[A sericin hydrogel scaffold for sustained dexamethasone release modulates macrophage polarization to promote mandibular bone defect repair in rats].

OBJECTIVE: To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization. METHODS: The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-ß-cyclodextrin (NH2-ß-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining. RESULTS: In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect. CONCLUSION: The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.

IL-6/gp130 signaling in CD4+ T cells drives the pathogenesis of pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by stenosis and occlusions of small pulmonary arteries, leading to elevated pulmonary arterial pressure and right heart failure. Although accumulating evidence shows the importance of interleukin (IL)-6 in the pathogenesis of PAH, the target cells of IL-6 are poorly understood. Using mice harboring the floxed allele of gp130, a subunit of the IL-6 receptor, we found substantial Cre recombination in all hematopoietic cell lineages from the primitive hematopoietic stem cell level in SM22α-Cre mice. We also revealed that a CD4+ cell-specific gp130 deletion ameliorated the phenotype of hypoxia-induced pulmonary hypertension in mice. Disruption of IL-6 signaling via deletion of gp130 in CD4+ T cells inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and suppressed the hypoxia-induced increase in T helper 17 cells. To further examine the role of IL-6/gp130 signaling in more severe PH models, we developed Il6 knockout (KO) rats using the CRISPR/Cas9 system and showed that IL-6 deficiency could improve the pathophysiology in hypoxia-, monocrotaline-, and Sugen5416/hypoxia (SuHx)-induced rat PH models. Phosphorylation of STAT3 in CD4+ cells was also observed around the vascular lesions in the lungs of the SuHx rat model, but not in Il6 KO rats. Blockade of IL-6 signaling had an additive effect on conventional PAH therapeutics, such as endothelin receptor antagonist (macitentan) and soluble guanylyl cyclase stimulator (BAY41-2272). These findings suggest that IL-6/gp130 signaling in CD4+ cells plays a critical role in the pathogenesis of PAH.

Efficacy of high-intensity interval and continuous endurance trainings on cecal microbiota metabolites and inflammatory factors in diabetic rats induced by high-fat diet.

Physical exercise is known to modulate the intestinal microbiota composition and control the symptoms of metabolic syndrome. In this research, we intend to investigate and compare the effect of high-intensity interval and continuous endurance trainings (HIIT and CET) on cecal microbiota metabolites and inflammatory factors in diabetic rats. A number of Wistar rats were made diabetic by a high-fat diet and trained under two types of exercise protocols, HIIT and CET. After taking samples from the cecal tissue and serum of rats to reveal the effect of exercise, three microbial species from the Firmicute and Bacteroid phyla, which are the main types of intestinal microbes, and their metabolites include two short-chain fatty acids (SCFAs), butyrate and propionate and also, the inflammatory factors TLR4 and IL6 were analyzed through quantitative polymerase chain reaction (qPCR), high-performance liquid chromatography (HPLC), and Enzyme-linked immunosorbent assay (ELISA) methods. In general, exercise while increasing the representative of Firmicute has caused a relative reduction of Bacteroides and improved the concentration of SCFAs. In this regard, HIIT outperforms CET in up-regulating Akkermansia and Butyrivibrio expression, and butyrate and propionate metabolites concentration. Also, both exercises significantly reduced cecal expression of TLR4 and sera concentration of IL6 compared to the diabetic group, although the reduction rate was higher in the CET group than in HIIT. Our findings suggest that some symptoms of metabolic syndrome such as intestinal dysbiosis and the resulting metabolic disorders are better controlled by HIIT and inflammation by CET. Certainly, more extensive research on other contributing factors could help clarify the results.

Modulation of Gut Microbial Metabolism by Cyanidin-3-O-Glucoside in Mitigating Polystyrene-Induced Colonic Inflammation: Insights from 16S rRNA Sequencing and Metabolomics.

