TGF-ß and TNF-α interaction promotes the expression of MMP-9 through H3K36 dimethylation: implications in breast cancer metastasis.

Increased MMP-9 expression in the tumor microenvironment (TME) plays a crucial role in the extracellular matrix remodeling to facilitate cancer invasion and metastasis. However, the mechanism of MMP-9 upregulation in TME remains elusive. Since TGF-ß and TNF-α levels are elevated in TME, we asked whether these two agents interacted to induce/augment MMP-9 expression. Using a well-established MDA-MB-231 breast cancer model, we found that the synergy between TGF-ß and TNF-α led to MMP-9 upregulation at the transcriptional and translational levels, compared to treatments with each agent alone. Our in vitro findings are corroborated by co-expression of elevated MMP-9 with TGF-ß and TNF-α in human breast cancer tissues. Mechanistically, we found that the MMP-9 upregulation driven by TGF-ß/TNF-α cooperativity was attenuated by selective inhibition of the TGF-ßRI/Smad3 pathway. Comparable outcomes were observed upon inhibition of TGF-ß-induced phosphorylation of Smad2/3 and p38. As expected, the cells defective in Smad2/3 or p38-mediated signaling did not exhibit this synergistic induction of MMP-9. Importantly, the inhibition of histone methylation but not acetylation dampened the synergistic MMP-9 expression. Histone modification profiling further identified the H3K36me2 as an epigenetic regulatory mark of this synergy. Moreover, TGF-ß/TNF-α co-stimulation led to increased levels of the transcriptionally permissive dimethylation mark at H3K36 in the MMP-9 promoter. Comparable outcomes were noted in cells deficient in NSD2 histone methyltransferase. In conclusion, our findings support a cooperativity model in which TGF-ß could amplify the TNF-α-mediated MMP-9 production via chromatin remodeling and facilitate breast cancer invasion and metastasis.

Inhibition of CEACAM1 expression in cytokine-activated neutrophils using JAK inhibitors.

OBJECTIVES: Carcinoembryonic-antigen-related cell-adhesion molecule 1 (CEACAM1) is an adhesion molecule that acts as a coinhibitory receptor in the immune system. We previously demonstrated that CEACAM1 is predominantly expressed on peripheral blood neutrophils in patients with RA. The aim of the present study was to investigate the effects of Janus kinase inhibitors (JAKi) on cytokine-activated human neutrophils and CEACAM1 expression. METHODS: Peripheral blood neutrophils were obtained from healthy subjects. Isolated neutrophils were stimulated with tumor necrosis factor-alpha (TNF-α) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of JAKi. The expression of CEACAM1 in peripheral blood neutrophils was analyzed by flow cytometry. Protein phosphorylation of signal transducer and activator of transcription (STAT)1, STAT3, and STAT5 was assessed by western blot using phospho-specific antibodies. RESULTS: We found that TNF-α-induced CEACAM1 expression was marginally suppressed after pretreatment with pan-JAK inhibitor, tofacitinib. Moreover, TNF-α induced STAT1 and STAT3 phosphorylation at the late stimulation phase (4 to 16 h). The expressions of CEACAM1 on neutrophils were markedly up-regulated by GM-CSF not by interleukin (IL)-6 stimulation. All JAKi inhibited GM-CSF-induced CEACAM1 expressions on neutrophils, however, the inhibitory effects of baricitinib were larger compared to those of tofacitinib or filgotinib. Moreover, CEACAM1 was marginally upregulated in interferon (IFN)-γ stimulated neutrophils. Similarly, JAKi inhibited IFN-γ-induced CEACAM1 expressions on neutrophils. CONCLUSIONS: We demonstrated that JAKi prevent GM-CSF-induced CEACAM1 expression in neutrophils, and JAKi-induced inhibition depends on their selectivity against JAK isoforms. These findings suggest that JAKi can modulate the expression of CEACAM1 in cytokine-activated neutrophils, thereby limiting their activation.

Role of endothelial dysfunction in sleep-disordered breathing in egyptian children with sickle cell disease.

