A strategy for enhancing bioactivity and osseointegration with antibacterial effect by incorporating magnesium in polylactic acid based biodegradable orthopedic implant.

Biodegradable orthopedic implants are essential for restoring the physiological structure and function of bone tissue while ensuring complete degradation after recovery. Polylactic acid (PLA), a biodegradable polymer, is considered a promising material due to its considerable mechanical properties and biocompatibility. However, further improvements are necessary to enhance the mechanical strength and bioactivity of PLA for reliable load-bearing orthopedic applications. In this study, a multifunctional PLA-based composite was fabricated by incorporating tricalcium phosphate (TCP) microspheres and magnesium (Mg) particles homogenously at a volume fraction of 40 %. This approach aims to enhance mechanical strength, accelerate pore generation, and improve biological and antibacterial performance. Mg content was incorporated into the composite at varying values of 1, 3, and 5 vol% (referred to as PLA/TCP-1 Mg, PLA/TCP-3 Mg, and PLA/TCP-5 Mg, respectively). The compressive strength and stiffness were significantly enhanced in all composites, reaching 87.7, 85.9, and 84.1 MPa, and 2.7, 3.0, and 3.1 GPa, respectively. The degradation test indicated faster elimination of the reinforcers as the Mg content increased, resulting in accelerated pore generation to induce enhanced osseointegration. Because PLA/TCP-3 Mg and PLA/TCP-5 Mg exhibited cracks in the PLA matrix due to rapid corrosion of Mg forming corrosion byproducts, to optimize the Mg particle content, PLA/TCP-1 Mg was selected for further evaluation. As determined by in vitro biological and antibacterial testing, PLA/TCP-1 Mg showed enhanced bioactivity with pre-osteoblast cells and exhibited antibacterial properties by suppressing bacterial colonization. Overall, the multifunctional PLA/TCP-Mg composite showed improved mechanobiological performance, making it a promising material for biodegradable orthopedic implants.

Clonal evolution of long-term expanding head and neck cancer organoid: Impact on treatment response for personalized therapeutic screening.

OBJECTIVES: In biobanking based on patient-derived organoids (PDO), the genetic stability of organoid lines is critical for the clinical relevance of PDO with parental tumors. However, data on mutational heterogeneity and clonal evolution of PDO and their effects on treatment response are insufficient. METHODS: To investigate whether head and neck cancer organoids (HNCOs) could maintain the genetic characteristics of their original tumors and elucidate the clonal evolution process during a long-term passage, we performed targeted sequencing, covering 377 cancer-related genes and adopted a sub-clonal fraction model. To explore therapeutic response variability between an early and late passage (>passage 6), we generated dose-response curves for drugs and radiation using two HNCO lines. RESULTS: Using 3D ex vivo organoid culture protocol, we successfully established 27 HNCOs from 39 patients with an overall success rate of 70% (27/39). Their mutational profiles were highly concordant, with three of the HNCOs analyzed showing greater than 70% concordance. Only one HNCO displayed less than 50% concordance. However, many of these organoid lines displayed clonal evolution during serial passaging, although major cancer driver genes and VAF distributions were shared between early and later passages. We also found that all late passages of HNCOs tended to be more sensitive to radiation than early passages, similar to drug response results. CONCLUSIONS: We report the establishment of HNCO lines derived from 27 patients and demonstrate their genetic concordance with corresponding parental tumors. Furthermore, we show serial changes in mutational profiles of HNCO along with long passage culture and the impact of these clonal evolutions on response to radiotherapy.

Antibacterial PLA/Mg composite with enhanced mechanical and biological performance for biodegradable orthopedic implants.