Microplastics derived from plastic waste have emerged as a pervasive environmental pollutant with potential transfer and accumulation through the food chain, thus posing risks to both ecosystems and human health. The gut microbiota, tightly intertwined with metabolic processes, exert substantial influences on host physiology by utilizing dietary compounds and generating bacterial metabolites such as tryptophan and bile acid. Our previous studies have demonstrated that exposure to microplastic polystyrene (PS) disrupts the gut microbiota and induces colonic inflammation. Meanwhile, intervention with cyanidin-3-O-glucoside (C3G), a natural anthocyanin derived from red bayberry, could mitigate colonic inflammation by reshaping the gut bacterial composition. Despite these findings, the specific influence of gut bacteria and their metabolites on alleviating colonic inflammation through C3G intervention remains incompletely elucidated. Therefore, employing a C57BL/6 mouse model, this study aims to investigate the mechanisms underlying how C3G modulates gut bacteria and their metabolites to alleviate colonic inflammation. Notably, our findings demonstrated the efficacy of C3G in reversing the elevated levels of pro-inflammatory cytokines (IL-6, IL-1ß, and TNF-α) and the upregulation of mRNA expression (Il-6, Il-1ß, and Tnf-α) induced by PS exposure. Meanwhile, C3G effectively inhibited the reduction in levels (IL-22, IL-10, and IL-4) and the downregulation of mRNA expression (Il-22, Il-10, and Il-4) of anti-inflammatory cytokines induced by PS exposure. Moreover, PS-induced phosphorylation of the transcription factor NF-κB in the nucleus, as well as the increased level of protein expression of iNOS and COX-2 in the colon, were inhibited by C3G. Metabolisms of gut bacterial tryptophan and bile acids have been extensively implicated in the regulation of inflammatory processes. The 16S rRNA high-throughput sequencing disclosed that PS treatment significantly increased the abundance of pro-inflammatory bacteria (Desulfovibrio, norank_f_Oscillospiraceae, Helicobacter, and Lachnoclostridium) while decreasing the abundance of anti-inflammatory bacteria (Dubosiella, Akkermansia, and Alistipes). Intriguingly, C3G intervention reversed these pro-inflammatory changes in bacterial abundances and augmented the enrichment of bacterial genes involved in tryptophan and bile acid metabolism pathways. Furthermore, untargeted metabolomic analysis revealed the notable upregulation of metabolites associated with tryptophan metabolism (shikimate, l-tryptophan, indole-3-lactic acid, and N-acetylserotonin) and bile acid metabolism (3b-hydroxy-5-cholenoic acid, chenodeoxycholate, taurine, and lithocholic acid) following C3G administration. Collectively, these findings shed new light on the protective effects of dietary C3G against PS exposure and underscore the involvement of specific gut bacterial metabolites in the amelioration of colonic inflammation.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

OBJECTIVES: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1ß, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

[Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages].

OBJECTIVE: To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. METHODS: Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. RESULTS: There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1ß (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-ß mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1ß, TNF-α and TGF-ß in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-ß mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). CONCLUSIONS: Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

The healing effect of a mixture of Arnebia euchroma and animal fat on burn wounds in rats in comparison with sulfadiazine.

OBJECTIVE: Thermal burn is a serious cause of morbidity and mortality that affects millions of people worldwide. The aim of this experimental study was to investigate the efficacy of Arnebia euchroma (AE) to treat burn wounds in a rat model. METHOD: A total of 80 male rats (200-250g) were shaved over the back of the neck (2×3cm2) and a second-degree burn wound was induced at this site under general anaesthesia. The rats were then randomly assigned to one of four groups (each n=20) and the burns were treated daily for 14 days as follows: (1) dressed with animal fat; (2) dressed with sulfadiazine; (3) dressed with a mixture of AE and animal fat; (4) no treatment (control). Five rats from each group were sacrificed on days 3, 5, 9 and 14 post-burn and the wounds were evaluated histologically and immunohistochemically for the expression of interleukin (IL)-1 and IL-6. RESULTS: There was a significant increase at day 3 and decrease on day 5 samples for the expression of IL-1 in the AE plus fat group and IL-6 in the AE plus fat and sulfadiazine groups, compared to the control and fat treatment groups, respectively. Both AE plus fat and sulfadiazine treatments reduced inflammation and granulation tissue formation by day 5 post-burn, while re-epithelialisation commenced by day 9 post-burn. In addition, burns treated with AE plus fat exhibited keratinised epidermis, associated with regular collagen fibres, compared to moderately dense collagen fibres without vascularisation in the sulfadiazine group. CONCLUSION: These findings suggested that AE plus fat was superior to sulfadiazine in enhancing burn wound healing in rats.

