Expression Levels Of Cytokines Research Articles

The role of IFN-γ/CXCL10 axis in Mycoplasma pneumonia infection

Mycoplasma pneumoniae caused lower respiratory tract infection in children and can exacerbate these infections through the production of various inflammatory factors, with chemokines playing a key role. However, the pathogenesis of this infection is complicated and thus has not been thoroughly studied. We clarified that cytokine expression levels were analyzed in both peripheral blood and bronchoalveolar lavage fluid (BALF), and in vitro assays were conducted using THP-1 macrophages. We discovered that, compared to control children, M. pneumoniae pneumonia (MPP) patients expressed significantly higher levels of CXCL10 both in the peripheral blood and BALF. Moreover, numbers of macrophages, predominantly with a M1 phenotype, were significantly increased in BALFs of children of MPP. In vitro, coculture with IFN-γ or activated CD4+ Th1 cells significantly promoted CXCL10 expressions in THP-1 derived macrophages, which was largely reversed by siRNA-mediated down regulation of STAT1. In addition, IFN-γ-stimulated macrophages greatly promoted the trans-migration of Th1 cells. our data show that Th1 cells-derived IFN-γ augments CXCL10 production in macrophages via the JAK-STAT1 pathway, which subsequently recruits more immune cells like Th1 cells into the infection sites, thereby constituting a positive feedback loop and aggravating the type I inflammatory responses in MPP patients.

Afobazole alleviates streptozotocin-induced diabetic nephropathy in rats via hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic properties: Role of the S1R/Nrf2 antioxidant axis.

Sigma-1 receptor (S1R) activation was recently identified as a promising target for preventing diabetic nephropathy (DN) by mitigating hypoxia, oxidative stress, and inflammation. This study aimed to investigate the potential reno-protective effect of the S1R agonist afobazole against streptozotocin (STZ)-induced DN in rats compared to metformin. Rats were split into six groups: the normal control group; the diabetic control group received STZ (55mg/kg i.p.); the other four groups received STZ and were treated with different doses of either afobazole (10, 15, and 20mg/kg) or metformin (200mg/kg). Metabolic parameters and renal function were assessed. Expression levels of oxidative stress markers and inflammatory cytokines were measured using ELISA, apoptosis-related proteins were evaluated using immunohistochemistry, and gene expression of S1R, Nrf2, NF-κB, and TLR-4 was determined. Histopathological analysis was performed on kidney tissues. Both afobazole and metformin exerted hypoglycemic effects, alleviating renal injury, reducing blood urea nitrogen (BUN) and serum creatinine, and restoring oxidant/antioxidant balance in diabetic rats. Both treatments boosted renal S1R and Nrf2 levels, suppressed inflammatory proteins and cytokines, and reduced apoptotic features. The study revealed that afobazole provided nephroprotection in STZ-induced DN through a hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic potential mediated by activating the S1R/Nrf2 antioxidant axis. The 15mg/kg dose elicited the most pronounced nephroprotective effects, outperforming other treatment groups.

Perfluorohexane Sulfonic Acid Disrupts the Immune Microenvironment for Spermatogenesis by Damaging the Structure of the Blood-Testis Barrier in Mice.

Perfluorohexane sulfonic acid (PFHxS) is extensively used in waterproof coatings and fire-fighting foams, and several studies have found it to be a potential health hazard, but there is still unknown about its effects on spermatogenesis. Our results showed that PFHxS-treated mice have significant reproductive toxicity, including a decrease in sperm count and motility, and the levels of sex hormones (P < 0.05). Concurrently, structural abnormalities are observed in sperm, affecting ≈60-75% of those in the PFHxS-treated group. Additionally, it is found that the structure of the blood-testis barrier (BTB) is damaged after PFHxS treatment, leading to higher expression levels of inflammatory cytokines in the microenvironment for spermatogenesis. Moreover, the expression of proteins associated with mitochondrial biogenesis, including PTEN-induced kinase 1 (PINK1) and NADPH oxidase 4 (NOX4), is dysregulated in the testes after PFHxS treatment. Based on metabolome data, the differential metabolite 3-hydroxybutanoic acid is identified in the PFHxS-treated group, which can regulate the histone Kac levels, especially H3K4ac and H3K9ac. In summary, the results of this study suggest that in the testes of PFHxS-treated mice, inflammatory factors disrupt the mitochondrial function and metabolic profiles and hinder the progress of gene transcription through histone Kac, ultimately causing sperm dysfunction.