BACKGROUND: Endothelial dysfunction is an integral pathophysiologic mechanism in sickle cell disease (SCD), and can lead to many complications. Sleep-disordered breathing (SDB) is a SCD complication with diverse incidence and pathophysiology. This study aimed to determine the prevalence of SDB in children with SCD and to assess its relation to endothelial dysfunction. METHODS: Sixty children with SCD and 60 healthy controls were enrolled. The levels of TNF-α, IL-6, and IL-17A were evaluated in the entire cohort using enzyme-linked immunosorbent assay (ELISA) kits. Polysomnography (PSG) was performed for all SCD patients after completion of the Pediatric Sleep Questionnaire (PSQ). RESULTS: TNF-α, IL-6, and IL-17A levels were significantly greater in children with SCD than in controls (p-values < 0.001, < 0.001, and 0.006, respectively). The PSQ revealed symptoms suggestive of SDB in 50 children with SCD (83.3%), and PSG revealed obstructive sleep apnea (OSA) in 44 children with SCD (73.3%); 22 patients had mild OSA, and 22 had moderate-to-severe OSA according to the apnea-hypopnea index (AHI). TNF-α was significantly greater in SCD children who reported heavy or loud breathing, trouble breathing or struggle to breathe, and difficulty waking up in the morning (p-values = 0.002, 0.002, and 0.031, respectively). The IL-6 levels were significantly greater in SCD children who stopped growing normally (p-value = 0.002). The levels of IL-6 and IL-17A were significantly greater in SCD children with morning headaches (p-values = 0.007 and 0.004, respectively). CONCLUSION: Children with SCD showed a high prevalence of SDB with significantly elevated levels of markers of endothelial function, highlighting the interplay of SDB and endothelial dysfunction in SCD.

Adalimumab Treatment Effects on Inflammation and Adipose Tissue Mitochondrial Respiration in Hidradenitis Suppurativa.

OBJECTIVE: Tumour necrosis factor (TNF)-α is a proinflammatory marker and has been shown to affect mitochondrial function in different tissues. We investigated the effect on adipose tissue (AT) inflammation and mitochondrial respiration in patients with hidradenitis suppurativa (HS) after 12 weeks of treatment with adalimumab, a TNF-α inhibitor. METHODS: We sampled blood and an AT biopsy from 13 patients with HS and 10 control subjects after an overnight fast. The patients were retested after at least 12 weeks of treatment with adalimumab (40 mg/week). We measured macrophage content and mitochondrial respiration in the AT and interleukin (IL)-1ß, IL-6, IL-10, high-sensitivity C-reactive protein (hsCRP), interferon-γ, TNF-α, adiponectin and leptin in plasma. Clinical scores and Dermatology Quality of Life Index (DLQI) were assessed. RESULTS: We found a higher anti-inflammatory macrophage content (CD206+) in the patient group compared with the control group, but no differences between before and after the intervention. No difference in mitochondrial respiration was observed. We observed higher plasma IL-6 and hsCRP concentrations in patients with HS compared to controls, with no differences before and after the intervention. The difference between controls and HS patients was abolished after the intervention. HS patients improved their DLQI after the intervention with no change in clinical scores. CONCLUSION: Treatment with adalimumab in patients with HS does not alter AT inflammation or mitochondrial respiratory capacity; however, we did see a higher content of anti-inflammatory macrophages in the patient group compared with the control group.

Effect of mitochondrial coenzyme-Q10 precursor solanesol in gentamicin-induced experimental nephrotoxicity: Evidence from restoration of ETC-complexes and histopathological alterations.

Nephrotoxicity occurs when the body is exposed to certain drugs or toxins. When kidney damage occurs, the kidney fails to eliminate excess urine and waste. Solanesol (C45H74O) is a tri-sesquiterpenoid alcohol first isolated from tobacco, and it is widely distributed in plants of the Solanaceae family. Solanesol (SNL) is an intermediate in the synthesis of coenzyme Q10 (CoQ10), an antioxidant which protects nerve cells. This study investigated the protective effect of SNL at doses of 30 and 60 mg/kg in gentamicin-induced nephrotoxicity in Wistar albino rats. Animals were distributed into six groups and administered 100 mg/kg gentamicin-intraperitoneal injection for 14 days. Biochemical assessments were performed on kidney homogenate, blood, and serum. Treatment with SNL was shown as lower serum levels of creatinine, blood urea nitrogen (BUN), thiobarbituric acid reactive substances (TBARS), and Tumor necrosis factor alpha)TNF-α ((p < .001). It also restored reduced glutathione (GSH) and mitochondrial complex enzymatic activity as protective measures against gentamicin-induced nephrotoxicity. SNL were shown to reduce inflammation and oxidative stress markers (p < .001). Histological findings furtherly augmented the protective effects of SNL. Long-term SNL therapy also restored mitochondrial electron transport chain complex enzymes, such as complex-I (p < .001). In conclusion, these findings suggest that SNL can represent a protective therapeutic option for drug-induced nephrotoxicity, a long-term adverse effect of aminoglycoside antibiotics such as gentamicin.