Sasanquasaponin from Camellia oleifera Abel Exerts an Anti-Inflammatory Effect in RAW 264.7 Cells via Inhibition of the NF-κB/MAPK Signaling Pathways.

Sasanquasaponin (SQS), a secondary metabolite that is derived from Camellia seeds, reportedly possesses notable biological properties. However, the anti-inflammatory effects of SQS and its underlying mechanisms remain poorly explored. Herein, we aimed to investigate the anti-inflammatory properties of SQS against lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells, focusing on the nuclear factor-κB (NF-κB) and MAPK signaling pathways. SQS was isolated using a deep eutectic solvent and D101 macroporous adsorption resin and analyzed using high-performance liquid chromatography. The viability of LPS-stimulated RAW264.7 was assessed using the CCK-8 assay. The presence of reactive oxygen species (ROS) was evaluated using 2',7'-dichlorofluorescein-diacetate. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were detected using reverse transcription-quantitative PCR and ELISA. Western blot was performed to analyze the protein expression of LPS-induced RAW264.7 cells. Herein, SQS exhibited anti-inflammatory activity: 30 µg/mL of SQS significantly reduced ROS generation, inhibited the LPS-induced expression of iNOS and COX-2, and attenuated the production of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α. The anti-inflammatory activity was potentially mediated by inhibiting the phosphorylation of IκBα and p65 in the NF-κB signaling pathway and the phosphorylation of ERK and JNK in the MAPK signaling pathway. Accordingly, SQS could inhibit inflammation in LPS-induced RAW264.7 cells by suppressing the NF-κB and MAPK signaling pathways. This study demonstrated the potential application of SQS as an anti-inflammatory agent.

Bombesin-like receptor 3 expression induced by bisphenol A is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing inflammation in liver cells.

BACKGROUND: Bisphenol A (BPA) is an exogenous endocrine disruptor mimicking hormones closely associated with health complications, such as cancer progression. BPA is also related to an increase in the prevalence of obesity-related diseases due to its obesogenic action. Bombesin-like receptor 3 (BRS3) is an important factor that should be considered in the adipogenic gene network, as depletion of this gene alters adiposity. METHODS: Therefore, the present study aimed to investigate the messenger ribonucleic acid (mRNA) expression of BRS3 in human liver THLE-2 cells post-BPA treatment by real-time polymerase chain reaction. The effects of BPA on the levels of pro-inflammatory proteins, interleukin 6 (IL6) and CC motif chemokine ligand 2 (CCL2), in conditioned media of BPA-treated THLE-2 cells and deoxyribonucleic acid (DNA) synthesis in replicating BPA-treated THLE-2 cells during the cell cycle were also examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. RESULTS: The study found that the mRNA expression of BRS3 was increased in THLE-2 cells treated with BPA. The study also showed that the expression levels of IL6 and CCL2 reached an optimum level in the conditioned media of BPA-treated THLE-2 cells after 48 h of treatment. Subsequently, the DNA synthesis analysis showed that bromodeoxyuridine/propidium iodide (BrdU/PI) stained positive cells were decreased in BPA-treated THLE-2 cells at 72 h of treatment. CONCLUSION: The study demonstrates that BRS3 expression induced by BPA is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing cellular inflammation in liver cells.

The anti-neoplastic effects of metformin modulate the acquired phenotype of fibroblast cells in the breast cancer-normal fibroblast co-culture system.

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFß, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFß in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFß secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer.

Fever-like temperature impacts on Staphylococcus aureus and Pseudomonas aeruginosa interaction, physiology, and virulence both in vitro and in vivo.

