Greenhouse and Pond Aquaculture.

The microbial community structure in aquaculture water plays an important role in the intestinal microbial diversity of aquatic animals. The Chinese soft-shelled turtle (SST) (Trionyx sinensis) is an important aquaculture species of high economic value in the Asia-Pacific region. An intuitive understanding of the microbial diversity and abundances of SST aquaculture is crucial for comprehending these ecosystems. Herein, the evolutionary characteristics of the bacterial communities in the SST and its aquaculture water systems were investigated using Illumina MiSeq sequencing. This experiment sampled nine SSTs from a pond outside a greenhouse and was repeated three times. The sequencing results revealed significant differences in the microflora composition at the phylum and genus levels in both the intestine and aquaculture water of the SSTs in the greenhouse and pond aquaculture environments. A total of 1039 genera belonging to 65 phyla were identified. At the phylum level, the relative abundances of Chloroflexi (24%), Acidobacteria (5%), and Nitrospira (3%) were higher in the greenhouse water than in the pond water. The relative abundances of Bacteroidetes (35%), Actinobacteria (8%), and Cyanobacteria (4%) were higher in the pond water than in the greenhouse water. The intestinal microorganisms in the SSTs experienced significant changes after the SSTs were transferred from a greenhouse culture to a pond culture environment for 28 days. After the SSTs were cultured in the ponds, we observed decreases in the relative abundances of Actinobacteria (39% to 25%), Cyanobacteria (24% to 0.8%), Chlorobacteria (9% to 3%), and Firmicutes (5.5% to 0.8%. However, we observed increases in the relative abundances of Bacteroidetes (2% to 35%) and Acidobacteria (0.3% to 25%). These results showed that the bacterial diversity and richness compositions in the intestinal tract and aquaculture water were the same. However, the relative abundances of bacterial communities varied. The results of this study are of great significance in understanding how the environment affects SST cultures. These data may provide valuable instructions for Chinese soft-shelled turtle aquaculture management.

Quercetin alleviates nanoparticle-induced osteolysis via deactivating pyroptosis.

As one of the most successful stories in modern medicine, total joint arthroplasty (TJA) is performed several million times worldwide every year. However, more than 20% of patients will suffer from aseptic loosening (AL) following periprosthetic osteolysis (PPO) in the next few years. Unfortunately, the only effective treatment for PPO, i.e., revision surgery, can cause great surgical trauma. It has been reported that the accumulation of reactive oxidative species (ROS) generated by exposure to wear particles could activate NLRP3 inflammasome in macrophages and accelerate the progress of osteolysis. Given that the conservative treatment is not effective and can be accompanied by apparent side effects, we, therefore, investigated the therapeutic effect of the natural compound quercetin (Que) on wear particle-induced osteolysis. Our results showed that Que could activate nuclear factor erythroid 2-related factor 2 (Nrf2) to remove ROS and deactivate inflammasome activation. Besides, the imbalance between osteoclastogenesis and osteogenesis induced by inflammatory cytokines was also rescued by Que. Collectively, our work demonstrates that Que may be a qualified candidate for the conservative treatment of wear particle-induced osteolysis.

Anti-Inflammatory and Antioxidant Properties of Squalene in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO4-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO4 treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO4-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Oral Vaccination of Largemouth Bass (Micropterus salmoides) against Largemouth Bass Ranavirus (LMBV) Using Yeast Surface Display Technology.