Biodegradability, bone-healing rate, and prevention of bacterial infection are critical factors for orthopedic implants. Polylactic acid (PLA) is a good candidate biodegradable material; however, it has insufficient mechanical strength and bioactivity for orthopedic implants. Magnesium (Mg), has good bioactivity, biodegradability, and sufficient mechanical properties, similar to that of bone. Moreover, Mg has an inherent antibacterial property via a photothermal effect, which generates localized heat, thus preventing bacterial infection. Therefore, Mg is a good candidate material for PLA composites, to improve their mechanical and biological performance and add an antibacterial property. Herein, we fabricated an antibacterial PLA/Mg composite for enhanced mechanical and biological performance with an antibacterial property for application as biodegradable orthopedic implants. The composite was fabricated with 15 and 30 vol% of Mg homogeneously dispersed in PLA without the generation of a defect using a high-shear mixer. The composites exhibited an enhanced compressive strength of 107.3 and 93.2 MPa, and stiffness of 2.3 and 2.5 GPa, respectively, compared with those of pure PLA which were 68.8 MPa and 1.6 GPa, respectively. Moreover, the PLA/Mg composite at 15 vol% Mg exhibited significant improvement of biological performance in terms of enhanced initial cell attachment and cell proliferation, whereas the composite at 30 vol% Mg showed deteriorated cell proliferation and differentiation because of the rapid degradation of the Mg particles. In turn, the PLA/Mg composites exerted an antibacterial effect based on the inherent antibacterial property of Mg as well as the photothermal effect induced by near-infrared (NIR) treatment, which can minimize infection after implantation surgery. Therefore, antibacterial PLA/Mg composites with enhanced mechanical and biological performance may be a candidate material with great potential for biodegradable orthopedic implants.

A novel 3D pillar/well array platform using patient-derived head and neck tumor to predict the individual radioresponse.

Predicting individual radiotherapy (RT) response is valuable in managing head and neck squamous cell carcinoma (HNSCC). We assessed the feasibility of our novel 3D culture platform to measure radioresponse using patient-derived cells (PDCs) from HNSCC patients. Cells from the FaDu line and tumor samples from 39 HNSCC patients were cultivated serially in MatrigelTM on a 3D pillar/well array culture system. The 3D tumor models were exposed to 0 to 8 Gy of radiation dose, and the radioresponse index (RTauc, area under the dose-response curve) was measured quantitatively with Calcein AM staining of live tumor cells. Calcein AM fluorescence showed reduced density and the number of FaDu colonies as radiation increased, implying a dose-dependent effect on cell viability in the 3D pillar/well culture system. 3D tumor models using PDCs were established successfully from 39 HNSCC patient tumor samples, maintaining original genomic and pathological characteristics. These 3D tumor models were exposed to ionizing radiation on a 3D pillar/well array, with a mean period of 12 days from tumor harvest to the measurement of RTauc. The RTauc of all PDCs varied from 3.5 to 9.4, and the lower 40th percentile (Z-score = -0.26) was considered a good radioresponse group with a threshold RTauc of 4.6. The good radioresponse group showed fewer adverse features than others. As of the last follow-up, recurrence-free survival was better in the good radioresponse group (p = 0.037). 3D pillar/well array platforms using PDC could rapidly quantify radioresponse index in patients with HNSCC, showing potential as a novel prognosticator.

Polydeoxyribonucleotide-delivering therapeutic hydrogel for diabetic wound healing.

Patients with diabetes experience delayed wound healing because of the uncontrolled glucose level in their bloodstream, which leads to impaired function of white blood cells, poor circulation, decreased production and repair of new blood vessels. Treatment using polydeoxyribonucleotide (PDRN), which is a DNA extracted from the sperm cells of salmon, has been introduced to accelerate the healing process of diabetic wounds. To accelerate the wound-healing process, sustained delivery of PDRN is critical. In this study, taking advantage of the non-invasive gelation property of alginate, PDRN was loaded inside the hydrogel (Alg-PDRN). The release behavior of PDRN was altered by controlling the crosslinking density of the Alg hydrogel. The amount of PDRN was the greatest inside the hydrogel with the highest crosslinking density because of the decreased diffusion. However, there was an optimal degree of crosslinking for the effective release of PDRN. In vitro studies using human dermal fibroblasts and diabetes mellitus fibroblasts and an in ovo chorioallantoic membrane assay confirmed that the Alg-PDRN hydrogel effectively induced cell proliferation and expression of angiogenic growth factors and promoted new blood vessel formation. Its effectiveness for accelerated diabetic wound healing was also confirmed in an in-vivo animal experiment using a diabetic mouse model.

A rapid quantification of invasive phenotype in head and neck squamous cell carcinoma: A novel 3D pillar array system.