BACKGROUND: Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) cause a wide variety of bacterial infections and coinfections, showing a complex interaction that involves the production of different metabolites and metabolic changes. Temperature is a key factor for bacterial survival and virulence and within the host, bacteria could be exposed to an increment in temperature during fever development. We analyzed the previously unexplored effect of fever-like temperatures (39 °C) on S. aureus USA300 and P. aeruginosa PAO1 microaerobic mono- and co-cultures compared with 37 °C, by using RNAseq and physiological assays including in vivo experiments. RESULTS: In general terms both temperature and co-culturing had a strong impact on both PA and SA with the exception of the temperature response of monocultured PA. We studied metabolic and virulence changes in both species. Altered metabolic features at 39 °C included arginine biosynthesis and the periplasmic glucose oxidation in S. aureus and P. aeruginosa monocultures respectively. When PA co-cultures were exposed at 39 °C, they upregulated ethanol oxidation-related genes along with an increment in organic acid accumulation. Regarding virulence factors, monocultured SA showed an increase in the mRNA expression of the agr operon and hld, pmsα, and pmsß genes at 39 °C. Supported by mRNA data, we performed physiological experiments and detected and increment in hemolysis, staphyloxantin production, and a decrease in biofilm formation at 39 °C. On the side of PA monocultures, we observed an increase in extracellular lipase and protease and biofilm formation at 39 °C along with a decrease in the motility in correlation with changes observed at mRNA abundance. Additionally, we assessed host-pathogen interaction both in vitro and in vivo. S. aureus monocultured at 39οC showed a decrease in cellular invasion and an increase in IL-8-but not in IL-6-production by A549 cell line. PA also decreased its cellular invasion when monocultured at 39 °C and did not induce any change in IL-8 or IL-6 production. PA strongly increased cellular invasion when co-cultured at 37 and 39 °C. Finally, we observed increased lethality in mice intranasally inoculated with S. aureus monocultures pre-incubated at 39 °C and even higher levels when inoculated with co-cultures. The bacterial burden for P. aeruginosa was higher in liver when the mice were infected with co-cultures previously incubated at 39 °C comparing with 37 °C. CONCLUSIONS: Our results highlight a relevant change in the virulence of bacterial opportunistic pathogens exposed to fever-like temperatures in presence of competitors, opening new questions related to bacteria-bacteria and host-pathogen interactions and coevolution.

New Derivatives of 1-(3-Methyl-1-Benzofuran-2-yl)Ethan-1-one: Synthesis and Preliminary Studies of Biological Activity.

A set of nine derivatives, including five brominated compounds, was synthesized and the structures of these novel compounds were confirmed using 1H and 13C NMR as well as ESI MS spectra. These compounds were tested on four different cancer cell lines, chronic myelogenous leukemia (K562), prostate cancer (PC3), colon cancer (SW620), human kidney cancer (Caki 1), and on healthy human keratocytes (HaCaT). MTT results reveal that two newly developed derivatives (6 and 8) exhibit selective action towards K562 cells and no toxic effect in HaCat cells. The biological activity of these two most promising compounds was evaluated by trypan blue assay, reactive oxygen species generation, and IL-6 secretion. To investigate the proapoptotic activity of selected compounds, the two following types of tests were performed: Annexin V Apoptosis Detection Kit I and Caspase-Glo 3/7 assay. The studies of the mechanism showed that both compounds have pro-oxidative effects and increase reactive oxygen species in cancer cells, especially at 12 h incubation. Through the Caspase-Glo 3/7 assay, the proapoptotic properties of both compounds were confirmed. The Annexin V-FITC test revealed that compounds 6 and 8 induce apoptosis in K562 cells. Both compounds inhibit the release of proinflammatory interleukin 6 (IL-6) in K562 cells. Additionally, all compounds were screened for their antibacterial activities using standard and clinical strains. Within the studied group, compound 7 showed moderate activity towards Gram-positive strains in antimicrobial studies, with MIC values ranging from 16 to 64 µg/mL.

Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

The radiopharmaceutical radium-223 has immunomodulatory effects in patients and facilitates anti-programmed death receptor-1 therapy in murine models of bone metastatic prostate cancer.