Safety, pharmacokinetics, and pharmacodynamics of ART-648, a PDE4 inhibitor in healthy subjects: A randomized, placebo-controlled phase I study.

Phosphodiesterase 4 (PDE4) inhibitor is associated with a broad-spectrum anti-inflammatory mechanism. However, securing clinically efficacious doses with sufficient safety margins remains challenging due to class specific adverse events that are often unavoidable in the clinic. ART-648 is an orally available PDE4 inhibitor being developed for the treatment of inflammatory diseases. According to the estimated clinical doses based on an in vitro whole-blood assay, a phase I study was designed. The purpose of this phase I study was to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) following single and multiple administration of ART-648 in healthy subjects. PD was assessed by suppression of lipopolysaccharide-induced TNFα release in ex vivo whole-blood assay. In the single rising dose study, ART-648 was safe and well tolerated with a dose-proportional increase in exposures up to 4 mg. Single doses of ART-648 demonstrated dose-dependent PD response, indicating target engagement at 2-8 mg doses. In the multiple rising dose study, doses up to 4 mg BID after careful titration were well tolerated, while doses up to 6 mg BID were tolerated not in all but the majority of subjects. In conclusion, ART-648 exhibits a favorable PK profile with robust target engagement at clinically safe and tolerated doses identified in healthy subjects.

Caffeine mitigates tamoxifen-induced fatty liver in Wistar rats.

PURPOSE: Tamoxifen, a widely used drug for breast cancer treatment, is associated with adverse effects on the liver, including the development of fatty liver. This study aimed to investigate the potential protective effect of caffeine against tamoxifen-induced fatty liver in Wistar rats. METHODS: Rats were divided into normal control, tamoxifen + saline, and tamoxifen + caffeine. Plasma samples were assessed for biochemical markers related to oxidative stress, inflammation, liver function, and cell damage. Additionally, liver histopathology was examined to quantify the extent of fatty infiltration. RESULTS: In the tamoxifen + saline group, elevated levels of plasma malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α), alanine aminotransferase (ALT), cytokeratin 18, and soluble ST2 were observed compared to the normal control group, indicating increased oxidative stress, inflammation, and liver injury (p < 0.01). Moreover, histopathological examination revealed a significant increase in fatty infiltration (p < 0.001). However, in the tamoxifen + caffeine group, these markers were markedly reduced (p < 0.05, p < 0.01), and fatty infiltration was significantly mitigated (p < 0.001). CONCLUSIONS: The findings suggest that caffeine administration attenuates tamoxifen-induced fatty liver in rats by ameliorating oxidative stress, inflammation, liver injury, and cell damage. Histopathological evidence further supports the protective role of caffeine. This study highlights the potential of caffeine as a therapeutic intervention to counter tamoxifen-induced hepatic complications, contributing to the optimization of breast cancer treatment strategies.

Enzymatic TET-1 inhibition highlights different epigenetic behaviours of IL-1ß and TNFα in tumour progression of OS cell lines.

Osteosarcoma (OS) is the most frequent primary malignant bone tumour, whose heterogeneity represents a major challenge for common antitumour therapies. Inflammatory cytokines are known to be necessary for OS progression. Therefore, to optimise therapy, it is important to discover reliable biomarkers by identifying the mechanism generating OS and investigating the inflammatory pathways that support the undifferentiated state. In this work, we highlight the differences of epigenetic activities of IL-1ß and TNFα, and the susceptibility of TET-1 enzymatic inhibition, in tumour progression of three different OS cell lines. Investigating DNA methylation of IL-6 promoter and determining its expression, we found that TET enzymatic inhibition influences proliferation induced by inflammatory cytokines in OS cell lines. Moreover, Bobcat 339 treatment blocks IL-1ß epigenetic action on IL-6 promoter, while only partially those of TNFα as well as inhibits IL-1ß-dependent epithelial-mesenchymal transition (EMT) process, but only partially those of TNFα. In conclusion, this work highlights that IL-1ß and TNFα have different effects on DNA demethylation in OS cell lines, making DNA methylation a potential biomarker of disease. Specifically, in IL-1ß treatment, TET-1 inhibition completely blocks tumour progression, while in TNFα actions, it is only partially effective. Given that these two inflammatory pathways can be therapeutic targets for treating these tumours, knowledge of their distinct epigenetic behaviours can be useful for developing precise and specific therapeutic strategies for this disease.