BACKGROUND: The widely used in vitro invasion assays for head and neck squamous cell carcinoma (HNSCC) are wound healing, transwell, and organotypic assays. However, these are still lab-intensive and time-consuming tasks. For the rapid detection and high throughput screening of invasiveness in 3D condition, we propose a novel spheroid invasion assay using commercially available pillar platform system. MATERIALS AND METHODS: Using the pillar-based spheroid invasion assay, migration and invasion was evaluated in three patient-derived cells (PDCs) of HNSCC. Immunofluorescence of live cells was used for the quantitative measurement of migratory and invaded cells attached to the pillar. Expression of epithelial-mesenchymal transition (EMT)-related gene (snai1/2) was measured by qRT-PCR. We also tested the impact of drug treatments (cisplatin, docetaxel) on the changes in the invasive phenotype. RESULTS: All PDCs successfully formed spheroid at 4 days and can be measured invasiveness within 7 days. Intriguingly, one PDC (#1) obtained from the advanced stage showed robust migration, invasion and higher transcription of snai1/2, compared with the other two PDCs. Furthermore, the invasion ratio of the control spheroids was about 70% while the invasion ratios of drug-treated spheroids were lower than 50%, and the difference showed statistical significance (p < 0.01). CONCLUSION: The presented spheroid invasion assay using pillar array could be useful for the evaluation of cancer cell behavior and physiology in response to diverse therapeutic drugs.

Fluorine-ion-releasing injectable alginate nanocomposite hydrogel for enhanced bioactivity and antibacterial property.

The creation of a moist environment and promotion of cell proliferation and migration together with antibacterial property are critical to the wound-healing process. Alginate (Alg) is an excellent candidate for injectable wound dressing materials because it can form a gel in a mild environment. Taking advantage of its gelation property, an injectable nano composite hydrogel containing nano-sized (about 90 nm) calcium fluoride (CaF2) particles was developed using in-situ precipitation process. The amount of released fluorine (F-) ion from the nanocomposite hydrogel increased with increasing CaF2 content inside the composite hydrogel and the ions stimulated both the proliferation and migration of fibroblast cells in vitro. The antibacterial property of the composite hydrogel against E. coli and S. aureus was confirmed through colony formation test where the number of bacterial colonies significantly decreased compared to Alg hydrogel. The in vivo results based on a full-thickness wound model showed that the nanocomposite hydrogel effectively enhanced the deposition of the extracellular matrix compared to that of the Alg hydrogel. This study demonstrates the potential of this nanocomposite hydrogel as a bioactive injectable wound-dressing material with the ability to inhibit bacterial growth and stimulate cell proliferation and migration for accelerated wound healing.

In vitro and in vivo evaluation of polylactic acid-based composite with tricalcium phosphate microsphere for enhanced biodegradability and osseointegration.

A biodegradable polylactic acid composite containing tricalcium phosphate microsphere was fabricated. The composite exhibited enhanced biocompatibility and a well-interconnected porous structure that enabled tissue ingrowth after degradation. The tricalcium phosphate microspheres had an average size of 106 ± 43 µm and were incorporated into the polylactic acid matrix using a high-shear mixer. The resulting bioactivity and hydrophilicity were enhanced to levels comparable to those of a polylactic acid composite containing tricalcium phosphate powder, which is a well-known material used in the medical field. An accelerated 30-day degradation test in HCl revealed successful generation of an open porous structure with ∼98% interconnectivity in the polylactic acid-tricalcium phosphate microsphere composite, demonstrating the potential of this material to induce enhanced osseointegration in the later stage of bone regeneration. The early stage osseointegration was also evaluated by implanting the composite in vivo using a rabbit femoral defect model. After 16 weeks of implantation, the bone-to-implant contact ratio of the polylactic acid-tricalcium phosphate microsphere composite was enhanced owing to tissue ingrowth through the generated pores near the surface.

Effective Wound Healing by Antibacterial and Bioactive Calcium-Fluoride-Containing Composite Hydrogel Dressings Prepared Using in Situ Precipitation.