Largemouth bass ranavirus (LMBV) infects largemouth bass, leading to significant mortality and economic losses. There are no safe and effective drugs against this disease. Oral vaccines that directly target the intestinal mucosal immune system play an important role in resisting pathogens. Herein, the B subunit of Escherichia coli heat-labile enterotoxin (LTB, a mucosal immune adjuvant) and the LMBV main capsid protein (MCP) were expressed using Saccharomyces cerevisiae surface display technology. The yeast-prepared oral vaccines were named EBY100-OMCP and EBY100-LTB-OMCP. The candidate vaccines could resist the acidic intestinal environment. After 7 days of continuous oral immunization, indicators of innate and adaptive immunity were measured on days 1, 7, 14, 21, 28, 35, and 42. High activities of immune enzymes (T-SOD, AKP, ACP, and LZM) in serum and intestinal mucus were detected. IgM in the head kidney was significantly upregulated (EBY100-OMCP group: 3.8-fold; BY100-LTB-OMCP group: 4.3-fold). IgT was upregulated in the intestines (EBY100-OMCP group: 5.6-fold; EBY100-LTB-OMCP group: 6.7-fold). Serum neutralizing antibody titers of the two groups reached 1:85. Oral vaccination protected against LMBV infection. The relative percent survival was 52.1% (EBY100-OMCP) and 66.7% (EBY100-LTB-OMCP). Thus, EBY100-OMCP and EBY100-LTB-OMCP are promising and effective candidate vaccines against LMBV infection.

Anti-inflammatory and antioxidant properties of rice bran oil extract in copper sulfate-induced inflammation in zebrafish (Danio rerio).

Tocotrienols have strong antioxidant properties; however, tocotrienol has not been investigated in detail in aquatic products. In this study, the anti-inflammatory and antioxidant activities of the tocotrienol-rich fraction from rice bran oil and its potential mechanism were verified in a zebrafish CuSO4 inflammation model. The in vitro antioxidant activity was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) stable radical method. The copper chelating activity was determined using the pyrocatechol violet method. Intracellular reactive oxygen species in zebrafish were detected using a fluorescent ROS probe. Transgenic Tg (lyz: DsRed2) zebrafish were used for neutrophil transmigration assays. The mRNA expression levels of antioxidant and pro-inflammatory factor genes were measured using quantitative real-time reverse transcription PCR. In the concentration range tested, 100 µg/mL TRF had the highest copper chelating activity (10%). TRF showed DPPH-free radical scavenging ability, which was 53% at 100 µg/mL TRF. TRF effectively repressed ROS generation and inhibited neutrophil migration to the inflamed site. Moreover, TRF upregulated the expression of antioxidant genes sod and gpx4b, inhibited the expression of pro-inflammatory factors tnfa and il8, and suppressed CuSO4-induced inflammation. In conclusion, TRF has significant anti-inflammatory and antioxidant properties, which supports the use of TRF as an aquatic feed additive to improve the anti-inflammatory and antioxidant capacity of aquatic products.

Anti-Inflammatory and Antioxidant Properties of ß-Sitosterol in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

ß-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of ß-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of ß-sitosterol and then exposed to a nonlethal concentration of CuSO4 to induce oxidative stress. Treatment with ß-sitosterol at 70 or 100 µg/mL significantly reduced CuSO4-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of ß-sitosterol. Treatment with ß-sitosterol protected zebrafish larvae against oxidative damage from CuSO4 by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with ß-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of ß-sitosterol and suggest that ß-sitosterol may be useful for the treatment of various inflammatory diseases.

Propionate and butyrate attenuate macrophage pyroptosis and osteoclastogenesis induced by CoCrMo alloy particles.