SARS-CoV-2 spike protein induces the cytokine release syndrome by stimulating T cells to produce more IL-2.

Introduction: Cytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. Methods: Using spike protein combined with IL-2, IFN-γ, and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines, the content of cytokines in the supernatant was detected, and the effects of NK, T, and monocytes were analyzed. Results: This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1ß, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as T cells to release IFN-γ Mechanistically, IFN-γ and TNF-α enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore, TNF-α also activates NF-κB signaling in monocytes, which further cooperates with IFN-γ and spike protein to modulate NF-κB-dependent transcription of CRS-related inflammatory cytokines. Discussion: Targeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.

A comparative study of the developability of full-length antibodies, fragments, and bispecific formats reveals higher stability risks for engineered constructs.

Engineered antibody formats, such as antibody fragments and bispecifics, have the potential to offer improved therapeutic efficacy compared to traditional full-length monoclonal antibodies (mAbs). However, the translation of these non-natural molecules into successful therapeutics can be hampered by developability challenges. Here, we systematically analyzed 64 different antibody constructs targeting Tumor Necrosis Factor (TNF) which cover 8 distinct molecular format families, encompassing full-length antibodies, various types of single chain variable fragments, and bispecifics. We measured 15 biophysical properties related to activity, manufacturing, and stability, scoring variants with a flag-based risk approach and a recent in silico developability profiler. Our comparative assessment revealed that overall developability is higher for the natural full-length antibody format. Bispecific antibodies, antibodies with scFv fragments at the C-terminus of the light chain, and single-chain Fv antibody fragments (scFvs) have intermediate developability properties, while more complicated formats, such as scFv- scFv, bispecific mAbs with one Fab exchanged with a scFv, and diabody formats are collectively more challenging. In particular, our study highlights the propensity for fragmentation and aggregation, both in bulk and at interfaces, for many current engineered formats.

Ginsenoside Rh2 Alleviates LPS-Induced Inflammatory Responses by Binding to TLR4/MD-2 and Blocking TLR4 Dimerization.

Lipopolysaccharide (LPS) triggers a severe systemic inflammatory reaction in mammals, with the dimerization of TLR4/MD-2 upon LPS stimulation serving as the pivotal mechanism in the transmission of inflammatory signals. Ginsenoside Rh2 (G-Rh2), one of the active constituents of red ginseng, exerts potent anti-inflammatory activity. However, whether G-Rh2 can block the TLR4 dimerization to exert anti-inflammatory effects remains unclear. Here, we first investigated the non-cytotoxic concentration of G-Rh2 on RAW 264.7 cells, and detected the releases of pro-inflammatory cytokines in LPS-treated RAW 264.7 cells, and then uncovered the mechanisms involved in the anti-inflammatory activity of G-Rh2 through flow cytometry, fluorescent membrane localization, Western blotting, co-immunoprecipitation (Co-IP), molecular docking and surface plasmon resonance (SPR) analysis in LPS-stimulated macrophages. Our results show that G-Rh2 stimulation markedly inhibited the secretion of LPS-induced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nitric oxide (NO). Additionally, G-Rh2 blocked the binding of LPS with the membrane of RAW 264.7 cells through direct interaction with TLR4 and MD-2 proteins, leading to the disruption of the dimerization of TLR4 and MD-2, followed by suppression of the TLR4/NF-κB signaling pathway. Our results suggest that G-Rh2 acts as a new inhibitor of TLR4 dimerization and may serve as a promising therapeutic agent against inflammation.

Protective Effects of Vitamin D on Proteoglycans of Human Articular Chondrocytes through TGF-ß1 Signaling.