In this study, we report the development of a hyaluronic acid (HA)-based composite hydrogel containing calcium fluoride (CaF2) with good biocompatibility and antibacterial properties for multifunctional wound dressing applications. CaF2 was newly selected for incorporation within HA because it can release both Ca2+ and F- ions, which are well-known ions for affecting cell proliferation and inhibiting bacterial growth, respectively. In particular, an in situ precipitation process enables easy control over the released amount of F- ions by simply adjusting the precursor solutions (calcium chloride (CaCl2) and ammonium fluoride (NH4F)) used for the CaF2 precipitation. CaF2 particles were uniformly embedded within a HA-based pure hydrogel using an in situ precipitation process. Through variation of the CaCl2 and NH4F concentrations used in the precipitation as well as the precipitation time, composite hydrogels with different ion-release profiles were obtained. By controlling the precipitation time, especially for 10 min and after 30 min, large differences in the ion-release profiles as a function of CaF2 concentration were observed. A shorter precipitation time resulted in faster release of fluoride, whereas for the 30 min and 1 h samples, sustained ion release was achieved. Colony tests and live/dead assays using Escherichia coli and Staphylococcus aureus revealed a lower density of bacteria on the CaF2 composite hydrogels than on the pure hydrogel for both strains. In addition, improved cellular responses such as cell attachment and proliferation were also observed for the CaF2 composite hydrogels compared to those for the pure hydrogel. Furthermore, the composite hydrogels exhibited excellent wound healing efficiency, as evidenced by an in vitro cell migration assay. Finally, monitoring of the wound closure changes using a full-thickness wound in a rat model revealed the accelerated wound healing capability of the CaF2 composite hydrogels compared with that of the pure hydrogel. Based on our findings, these CaF2 composite hydrogels show great potential for application as advanced hydrogel wound dressings with antibacterial properties and accelerated wound-healing capabilities.

Postoperative anti-PD-1 antibody treatment to reduce recurrence in a cancer ablation surgical wound.

BACKGROUND: Postoperative radiation and chemotherapy are routinely applied for microscopic residual diseases; however, treatment outcomes are not optimal, and patients frequently suffer from treatment-related toxicities. To search for an effective and less-toxic adjuvant treatment for patients with high risk of recurrence, the preventive effect of anti-programmed cell death protein 1 (PD-1) treatment was evaluated in an in vivo animal model of post-surgical tumor recurrence. MATERIALS AND METHODS: An animal model of postsurgical tumor recurrence (SCCVII tumors in C3H mice) was established by reinoculating tumor cells (105 cells) into surgical wound of primary tumor resection. Initial and recurrent tumors were compared by an immunohistochemistry and complementary DNA microarray. Using this in vivo model, tumor recurrence rates were evaluated in the animals receiving anti-PD-1 treatments. Animals were rechallenged with tumor cells, and interferon gamma secretion from spleen cells was analyzed to determine tumor-specific antitumor immunity. RESULTS: FoxP3high cell population was significantly elevated in recurrent tumors compared with that in primary tumors. Some immune response-related factors (granzyme F, neuronal leucine-rich repeat protein 1, myosin heavy chain 3, and transmembrane protein 8C) showed significant differences between primary and recurrent tumors. In this animal model, anti-PD-1 treatments significantly suppressed tumor recurrence. Importantly, tumor induction was significantly reduced when anti-PD-1-treated mice were rechallenged with tumor cells. Tumor cell-specific interferon gamma production was increased in these animals. CONCLUSIONS: Postoperative anti-PD-1 treatment significantly reduced recurrence in a cancer ablation surgical wound in an in vivo model of tumor recurrence. Our data lay the preclinical groundwork for the future clinical validation of adjuvant anti-PD-1 treatments in patients.

Prognostic significance of epithelial-mesenchymal transition of extracapsular spread tumors in lymph node metastases of head and neck cancer.

BACKGROUND AND PURPOSE: The extracapsular spread (ECS) of lymph node metastasis (LNM) reflects tumor aggressiveness and is associated with poor survival and risk of distant metastasis. In this study, we aimed to explore the prognostic significance of epithelial-mesenchymal transition (EMT) of ECS tumors in LNM of head and neck cancers. METHODS: We collected LNM samples from head and neck cancer patients (follow-up >2 years) and made 20 ECS(-): ECS(+) pairs (1:2) of LNM (N = 60), matched by the primary sites and by T and N classifications. Immunostaining of cytokeratin, E-cadherin, vimentin, and CD31 were performed and quantified to determine the epithelial-mesenchymal transition percent (EMT%), defined as vimentin(+)/cytokeratin(+) area of ECS. Univariate and multivariable analyses of clinic-pathologic factors, including EMT% of ECS, were conducted to identify the significant prognosticators. In addition, the anatomical relationship between CD31 vessels and ECS tumors was analyzed. RESULTS: Rather than the presence of ECS in LNM, higher EMT% (>50 %) of ECS strongly correlated with the worse overall and disease-free survival and had more frequent recurrence and distant dissemination in their clinical courses. ECS tumors intermingled closely with Ki-67(-) CD31(+) non-proliferating perinodal blood vessels. Particularly, vimentin(+) ECS areas exhibited a higher density of CD31(+) perinodal vessels than did vimentin(-) ECS. CONCLUSION: High EMT scores of ECS tumors in LNM predict an unfavorable prognosis and systemic dissemination more accurately than the simple presence of ECS in LNM in head and neck cancer patients.