BACKGROUND: Wear particles-induced osteolysis is a major long-term complication after total joint arthroplasty. Up to now, there is no effective treatment for wear particles-induced osteolysis except for the revision surgery, which is a heavy psychological and economic burden to patients. A metabolite of gut microbiota, short chain fatty acids (SCFAs), has been reported to be beneficial for many chronic inflammatory diseases. This study aimed to investigate the therapeutic effect of SCFAs on osteolysis. METHODS: A model of inflammatory osteolysis was established by applying CoCrMo alloy particles to mouse calvarium. After two weeks of intervention, the anti-inflammatory effects of SCFAs on wear particle-induced osteolysis were evaluated by Micro-CT analysis and immunohistochemistry staining. In vitro study, lipopolysaccharide (LPS) primed bone marrow-derived macrophages (BMDMs) and Tohoku Hospital Pediatrics-1 (THP-1) macrophages were stimulated with CoCrMo particles to activate inflammasome in the presence of acetate (C2), propionate (C3), and butyrate (C4). Western blotting, Enzyme-linked immunosorbent assay, and immunofluorescence were used to detect the activation of NLRP3 inflammasome. The effects of SCFAs on osteoclasts were evaluate by qRT-PCR, Western blotting, immunofluorescence, and tartrate-resistant acid phosphatase (TRAP) staining. Additionally, histone deacetylase (HDAC) inhibitors, agonists of GPR41, GPR43, and GPR109A were applied to confirm the underlying mechanism of SCFAs on the inflammasome activation of macrophages and osteoclastogenesis. RESULTS: C3 and C4 but not C2 could alleviate wear particles-induced osteolysis with fewer bone erosion pits (P < 0.001), higher level of bone volume to tissue volume (BV/TV, P < 0.001), bone mineral density (BMD, P < 0.001), and a lower total porosity (P < 0.001). C3 and C4 prevented CoCrMo alloy particles-induced ASC speck formation and nucleation-induced oligomerization, suppressing the cleavage of caspase-1 (P < 0.05) and IL-1ß (P < 0.05) stimulated by CoCrMo alloy particles. C3 and C4 also inhibited the generation of Gasdermin D-N-terminal fragment (GSDMD-NT) to regulate pyroptosis. Besides, C3 and C4 have a negative impact on osteoclast differentiation (P < 0.05) and its function (P < 0.05), affecting the podosome arrangement and morphologically normal podosome belts formation. CONCLUSION: Our work showed that C3 and C4 are qualified candidates for the treatment of wear particle-induced osteolysis.

The ketone body ß-hydroxybutyrate alleviates CoCrMo alloy particles induced osteolysis by regulating NLRP3 inflammasome and osteoclast differentiation.

BACKGROUND: Aseptic Loosening (AL) following periprosthetic osteolysis is the main long-term complication after total joint arthroplasty (TJA). However, there is rare effective treatment except for revision surgery, which is costly and painful to the patients. In recent years, the ketone body ß-hydroxybutyrate (BHB) has attracted much attention and has been proved to be beneficial in many chronic diseases. With respect to the studies on the ketone body ß-hydroxybutyrate (BHB), its anti-inflammatory ability has been widely investigated. Although the ketone body ß-hydroxybutyrate has been applied in many inflammatory diseases and has achieved considerable therapeutic efficacy, its effect on wear particles induced osteolysis is still unknown. RESULTS: In this work, we confirmed that the anti-inflammatory action of ß-hydroxybutyrate (BHB) could be reappeared in CoCrMo alloy particles induced osteolysis. Mechanistically, the ketone body ß-hydroxybutyrate (BHB) deactivated the activation of NLRP3 inflammasome triggered by CoCrMo alloy particles. Of note, this inhibitory action was independent of Gpr109a receptor as well as histone deacetylase (HDAC) suppression. Furthermore, given that butyrate, one kind of short chain fatty acid (SCFA) structurally related to ß-hydroxybutyrate (BHB), has been reported to be an inhibitor of osteoclast, thus we also investigate the effect of ß-hydroxybutyrate (BHB) on osteoclast, which was contributed to bone resorption. It was found that ß-hydroxybutyrate (BHB) did not only affect osteoclast differentiation, but also inhibit its function. Unlike the inflammasome, the effect of ß-hydroxybutyrate (BHB) on osteoclast may mainly rely on histone deacetylase (HDAC) suppression. CONCLUSIONS: In general, our study showed that the alleviation of osteolysis may owe to the effect of ß-hydroxybutyrate (BHB) on inflammasome deactivation and osteoclast.

The effect of traumatic brain injury on bone healing from a novel exosome centered perspective in a mice model.