The extracellular matrix of cartilage primarily constitutes of collagen and aggrecan. Cartilage degradation starts with aggrecan loss in osteoarthritis (OA). Vitamin D (VD) plays an essential role in several inflammation-related diseases and can protect the collagen in cartilage during OA. The present study focused on the role of VD in aggrecan turnover of human articular chondrocytes treated with tumor necrosis factor α (TNF-α) and the possible mechanism. Treatment with different doses of VD and different periods of intervention with TNF-α and TGF-ß1 receptor (TGFßR1) inhibitor SB525334 were investigated. The viability of human chondrocytes and extracellular secretion of TGF-ß1 were measured. The expression of intracellular TGFßR1 and VD receptor was examined. Transcriptional and translational levels of aggrecan and the related metabolic factors were analyzed. The results showed that TNF-α markedly reduced the viability, TGFßR1 expressions and aggrecan levels of human chondrocytes, and increased disintegrin and metalloproteinase with thrombospondin motifs. The alterations were partially inhibited by VD treatment. Furthermore, the effects of VD were blocked by the TGFßR1 inhibitor SB525334 in TNF-α-treated cells. VD may prevent proteoglycan loss due to TNF-α via TGF-ß1 signaling in human chondrocytes.

Lupeol stimulates iNOS, TNF-α, and IL-10 expression in the U937 cell line infected with old-world Leishmania donovani.

OBJECTIVE: Visceral leishmaniasis is a neglected tropical disease that can be lethal if not treated. The available medicines have severe side effects, such as toxicity and drug resistance. Various investigations are looking into new anti-leishmanial compounds from natural products that have little impact on host cells. Lupeol, a triterpenoid present in the flora of many edible plants, has been shown to have antimicrobial properties. The present study investigated the immunomodulatory effects of lupeol on U937 macrophages infected with Leishmania donovani, focusing on the expression of key cytokines and enzymes involved in the immune response. METHODS: U937 macrophages were infected with Leishmania donovani amastigotes and treated with varying concentrations of lupeol throughout three days. The expression levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10) were measured using real-time polymerase chain reaction (RT-PCR). A positive simulation of gene expression was estimated using ΔΔCT to assess relative expression. RESULTS: The results demonstrated that lupeol significantly upregulated iNOS and TNF-α expression, especially at higher concentrations, indicating enhanced pro-inflammatory and anti-leishmanial activity. Interestingly, IL-10 expression also increased, suggesting a complex immunomodulatory role of lupeol that involves both pro-inflammatory and anti-inflammatory pathways. Pearson correlation analysis revealed a strong association between iNOS and TNF-α (0.97692), as well as a moderate correlation between iNOS and IL-10 (0.51603). CONCLUSION: These findings suggest that lupeol may promote a balanced immune response, enhancing the body's ability to combat L. donovani while potentially mitigating excessive inflammation. Lupeol can potentially serve as a novel therapeutic agent against visceral leishmaniasis.

[Protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on damage to hippocampal synaptic microenvironment in rats with diabetes-related depression based on microglia-neuron crosstalk signal CD300f/TLR4].

This study aims to reveal the protective effect and mechanism of Zuogui Jiangtang Jieyu Formula on the damage to hippo-campal synaptic microenvironment in rats with diabetes-related depression(DD) via regulating microglia immune receptor molecule-like family member f(CD300f)/Toll-like receptor 4(TLR4) signal. Firstly, the model of DD rats was established by a two-week high-fat diet+STZ injection+chronic mild and unpredictable stress plus isolation for 28 days. The rats were randomly divided into normal group, model group, CD300f blocker(CLM1, 2 µg·kg~(-1)) group, CD300f agonist(Fcγ, 5 µg·kg~(-1)) group, positive drug(0.18 g·kg~(-1) metformin+1.8 mg·kg~(-1) fluoxetine) group, and high-dose and low-dose(20.52 and 10.26 g·kg~(-1)) Zuogui Jiangtang Jieyu Formula groups. Depression-like behavior of rats was evaluated by open field and forced swimming experiments. The levels of blood glucose and insulin were detected by biochemical analysis. The levels of tumor necrosis factor α(TNF-α), interleukin-1ß(IL-1ß), indoleamine 2, 3-dioxygenase(IDO), 5-hydroxytryptamine(5-HT), and dopamine(DA) in the hippocampus were detected by enzyme-linked immunosorbent assay. The changes in the synaptic ultrastructure in hippocampal neurons of rats were observed by transmission electron microscopy. The protein expressions of CD300f, TLR4, synaptophysin(SYN), and postsynaptic density protein 95(PSD-95) in microglial cells of the hippocampus were detected by immunofluorescence and Western blot. The results indicated that compared with that in the normal group, the total movement distance in open field experiments was reduced in the model group, and the immobility time in forced swimming experiments increased, with an elevated insulin level in serum, as well as TNF-α, IL-1ß, and IDO levels in the hippocampus. The 5-HT and DA levels in the hippocampus were reduced. In addition, the CD300f expression was down-regulated in microglial cells of the hippocampus, and the TLR4 expression was up-regulated. Moreover, the expression of synapse-related proteins SYN and PSD-95 in hippocampal neurons decreased, and the synaptic ultrastructure of hippocampal neurons was significantly damaged. Compared with the model group, the CD300f blocker and agonist aggravated and alleviated the above abnormal changes, respectively. High-dose and low-dose Zuogui Jiangtang Jieyu Formula could significantly improve the above depression-like beha-vior in rats, inhibit the abnormal increase of TNF-α, IL-1ß, and IDO and the decrease of 5-HT and DA, effectively increase the expression of CD300f in microglial cells, and decrease the expression of TLR4. They could up-regulate the protein expression of presyna-ptic membrane SYN and postsynaptic membrane PSD-95 in hippocampal neurons and finally improve the damage to the hippocampal synaptic microenvironment. In conclusion, this research confirmed that Zuogui Jiangtang Jieyu Formula effectively alleviated the depression-like behavior and inhibited inflammatory activation of microglial cells in the hippocampus of rats with DD, and the mechanism might be related to the regulation of CD300f/TLR4 signal to alleviate the damage to hippocampal synaptic microenvironment.