Comparison of adenoid cystic carcinomas arising from the parotid gland vs. the submandibular gland: focus on systemic metastasis and tumor-associated blood vessels.

BACKGROUND: Although several studies reported that distant metastasis occurs more frequently in the tumors of submandibular gland (SMG) than parotid gland (PG), why SMG tumors preferentially metastasize to distant organs is not fully understood. We aimed to identify the differential tumor microenvironment for distant metastasis and possible underlying mechanisms. METHODS: We retrieved 27 cases of 1-4-cm-sized adenoid cystic carcinomas (ACCs) arising from the PG (n = 12) and SMG (n = 15). c-KIT, VEGF-R2, and CD31 staining were quantified by image-based analysis to define the positive expression or tumor-associated vessel areas in two representative sections per case. In addition, angiogenesis-related genomic expression profiling was carried out to explore the underlying mechanism, which was confirmed by RT-PCR and immunohistochemistry. RESULTS: Earlier systemic dissemination within 2 years was detected exclusively in SMG ACCs (5/15). The area of tumor-associated blood vessels was larger in SMG ACCs than PG ACCs, and ACCs showing distant metastasis had greater blood vessel area than those without metastasis. Interestingly, normal SMG had more blood vessels per area than PG. Among angiogenesis-related signals, the level of IL-6 was significantly lower in SMG ACCs than PG ACCs. Moreover, IL-6 expression decreased significantly in SMG ACCs compared with that in normal SMG, whereas it was up-regulated in PG ACCs. CONCLUSION: ACCs in the SMG microenvironment have more abundant tumor-associated blood vessels than PG ACCs, which may explain the higher risk of distant metastasis from SMG tumors.

Human retinal pigment epithelial cells express the long pentraxin PTX3.

PURPOSE: To determine whether the long pentraxin 3 (PTX3) is expressed in human retinal pigment epithelial cells and is induced by inflammatory cytokines, interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), expression of PTX3 was investigated in the human retinal pigment epithelial cell line, ARPE-19 cells. METHODS: In ARPE-19 cells, we first analyzed PTX3 production in the presence or absence of inflammatory cytokines, IL-1ß, TNF-α, and IFN-γ, dose- and time-dependently using enzyme-linked immunosorbent assay. Protein and mRNA expression of PTX3 was measured with western blotting analysis and real-time reverse transcription-polymerase chain reaction. Specific inhibitors were used to determine the signaling pathways of inflammatory cytokine-induced PTX3 expression. RESULTS: In this study, production of PTX3 was induced by IL-1ß and TNF-α dose- and time-dependently, but not by IFN-γ in ARPE-19 cells. Protein and mRNA expression of PTX3 was significantly upregulated in the presence of IL-1ß and TNF-α. Furthermore, pretreatment with extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells specific inhibitor abolished IL-1ß and TNF-α-induced PTX3 production, but the other inhibitors had no effect. CONCLUSIONS: These results suggested that human retinal pigment epithelial cells may be a major source of PTX3 production in the presence of proinflammatory cytokines, IL-1ß and TNF-α, and could be an important mediator for host defense and inflammatory response in the retina. The importance of the mitogen-activated protein kinase/extracellular signal-regulated kinase1/2 and nuclear factor kappa-light-chain-enhancer of activated B cells pathways for regulated PTX3 expression may be a potential target for PTX3 regulation in the retina.

Curcumin protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression and reduction of reactive oxygen.