BACKGROUND: In patients with traumatic brain injury (TBI) combined with long bone fracture, the fracture healing is always faster than that of patients with single fracture, which is characterized by more callus growth at the fracture site and even ectopic ossification. Exosomes are nanoscale membrane vesicles secreted by cells, which contain cell-specific proteins, miRNAs, and mRNAs. METHODS: In this study, we used exosomes as the entry point to explore the mechanism of brain trauma promoting fracture healing. We established a model of tibia fracture with TBI in mice to observe the callus growth and expression of osteogenic factors at the fracture site. Blood samples of model mice were further collected, exosomes in plasma were extracted by ultra-centrifugation method, and then identified and acted on osteoblasts cultured in vitro. The effects of exosomes on osteoblast differentiation at the cell, protein and gene levels were investigated by Western Blot and q-PCR, respectively. Furthermore, miRNA sequencing of exosomes was performed to identify a pattern of miRNAs that were present at increased or decreased levels. RESULTS: The results suggested that plasma exosomes after TBI had the ability to promote the proliferation and differentiation of osteoblasts, which might be due to the increased expression of osteoblast-related miRNA in exosomes. They were transmitted to the osteoblasts at the fracture site, so as to achieve the role of promoting osteogenic differentiation. CONCLUSION: The TBI-derived exosomes may have potential applications for promoting fracture healing in future. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: Plasma exosomes early after TBI have the ability to promote osteoblast proliferation and differentiation. The mechanism may be achieved by miRNA in exosomes. Plasma exosomes may be used as breakthrough clinical treatment for delayed or non-union fractures.

Hydrogen sulfide protects against particle-induced inflammatory response and osteolysis via SIRT1 pathway in prosthesis loosening.

Wear debris-induced osteolysis and ensuing aseptic loosening is the main cause of implant failure and revision surgery. Wear debris-induced inflammatory response plays key roles in peri-implant osteolysis. Recently, substantial of evidence suggests that hydrogen sulfide (H2 S), the third gasotransmitter, is a critical player regulating inflammation. However, the role and therapeutic potential of H2 S in wear debris-induced inflammation and osteolysis remains to be defined. In the present study, we investigated the effect of H2 S on wear debris-induced pro-inflammatory cytokines expression and osteolysis in vitro and in vivo. With a slow-releasing H2 S donor GYY4137, our study demonstrated that H2 S attenuated wear debris-induced osteolysis and osteoclastogenesis in murine calvaria resorption models. The expression of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) that stimulated by wear particles were significantly reduced by GYY4137. Further, the level of sirtuin 1 (SIRT1), which possesses anti-inflammation property, was examined in vivo and in macrophages. And we found that wear debris decreased the expression of SIRT1. Cotreated macrophages with GYY4137 in part reversed the decline of SIRT1. More importantly, with the SIRT1 recombinant lentivirus and small interfering RNAs (siRNA) against SIRT1, our data indicated that SIRT1 mediated the inhibitory effects of GYY4137 on wear debris-induced inflammation. Collectively, these results suggested that exogenous H2 S production (via H2 S donors) may represent a potential approach for the treatment of wear particle-induced osteolysis.

ASIC1a activation induces calcium-dependent apoptosis of BMSCs under conditions that mimic the acidic microenvironment of the degenerated intervertebral disc.

PURPOSE: In the degenerated intervertebral disc (IVD), matrix acidity challenges transplanted bone marrow mesenchymal stem cells (BMSCs). The Ca2+-permeable acid-sensing ion channel 1a (ASIC1a) is responsible for acidosis-mediated tissue injury. The aim of our study was to confirm whether ASIC1a activation induces BMSC apoptosis under conditions that mimic the acidic microenvironment of the degenerated IVD. METHODS: ASIC1a expression in rat BMSCs was investigated by real time-PCR, Western blot (WB) and immunofluorescence. The proliferation and apoptosis of BMSCs under acidic conditions were analyzed by MTT and TUNEL assays. Ca2+-imaging was used to assess the acid-induced increase in the intracellular Ca2+ concentration ([Ca2+]i). The activation of calpain and calcineurin was analyzed using specific kits, and WB analysis was performed to detect apoptosis-related proteins. Ultrastructural changes in BMSCs were observed using transmission electron microscopy (TEM). RESULTS: Acid exposure led to the activation of ASIC1a and increased BMSC apoptosis. The Ca2+ imaging assay showed a significant increase in the [Ca2+]i in response to a solution at pH 6.0. However, BMSC apoptosis and [Ca2+]i elevation were alleviated in the presence of an ASIC1a inhibitor. Moreover, ASIC1a mediated the Ca2+ influx-induced activation of calpain and calcineurin in BMSCs. WB analysis and TEM revealed mitochondrial apoptosis, which was inhibited by an ASIC1a inhibitor, in BMSCs under acidic conditions. CONCLUSIONS: The mimical acidic microenvironment of the degenerated IVD can induce BMSC apoptosis by activating Ca2+-permeable ASIC1a. An acid-induced elevation of [Ca2+]i in BMSCs leads to the subsequent activation of calpain and calcineurin, further resulting in increased mitochondrial permeability and mitochondrial-mediated apoptosis.