[Daidzein attenuates high glucose-induced inflammatory injury in macrophages by regulating NLRP3 inflammasome signaling pathway].

This study aims to explore the inhibitory effect of daidzein on macrophage inflammation induced by high glucose via regulating the NOD-like receptor protein 3(NLRP3) inflammasome signaling pathway. The cell counting kit-8(CCK-8) assay was employed to detect the effects of daidzein at different concentrations on the viability of RAW264.7 cells. Western blot was employed to determine the protein level of tumor necrosis factor(TNF)-α in macrophages exposed to different concentrations of glucose for different time periods as well as the expression levels of proteins involved in the polarization and Toll-like receptor 4(TLR4)-myeloid differentiation factor(MyD88)-NLRP3 inflammasome pathway of the macrophages exposed to high glucose. Enzyme-linked immunosorbent assay was employed to measure the levels of TNF-α, interleukin(IL)-18, and IL-1ß secreted by macrophages. The expression level of nuclear factor-kappa B(NF-κB) p65 in macrophages exposed to high glucose was detected by immunofluorescence, and the level of intracellular reactive oxygen species(ROS) was detected by the DCFH-DA fluorescent probe. The mRNA levels of NLRP3, TNF-α, and IL-18 in macrophages were determined by qRT-PCR. The results showed that treatment with 30 mmol·L~(-1) glucose for 48 h was the best condition for the modeling of macrophage injury. Compared with the blank group, the model group showed improved polarization of macrophages, increased secretion of TNF-α, IL-18, and IL-1ß, elevated ROS level, and up-regulated expression of NF-κB p65. In addition, the modeling up-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 and the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. Compared with the model group, daidzein(10, 20, and 40 µmol·L~(-1)) lowered the levels of inflammatory cytokines and down-regulated the mRNA levels of NLRP3, TNF-α, and IL-18 as well as the protein levels of TLR4, MyD88, NLRP3, NF-κB p65, p-NF-κB p65, I-κB, p-I-κB, ASC, pro-caspase-1, pro-IL-1ß, cleaved IL-1ß, and pro-IL-18. In addition, daidzein reduced intracellular ROS. According to the available reports and the experimental results, high glucose can induce the polarization of macrophages and promote the secretion of inflammatory cytokines. Daidzein can inhibit the expression of ROS in macrophages by regulating the NLRP3 inflammasome signaling pathway, thereby reducing the inflammation of macrophages exposed to high glucose.

[Effects of electroacupuncture at "Jiaji" (EX-B 2) on extracellular matrix of chondrocytes and inflammatory reaction in rabbits with Modic changes of cartilage endplate].