Zebrafish as a Visible Neuroinflammation Model for Evaluating the Anti-Inflammation Effect of Curcumin-Loaded Ferritin Nanoparticles.

It is crucial to inhibit the neuroinflammation response as it is a prominent factor contributing to the pathogenesis of neurodegenerative disorders. However, the limited development of neuroinflammation models dramatically hinders the efficiency of nanomedicine discovery. In recent years, the optically transparent zebrafish model provided unique advantages for in vivo imaging of the whole body, allowing the progression of the disease to be visualized. In this study, a lipopolysaccharide (LPS)-mediated zebrafish neuroinflammation model was established to visualize the brain distribution and quickly evaluate the anti-inflammation effect of human ferritin-loaded curcumin (Cur@HFn) nanoparticles. The Cur@HFn drug delivery system was successfully prepared and characterized. The HFn nanocage demonstrated significant brain accumulation and prolonged circulation in a zebrafish larval model. In the LPS-induced zebrafish model, Cur@HFn significantly reduced neutrophil recruitment within the brain region of the LPS-treated zebrafish. Additionally, Cur@HFn mitigated nitric oxide (NO) release and downregulated the mRNA expression levels of proinflammatory cytokines, including TNF-α and IL-1β. Lastly, Cur@HFn significantly reduced the damage of raphe nucleus neurons and alleviated the locomotion deficiency caused by LPS. Overall, our findings highlight that Cur@HFn is a promising drug delivery system for the targeted treatment of brain disorders. This zebrafish neuroinflammation model could be used for high-throughput in vivo drug screening and discovery.

Regulation of LPS-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells via TLR4-Mediated NF-κB and MAPK Signaling Pathways by Lactoferrin.

Lactoferrin (LF), a member of the transferrin family, is widely present in mammalian milk and other secretions, exhibiting anti-inflammatory, antibacterial, and anti-infective properties. Although the biological functions of LF have been extensively studied, there are few reports on its effects and molecular mechanisms concerning bovine mastitis caused by bacterial infection. This study used bovine mammary epithelial cells (BMECs) cultured in vitro as the research model. An inflammatory injury model was established by stimulating BMECs with LPS to investigate whether LF at different concentrations (10, 50, 100, and 200 μg·mL-1) could inhibit the inflammatory response before and after the onset of inflammation. The expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels was detected using RT-qPCR and ELISA. Western blotting was employed to evaluate the phosphorylation levels in the inflammatory signaling pathways MAPK/P38/ERK and NF-κB/P65, while RT-qPCR was used to examine the impact on TLR4 receptor gene expression. The results display that pretreatment with LF prior to LPS-induced inflammation in BMECs reduced the expression of inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α at both the gene and protein levels (p < 0.05). LF also inhibited the phosphorylation of NF-κB/P65 and MAPK/P38/ERK signaling pathways and downregulated TLR4 receptor gene expression (p < 0.05). However, when LF was added after the onset of LPS-induced inflammation, inflammatory cytokine expression and phosphorylation levels in the NF-κB/P65 and MAPK/P38/ERK pathways remained elevated, along with high expression of the TLR4 receptor gene (p < 0.05). These findings show that LF can antagonize LPS-induced inflammatory responses in BMECs and reduce cytokine expression, exhibiting anti-inflammatory effects when administered before inflammation. Conversely, when LF is added post-inflammation, it appears to enhance cytokine expression, potentially promoting the recruitment of more cells or factors to resolve inflammation rapidly. Both effects are mediated through the TLR4 receptor and the NF-κB/P65 and MAPK/P38/ERK signaling pathways.

Decursinol angelate relieves inflammatory bowel disease by inhibiting the ROS/TXNIP/NLRP3 pathway and pyroptosis.