BACKGROUND & PURPOSE: Radium-223 (Ra223) improves survival in metastatic prostate cancer (mPC), but its impact on systemic immunity is unclear, and biomarkers of response are lacking. We examined markers of immunomodulatory activity during standard clinical Ra223 and studied the impact of Ra223 on response to immune checkpoint inhibition (ICI) in preclinical models. MATERIALS & METHODS: We conducted a single-arm biomarker study of Ra223 in 22 bone mPC patients. We measured circulating immune cell subsets and a panel of cytokines before and during Ra223 therapy and correlated them with overall survival (OS). Using two murine mPC models-orthotopic PtenSmad4-null and TRAMP-C1 grafts in syngeneic immunocompetent mice-we tested the efficacy of combining Ra223 with ICI. RESULTS: Above-median level of IL-6 at baseline was associated with a median OS of 358 versus 947 days for below levels; p = 0.044, from the log-rank test. Baseline PlGF and PSA inversely correlated with OS (p = 0.018 and p = 0.037, respectively, from the Cox model). Ra223 treatment was associated with a mild decrease in some peripheral immune cell populations and a shift in the proportion of MDSCs from granulocytic to myeloid. In mice, Ra223 increased the proliferation of CD8+ and CD4+ helper T cells without leading to CD8+ T cell exhaustion in the mPC lesions. In one of the models, combining Ra223 and anti-PD-1 antibody significantly prolonged survival, which correlated with increased CD8+ T cell infiltration in tumor tissue. CONCLUSION: The inflammatory cytokine IL-6 and the angiogenic biomarker PlGF at baseline were promising outcome biomarkers after standard Ra223 treatment. In mouse models, Ra223 increased intratumoral CD8+ T cell infiltration and proliferation and could improve OS when combined with anti-PD-1 ICI.

Blocking IL-17A prevents oxycodone-induced depression-like effects and elevation of IL-6 levels in the ventral tegmental area and reduces oxycodone-derived physical dependence in rats.

Oxycodone is the most prescribed opioid for pain management and has been available in clinics for almost a century, but effects of chronic oxycodone have been studied less than morphine in preclinical and clinical studies. Newly developed depression has been coupled with chronic oxycodone use in a few clinical studies, but no preclinical studies have investigated the pathogenesis of oxycodone-induced depression. Gut microbiome changes following oxycodone use is an understudied area, and interleukin-17A (IL-17A) is linked to both the development of mood disorders and regulation of gut microbiome. The present study investigated effects of chronic oxycodone exposure on mood-related behaviors (depression and anxiety), pain hypersensitivity, physical dependence, immune markers, and the gut microbiome and tested the hypothesis that blocking IL-17A with a systemically administered monoclonal antibody reduces oxycodone-derived effects. Oxycodone (using an incremental dosing regimen) or saline was injected twice a day for 12 days. IL-17A Ab (200 µg/100 µl) or saline was administered every 3rd day during the 12-day interval. Chronic oxycodone induced a depression-like effect, but not anxiogenic- or anxiolytic-like effects; promoted hyperalgesia; increased IL-17A and IL-6 levels in the ventral tegmental area (VTA); and induced physical dependence. IL-17A Ab co-administration with oxycodone prevented the depression-like effect and hyperalgesia, reduced naloxone-precipitated withdrawal signs, and normalized the increase in cytokine levels. Chronic oxycodone exposure did not affect gut microbiome and integrity. Our results identify a role for IL-17A in oxycodone-related behavioral and neuroimmune effects and show that IL-17A Ab has potential therapeutic value in blocking these effects. Given that humanized IL-17A Ab is approved for treatment of psoriasis and psoriatic arthritis, our findings point toward studying it for use in the treatment of oxycodone use disorder.

Are silymarin and N-acetylcysteine able to prevent liver damage mediated by multiple factors? Findings against ethanol plus LPS-induced liver injury in mice.