OBJECTIVE: To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) on extracellular matrix (ECM) of chondrocytes and inflammatory reaction in rabbits with Modic changes (MC) of cartilage endplate, and to explore the mechanism of EA in treating MC of endplate cartilage. METHODS: Eighteen male New Zealand white rabbits were randomly divided into a sham operation group, a model group and an EA group, 6 rabbits in each group. Based on the autoimmune theory, MC model was established by embedding autogenous nucleus pulposus in the rabbits of the model group and the EA group, based on autoimmunity. After successful modeling, EA was applied at bilateral "Jiaji" (EX-B 2) of L5 and L6 in the EA group, with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, 20 min a time, once a day, 1-day interval was taken after continuous 6-day intervention, for 4 weeks totally. Before and after modeling, as well as before and after intervention, the comprehensive response score was observed. After modeling and intervention, magnetic resonance imaging (MRI) was used to observe the signal intensity of intervertebral disc and cartilage endplate. After intervention, the morphology of chondrocytes of cartilage endplate was observed by HE staining; the positive expression of a disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS5) and Aggrecan in the cartilage endplate was detected by immunohistochemistry; the levels of inflammatory factors i.e. interleukin-1ß (1L-1ß) and tumor necrosis factor-α (TNF-α) in the cartilage endplate were detected by ELISA; the protein expression of ADAMTS5, Aggrecan, matrix metalloproteinase-13 (MMP-13), IL-1ß and TNF-α in the cartilage endplate was detected by Western blot. RESULTS: Compared with the sham operation group, in the model group, the comprehensive response score was decreased (P<0.01); L5/L6 intervertebral disc and the cancellous bones of endplate vertebral body showed low signal and unclear boundary; the chondrocytes of the cartilage endplate increased significantly, the cells were enlarged and hypertrophic, and the nuclei were wrinkled and clustered; the positive expression of ADAMTS5 as well as the levels of IL-1ß and TNF-α were increased (P<0.01), while the positive expression of Aggrecan was decreased (P<0.01) in the cartilage endplate; the protein expression of ADAMTS5, MMP-13, IL-1ß and TNF-α was increased (P<0.01), while that of Aggrecan was decreased (P<0.01) in the cartilage endplate. Compared with the model group, in the EA group, the comprehensive response score was increased (P<0.01); the signal of L5/L6 intervertebral disc and the cancellous bones of endplate vertebral body was enhanced; the chondrocytes of the cartilage endplate were reduced, the nuclei were slightly crumpled and scattered; the positive expression of ADAMTS5 as well as the levels of IL-1ß and TNF-α were decreased (P<0.05, P<0.01), while the positive expression of Aggrecan was increased (P<0.01) in the cartilage endplate; the protein expression of ADAMTS5, MMP-13, IL-1ß and TNF-α was decreased (P<0.05, P<0.01), while that of Aggrecan was increased (P<0.05) in the cartilage endplate. CONCLUSION: EA at "Jiaji" (EX-B 2) can delay the MC of cartilage endplate. The mechanism may be related to inhibiting the degradation of ECM of chondrocytes and the secretion of inflammatory factors, and repairing the degeneration of endplate cartilage.

Observation of clinical efficacy of acupuncture combined with bloodletting therapy and Qingwen Xiere decoction in mild to moderate COVID-19 patients. / é’ˆåˆºè”åˆåˆºè¡€åŠæ¸ ç˜Ÿæ³„çƒ­æ±¤å¯¹æ–°åž‹å† çŠ¶ç— æ¯’æ„ŸæŸ“è½»ä¸­åž‹æ‚£è€ ä¸´åºŠç–—æ•ˆè§‚å¯Ÿ.