Treatment outcomes of the posterolateral approach of plate fixation for posterior pilon fractures.

Posterior pilon fracture is a common type of intraarticular fracture encountered in clinical practice. The treatment of this fracture pattern has been increasingly reported. However, methods for minimizing the associated surgical trauma and achieve effective fixation still require to be established. The present study involved 23 patients with posterior pilon fracture treated at the First Affiliated Hospital of Soochow University (Suzhou, China) between March 2013 and October 2017. Klammer's classification system was used to divide the posterior pilon fractures into 3 types. The surgical procedure, reduction of post-operative fracture, peri-operative complications and post-operative functional recovery were reviewed and analyzed. The reduction in post-operative fractures was evaluated by determining the Burwell-Charnley scores at the last follow-up. Anatomical reduction was confirmed in 17 patients, and an acceptable reduction was reported in 6 patients. The American Orthopedic Foot and Ankle Score was used to assess ankle function recovery; the average score was 82.3 points (range, 44-97 points). In conclusion, the posterolateral approach is able to achieve anatomical exposure of the operative field. In addition, the posterior fracture fragment of the tibia may be fully exposed through the fibula fracture gap by retraction. Posterior placement of the plate may serve a definitive role in the fixation of the posterior fracture fragment and maintain stability in the anatomical reduction of the fracture, which is helpful in early functional rehabilitation.

Hydrogen treatment reduces tendon adhesion and inflammatory response.

A rat model of tendon repair was established to investigate the effects of hydrogen water on tendon adhesion reduction. Thirty-six Sprague Dawley rats were randomly divided into a normal saline (NS) group and a hydrogen water (HS) group according to the processing reagents after a tendon repairing operation. Pre- and postoperative superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in subjects' serum were observed. Skin fibroblasts were grouped into an NS group, H2 O2 group, H2 group, and H2 O2 H2 group. Expressions of Nrf2, CATA, and γ-GCS were also tested by Western blot analysis. 8-OHdG, GSH, MDA, and SOD of the cells were analyzed by the enzyme-linked immunosorbent assay method. The postoperative SOD activity and GSH contents were significantly reduced (P < 0.05), whereas the postoperative MDA level was significantly increased (P < 0.05). Similarly, the postoperative HS group showed significantly higher SOD activity and GSH contents (P < 0.05) but lower MDA (P < 0.05) compared with the postoperative NS group. MDA and 8-OHdG were significantly decreased in hydrogen-rich medium, while SOD and GSH were increased. The expression of Nrf2, CATA, and γ-GCS in antioxidant system were reduced after H2 O2 processing, which were restored after the application of hydrogen-rich medium. Hydrogen water can reduce tendon adhesion after tendon repairing and prohibit excessive inflammatory response, which could be associated with the activation of the Nrf2 pathway.

Enhanced Uptake of Fe3O4 Nanoparticles by Intestinal Epithelial Cells in a State of Inflammation.