This study investigated the effect of N-acetylcysteine (NAC) and silymarin (SIL) in the liver of mice exposed to ethanol and lipopolysaccharides (LPS). Mice were divided into four groups (n = 6): naive, vehicle, NAC (200 mg/kg), and SIL (200 mg/kg). Treatments were given orally (po) once daily for 10 days. Liver injury was induced by administration of ethanol (30%, po) for 10 days, once daily, followed by a single administration of LPS (2 mg/kg, ip) 24 h before euthanasia. After the treatment period, animals were euthanized, and liver and blood samples were collected. NAC, but not SIL, prevented the increase in oxalacetic glutamic transaminase (OGT) and pyruvic glutamic transaminase (PGT) serum levels. NAC and SIL did not restore levels of reduced glutathione or hepatic malonaldehyde. The treatments with NAC or SIL showed no difference in the activity of glutathione S-transferase, superoxide dismutase, and catalase compared to vehicle group. Myeloperoxidase and N-acetylglucosaminidase activities are increased, as well as the IL-6 and IL-10 levels in the liver. The treatment with NAC, but not SIL, reduced the N-acetylglucosamines activity and the IL-6 and IL-10 amount in the liver. Histological findings revealed microsteatosis in the vehicle group, which was not prevented by SIL but was partially reduced in animals receiving NAC. Unlike other liver injury models, NAC (200 mg/kg) or SIL (200 mg/kg) did not positively affect antioxidant patterns in liver tissue of animals exposed to ethanol plus LPS, but NAC treatment displays anti-inflammatory properties in this model.

An in vitro coculture approach to study the interplay between dental pulp cells and Streptococcus mutans.

AIM: To develop a new coculture system that allows exposure of dental pulp cells (DPCs) to Streptococcus mutans and dentine matrix proteins (eDMP) to study cellular interactions in dentine caries. METHODOLOGY: Dental pulp cells and S. mutans were cocultured with or without eDMP for 72 h. Cell proliferation and viability were assessed by cell counting and MTT assays, while bacterial growth and viability were determined by CFU and LIVE/DEAD staining. Glucose catabolism and lactate excretion were measured photometrically as metabolic indicators. To evaluate the inflammatory response, the release of cytokines and growth factors (IL-6, IL-8, TGF-ß1, VEGF) was determined by ELISA. Non-parametric statistical analyses were performed to compare all groups and time points (Mann-Whitney U test or Kruskal-Wallis test; α = .05). RESULTS: While eDMP and especially S. mutans reduced the number and viability of DPCs (p ≤ .0462), neither DPCs nor eDMP affected the growth and viability of S. mutans during coculture (p > .0546). The growth of S. mutans followed a common curve, but the death phase was not reached within 72 h. S. mutans consumed medium glucose in only 30 h, whereas in the absence of S. mutans, cells were able to catabolize glucose throughout 72 h, resulting in the corresponding amount of l-lactate. No change in medium pH was observed. S. mutans induced IL-6 production in DPCs (p ≤ .0011), whereas eDMP had no discernible effect (p > .7509). No significant changes in IL-8 were observed (p > .198). TGF-ß1, available from eDMP supplementation, was reduced by DPCs over time. VEGF, on the other hand, was increased in all groups during coculture. CONCLUSIONS: The results show that the coculture of DPCs and S. mutans is possible without functional impairment. The bacterially induced stimulation of proinflammatory and regenerative cytokines provides a basis for future investigations and the elucidation of molecular biological relationships in pulp defence against caries.

Targeting HIF-1α alleviates the inflammatory responses and rebuilds the CD4+ T cell subsets balance in the experimental autoimmune myasthenia gravis inflammation model via regulating cellular and humoral immunity.

BACKGROUND: Cells and tissues in an inflammatory state are usually hypoxic. The hypoxic environment can affect the differentiation of immune cells and produce Hypoxia-inducible Factor-1α (HIF-1α). Inflammation is also a major contributor to the development and deterioration of Myasthenia Gravis (MG). There are limited studies on the immunopathological mechanism and targeted therapy associated with MG exacerbated with inflammation. This research aimed to explore whether BAY 87-2243 (HIF-1α inhibitor) ameliorates the symptoms of the Experimental Autoimmune Myasthenia Gravis (EAMG) inflammation model and study its regulatory mechanism on cellular immunity and humoral immunity. METHODS: We first establish the EAMG inflammation model using Lipopolysaccharide (LPS), BAY 87-2243 was applied to the EAMG inflammation model and its therapeutic effects were evaluated in vivo and in vitro experiments. RESULTS: The proportion of Treg cells was increased whereas Th1, Th17, and Th1/17 cells were decreased in BAY 87-2243-treated EAMG inflammation model. BAY 87-2243 ameliorated the acetylcholine receptors (AChRs) loss and the complement deposited at the neuromuscular junction of the EAMG inflammation model, declined the levels of IFN-γ, IL-17, and IL-6 in serum, and further attenuated responses in the germinal center and reduced the antibody levels by inhibiting the IL-6-dependent STAT3 axis. CONCLUSION: BAY 87-2243 restored the balance of CD4+T cell subsets and reduced the production of the pro-inflammatory cytokines, thus acting as both an immune imbalance regulator and anti-inflammatory. The current study suggests that HIF-1α might be a potential target for the treatment of MG exacerbated with inflammation.