PURPOSE: To determine whether curcumin induces expression of the defensive enzyme heme oxygenase-1 (HO-1) and protects cells against oxidative stress in cultured human retinal pigment epithelial cells. METHODS: Effective concentrations and toxicities of curcumin were determined after 3 h of curcumin treatment with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Confluent human retinal pigment epithelium cell lines (ARPE-19) were preincubated with curcumin and oxidatively challenged with H(2)O(2). HO-1 expression was determined with western blot analysis. To confirm the protective role of HO-1 in oxidative stress, small interfering RNA (siRNA) against HO-1 or inhibitor of HO-1 was treated with curcumin in retinal pigment epithelium cells. Intracellular levels of reactive oxygen species (ROS) were measured with flow cytometry and confocal microscopy. Apoptosis was evaluated with Annexin V-fluoroscein isothiocyanate staining. RESULTS: Curcumin had little cytotoxicity at concentrations less than 30 µM, and HO-1 expression was the highest at the 15 µM concentration. At this concentration, curcumin also increased the cytoprotective effect against the oxidative stress of H(2)O(2) through the reduction of ROS levels in human retinal pigment epithelial cells. Curcumin's effect on the reduction of ROS was mediated by the increase in HO-1 expression. CONCLUSIONS: Curcumin upregulated the oxidative stress defense enzyme HO-1 and may protect human retinal pigment epithelial cells against oxidative stress by reducing ROS levels.

Pretreatment with CO-releasing molecules suppresses hepcidin expression during inflammation and endoplasmic reticulum stress through inhibition of the STAT3 and CREBH pathways.

The circulating peptide hormone hepcidin maintains systemic iron homeostasis. Hepcidin production increases during inflammation and as a result of endoplasmic reticulum (ER) stress. Elevated hepcidin levels decrease dietary iron absorption and promote iron sequestration in reticuloendothelial macrophages. Furthermore, increased plasma hepcidin levels cause hypoferremia and the anemia associated with chronic diseases. The signal transduction pathways that regulate hepcidin during inflammation and ER stress include the IL-6-dependent STAT-3 pathway and the unfolded protein response-associated cyclic AMP response element-binding protein-H (CREBH) pathway, respectively. We show that carbon monoxide (CO) suppresses hepcidin expression elicited by IL-6- and ER-stress agents by inhibiting STAT-3 phosphorylation and CREBH maturation, respectively. The inhibitory effect of CO on IL-6-inducible hepcidin expression is dependent on the suppressor of cytokine signaling-3 (SOCS-3) protein. Induction of ER stress in mice resulted in increased hepatic and serum hepcidin. CO administration inhibited ER-stress-induced hepcidin expression in vivo. Furthermore, ER stress caused iron accumulation in splenic macrophages, which could be prevented by CO. Our findings suggest novel anti-inflammatory therapeutic applications for CO, as well as therapeutic targets for the amelioration of anemia in the hypoferremic condition associated with chronic inflammatory and metabolic diseases.

Carbon monoxide, a reaction product of heme oxygenase-1, suppresses the expression of C-reactive protein by endoplasmic reticulum stress through modulation of the unfolded protein response.

The expression of C-reactive protein (CRP) rises rapidly in response to inflammation. The endoplasmic reticulum (ER) stress has been reported to cause CRP expression. Carbon monoxide (CO), a reaction product of heme oxygenase, exerts anti-inflammatory effects. In this study, we aimed to examine the role of CO in modulating ER stress-induced CRP expression. In HepG2 cells, ER stress triggered by tunicamycin, thapsigargin and homocysteine markedly induced CRP expression and the activation of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring transmembrane kinase/endonuclease 1α (IRE1α), activating transcription factor 6 (ATF6), and hepatocyte-specific cyclic AMP response element binding protein H (CREBH). A CO-releasing molecule (CORM) inhibited ER stress-induced CRP expression. While CORM attenuated ER stress-induced activation of IRE1α, ATF6 and CREBH, it augmented PERK activation, which was associated with its inhibition of CRP expression. CORM also inhibited CRP expression in response to the pro-inflammatory cytokine IL-6 that was found to induce ER stress response in HepG2 cells. Moreover, in mice treated with the ER stress inducer tunicamycin, CORM administration reduced serum levels of CRP and the expression of CRP mRNA in the liver. Collectively, our findings suggest that CO may attenuate ER stress-induced CRP expression through modulation of the unfolded protein response.