Fe3O4 nanoparticles (Fe3O4 NPs) have been used for medical and drug applications, although the mechanisms of cellular uptake and transport need to be further evaluated under inflammatory conditions. In the present study, we investigated the uptake of Fe3O4 NPs (20, 50, 100, and 200 nm) by intestinal epithelial cells under inflammatory conditions via the light scattering of flow cytometry and inductively coupled plasma mass spectrometry (ICP-MS) techniques. The results of the correlation analysis indicated that the uptake ratios of Fe3O4 NPs by intestinal epithelial cells under inflammatory conditions were higher than those under the control conditions. The transportation ratios of NPs by inflammatory Caco-2 cells increased almost 0.8-1.2 fold compared to the control. The internalization of the Fe3O4 NPs in Caco-2 cells was mediated by clathrin-related routes in both the control and an interleukin-1ß (IL-1ß)-induced inflammatory condition. The level of mRNA of clathrin expressed in Caco-2 cells that were stimulated by IL-1ß was almost three times more than the control. Consistently with the mRNA expression, the level of protein in the clathrin was upregulated. Additionally, it was verified for the first time that the expression of clathrin was upregulated in IL-1ß-stimulated Caco-2 cells. Collectively, these results provided a further potential understanding about the mechanism of Fe3O4 NPs' uptake by intestinal epithelial cells under inflammatory conditions.

Apoptotic pathways of macrophages within osteolytic interface membrane in periprosthestic osteolysis after total hip replacement.

Macrophage apoptosis in interface membrane, which occurs through either death receptor, mitochondrion, or endoplasmic reticulum (ER) stress pathways, has been suggested to play an important role in promoting osteolysis. However, how and why macrophage apoptosis originates and the correlation among these apoptotic pathways is not yet clear. The objective of this study was to identify the apoptotic mechanism of macrophages, and to explore the relationship between the apoptotic pathways and progression of osteolysis. Transmission electron microscopy (TEM) was utilized to analyze the tissue ultrastructure of wear particles, and in situ apoptotic macrophage identification was performed by TUNEL staining. We analyzed the expression of the key biomarkers of apoptotic pathways via immunohistochemistry and Western blotting. Our results demonstrated that the majority of wear particles within osteolytic interface membrane was in the 30-60 nm range, and that macrophage apoptotic ratio increased along with osteolysis progression. Normal hip dysplasia and mechanical loosening of tissues showed low expression levels of biomarkers for ER stress (Ca2+ , JNK, cleaved Caspase-4, IRE1-α, Grp78/Bip, and CHOP), mitochondrion (Bcl-2, Bax, and Cytochrome c), and death receptor (Fas and cleaved Caspase-8) pathways, while osteolytic interface membrane tissues expressed high levels of these biomarkers. In addition, we found that the ER stress intensity was in complete conformity with mitochondrial dysfunction and was consistent with the results of death receptor activation. Thus, our findings suggested that wear particles generated at implant interface can accelerate macrophage apoptosis through changes in apoptotic pathways and ultimately aggravate the symptom of osteolysis. These data represent a preferential apoptotic signaling pathway of macrophages as specific target points for the prevention and therapeutic modulation of periprosthetic osteolysis.

Expression of XBP1s in fibroblasts is critical for TiAl6 V4 particle-induced RANKL expression and osteolysis.

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)-kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle-induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl6 V4 particle (TiPs)-induced XBP1s (spliced form of X-box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri-implant membrane, animal models, and TiPs-stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate-resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro-computed tomography (micro-CT) with three-dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs-stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs-induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle-induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752-759, 2017.

Molecular hydrogen decelerates rheumatoid arthritis progression through inhibition of oxidative stress.

Rheumatoid arthritis (RA) is a chronic inflammatory disease which results in progressive destruction of the joint. In this study, we examined if the hydrogen could inhibit inflammation in a mouse model of collagen-induced arthritis (CIA) via oxidative stress on RA-FLSs. Moreover, to identify the mechanisms of action, we evaluated the effect of hydrogen on RA-FLSs development and the expression of pro-inflammatory cytokines and signaling pathways. Based on our result, H2 enriched medium can increase super oxide dismutase (SOD) level following H2O2 treatment and decrease 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. Since H2O2 treatment activates MAPK, NF-κB and TGF-ß1 in cells, our study suggested that H2 could inhibit H2O2 activated MAPK and NF-κB activation as well as TGF-ß1 expression in treated cells. Taken together, our data suggested that H2 can directly neutralize OH and ONOO- to reduce oxidative stress. Moreover, MAPK and NF-κB pathway also play roles in oxidative damage caused by H2O2 in RA-FLSs. H2 can provide protection to cells against inflammation, which may be related to inhibition of the activation of MAPK and NF-κB.