Role of microbiota in radiation-induced small-bowel damage.

Radiation-induced gastrointestinal damage is a common acute radiation syndrome. Previous studies have highlighted that Galectin-1 and Interleukin-6 (IL-6) are associated with flaking of small intestinal villi and intestinal radioresistance. Therefore, our goal is to study whether gut bacteria regulated by galectin-1 or IL-6 can mitigate radiation-induced small intestine damage. In this study, differences between galectin-1, sgp130-regulated and wild-type (WT) mice were analyzed by microbiome array. The effects of the Firmicutes/Bacteroidetes (F/B) ratio and the proportion of bacterial distribution at the phylum level were observed after 18 Gy whole abdomen radiation. Fecal microbiota transplantation was used to implant radioresistant gut flora into WT mice, and the number of viable small intestinal crypt foci was observed by immunohistochemistry. Fecal transplantation from galectin-1 knockout and sgp130 transgenic mice, with higher radiation resistance, into WT mice significantly increased the number of surviving small intestinal crypts. This radiation resistance, generated through gene regulation, was not affected by the F/B ratio. We initially found that the small intestinal villi of WT mice receiving radioresistant mouse fecal bacteria demonstrated better repair outcomes after radiation exposure. These results indicate the need for a focus on the identification and application of superior radioresistant bacterial strains. In our laboratory, we will further investigate specific radioresistant bacterial strains to alleviate acute side effects of radiation therapy to improve the patients' immune ability and postoperative quality of life.

METTL14 promotes IL-6-induced viability, glycolysis and inflammation in HaCaT cells via the m6A modification of TRIM27.

Interleukin-6 (IL-6) is a cytokine generated by healthy constituents of the skin, but is also up-regulated by a wide range of skin lesions and inflammatory conditions to trigger cytopathy of skin cells. TRIM27 was identified to contribute to the functional effects of IL-6 on skin cells. However, the underlying mechanism was not clear. Lentivirus infection was used for gene overexpression or silencing. RT-PCR and Western blot were used to respectively assess mRNA and protein levels. Cell viability was assessed by CCK-8 assay. Extracellular flux analysis was used to assess the levels of oxygen consumption rate and extracellular acidification rate. Mouse back skin was treated with imiquimod to produce psoriasis-like inflammation in vivo. Histological assessment and immunohistochemistry staining were respectively applied to analyse lesioned mouse and human skin samples. IL-6-induced increased viability, glycolysis and inflammation in keratinocytes was inhibited both by a chemical methylation inhibitor and by METTL14 knockdown. Further investigation found that METTL14 induces m6A methylation of TRIM27, which is recognized by a m6A reader, IGF2BP2. Elevation of TRIM27 level and activation of IL-6/STAT3 signalling pathway were found in an in vivo psoriasis-like inflammation model, whereas inhibition m6A methylation strongly alleviated the inflammation. Finally, METTL14, TRIM27, STAT3, p-STAT3 and IL-6 expressions were all found to be increased in clinical skin samples of psoriatic patients. Our results unravelled METTL14/TRIM27/IGF2BP2 signalling axis in keratinocyte cytopathy, which plays a critical role in facilitating the activation of IL-6/STAT3 signalling pathway. Our findings should provide inspirations for the design of new therapeutics for skin inflammatory diseases including psoriasis.