OBJECTIVES: To evaluate the clinical efficacy and safety of acupuncture combined with bloodletting therapy and Qingwen Xiere decoction in mild to moderate COVID-19 patients. METHODS: A total of 100 mild to moderate COVID-19 patients collected from December 2022 to February 2023 were randomly divided into control and observation groups, with 50 patients in each group. Patients in the control group received oral Qingwen Xiere decoction for 6 days. The observation group received acupuncture combined with bloodletting therapy in addition to oral Qingwen Xiere decoction, with the acupuncture (at Kongzui ï¼»LU6ï¼½, Hegu ï¼»LI4ï¼½, Quchi ï¼»LI11ï¼½, Feishu ï¼»BL13ï¼½, Zhongwan ï¼»CV12ï¼½, Qihai ï¼»CV6ï¼½, Yinlingquan ï¼»SP9ï¼½) administered 30 min each day for 6 days, and bloodletting (at Shaoshang ï¼»LU11ï¼½, Shangyang ï¼»LI1ï¼½, Dazhui ï¼»GV14ï¼½) administered every other day for 3 times. Traditional Chinese medicine syndrome scores and pulmonary CT scores were recorded before and after treatment. Serum contents of C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were measured using ELISA. Anxiety and depression degree were assessed using the Hamilton Depression Scale (HAMD) and Hamilton Anxiety Scale (HAMA). Safety of the treatments was evaluated in both groups. RESULTS: Compared with before treatment, after treatment, the control group showed improvement in fever, dry cough, sore throat, and total traditional Chinese medicine syndrome scores (P<0.01), but no significant improvement in muscle pain or fatigue；the observation group showed significant improvement in total traditional Chinese medicine syndrome and individual symptoms scores (P<0.01)；both groups demonstrated reductions in pulmonary CT scores, HAMA score, HAMD score and serum contents of CRP and IL-6 (P<0.01)；serum TNF-α content significantly decreased in the observation group (P<0.01). All outcome measures were superior in the observation group to the control group (P<0.01, P<0.05). No adverse reactions were reported in either group. CONCLUSIONS: Acupuncture combined with bloodletting therapy and oral Qingwen Xiere decoction effectively improves clinical symptoms, alleviates pulmonary inflammatory injury, reduces inflammatory cytokine contents, and mitigates anxiety and depression in mild to moderate COVID-19 patients, and without adverse effects.

Cellular and In Vivo Response to Industrial, Food Grade, and Photocatalytic TiO2 Nanoparticles.

We encounter titanium dioxide nanoparticles (TiO2 NPs) throughout our daily lives in the form of food coloring, cosmetics, and industrial materials. They are used on a massive industrial scale, with over 1 million metric tons in the global market. For the workers who process these materials, inhalation is a major concern. The goal of our current research is to provide a direct comparison of the three major types of TiO2 NPs (P25, E171, R101) in terms of surface characterization, cellular response, and in vivo response following introduction into the lungs of mice. In both cellular and in vivo experiments, we observe a pro-inflammatory response to the P25 TiO2 NPs that is not observed in the E171 or R101 TiO2 NPs at mass-matched concentrations. Cellular experiments measured a cytokine, TNF-α, as a marker of a pro-inflammatory response. In vivo experiments in mice measured the number of immune cells and four pro-inflammatory cytokines (IL-6, MIP-2, IP-10, and MCP-1) present in bronchoalveolar lavage fluid. A detailed physical and chemical characterization of the TiO2 NPs shows that the P25 TiO2 NPs are distinguished by smaller primary particles suggesting that samples matched by mass contain a larger number of P25 TiO2 NPs. Cellular dose-response measurements with the P25, E171, and R101 TiO2 NPs support this hypothesis showing increased TNF-α release by macrophages as a function of TiO2 NP dose. Overall, this direct comparison of the three major types of TiO2 NPs shows that the number of particles in a dose, which is dependent on the particle diameter, is a key parameter in TiO2 NP-induced inflammation.

Immune system adaptation during gender-affirming testosterone treatment.

Infectious, inflammatory and autoimmune conditions present differently in males and females. SARS-CoV-2 infection in naive males is associated with increased risk of death, whereas females are at increased risk of long COVID1, similar to observations in other infections2. Females respond more strongly to vaccines, and adverse reactions are more frequent3, like most autoimmune diseases4. Immunological sex differences stem from genetic, hormonal and behavioural factors5 but their relative importance is only partially understood6-8. In individuals assigned female sex at birth and undergoing gender-affirming testosterone therapy (trans men), hormone concentrations change markedly but the immunological consequences are poorly understood. Here we performed longitudinal systems-level analyses in 23 trans men and found that testosterone modulates a cross-regulated axis between type-I interferon and tumour necrosis factor. This is mediated by functional attenuation of type-I interferon responses in both plasmacytoid dendritic cells and monocytes. Conversely, testosterone potentiates monocyte responses leading to increased tumour necrosis factor, interleukin-6 and interleukin-15 production and downstream activation of nuclear factor kappa B-regulated genes and potentiation of interferon-γ responses, primarily in natural killer cells. These findings in trans men are corroborated by sex-divergent responses in public datasets and illustrate the dynamic regulation of human immunity by sex hormones, with implications for the health of individuals undergoing hormone therapy and our understanding of sex-divergent immune responses in cisgender individuals.