Despite evidence of the efficacy of decursinol angelate (DA), a prescription medication derived farom traditional Chinese medicine, in alleviating inflammatory bowel disease (IBD), the precise mechanisms behind its action remain unclear. Lipopolysaccharides (LPS) and dextran sodium sulfate (DSS) induction were used as in vitro and in vivo models of IBD, respectively, to assess the role of DA in alleviating IBD. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression levels of pro-inflammatory cytokines in mouse serum, Western blot was performed to detect the expression of TXNIP/NLRP3 pathway tight junction (TJ) proteins in colon tissues and cells, and immunohistochemistry, immunofluorescence and immunohistochemistry, immunofluorescence and qRT-PCR were used to validate the proteins related to this signaling pathway. Molecular docking technique and co-immunoprecipitation (Co-IP) method assay were applied to evaluate the targeting effect of DA on NLRP3 proteins, and MCC950, a specific inhibitor of NLRP3, was used as a positive control for validation. Our research indicates that DA's distinctive molecular mechanism could entail binding to the NLRP3 protein, thereby suppressing the activation of the NLRP3 pathway and diminishing the assembly and activation of the NLRP3 inflammasome, thus functioning as an anti-inflammatory agent. DA may play a role in improving BD by inhibiting the activation of the ROS/TXNIP/NLRP3 signaling pathway and the release of inflammatory mediators, and by repairing the intestinal barrier function.

Neuroprotective Effects of Eugenol Acetate Against Ischemic Stroke.

To explore the neuroprotective effect of Eugenol Acetate (EA) on post-stroke neuroinflammation and investigate the underlying mechanisms. For in vitro experiments, primary microglia were pre-incubated with EA for 2hours, followed by lipopolysaccharide (LPS) stimulation for 24hours or Oxygen-Glucose Deprivation (OGD) treatment for 4hours. Real-time quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and Western blot were performed to examine the expression levels of inflammatory cytokines in primary microglia. The activation of NF-κB signaling pathway was evaluated by immunofluorescence staining and Western blot. For in vivo experiments, middle cerebral artery occlusion (MCAO) was constructed to mimic ischemic brain injury on 8-week-old male C57BL/6J mice. The mice were continuously injected intraperitoneally with EA or vehicle after MCAO. Neurobehavioral tests and TTC staining were conducted to estimate the neurological deficits and infarct area. Moreover, the white matter integrity after MCAO was observed via immunofluorescence staining. EA significantly reduced the expression of pro-inflammatory cytokines in LPS or OGD treated primary microglia, and inhibited LPS-induced activation of the NF-κB signaling pathway. In addition, EA alleviated ischemic brain injury and improved neuromotor function of MCAO mice. Furthermore, long-term neurological deficits and white matter integrity were improved by EA treatment after MCAO. EA alleviated ischemic injury and restored white matter integrity in MCAO mice, which might be associated with the inhibition of NF-κB signaling pathway in microglia. Therefore, EA might be a promising candidate for the treatment of ischemic stroke.

Dorzolamide Intermediates with Potential Anti-Inflammatory Activity

Dorzolamide (DZD), a Carbonic anhydrase (CA) inhibitor clinically used to lower intraocular pressure, exhibits anti-inflammatory effects owing to the drug’s ability to inhibit the TIR domain-containing adaptor protein (TIRAP)-mediated signalling in macrophages. Here, we investigated whether DZD intermediates also demonstrate any anti-inflammatory property like DZD but with a reduced inhibition of CA. We found that several intermediates of DZD show increased binding to TIRAP at the common interface of kinases, such as Protein kinase C-delta (PKCδ) and Bruton's tyrosine kinase (BTK). Such binding results in a decreased activity of TIRAP, p38 Mitogen-activating protein kinases (MAPK), and p65, which are essential for major inflammatory signaling pathways. Remarkably, the DZD intermediates were more effective than DZD in decreasing the mRNA expression levels of pro-inflammatory cytokines in Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The DZD intermediates also exhibit a reduced binding energy to CA II and CA IV, highlighting their improved specificity as anti-inflammatory compounds with decreased unwanted biological effects. Furthermore, we validated the anti-inflammatory effect of the most efficient DZD intermediate, DRZ V, in a model of mouse sepsis. DRZ V-treated septic mice exhibited improved survival compared to DZD-treated septic mice. Our data indicate that the tested DZD intermediates are more effectual in dampening TIRAP-mediated inflammatory signaling as compared to DZD. Thus, DZD intermediates may be a promising option for developing novel anti-inflammatory therapeutics.

Vaccination of mice with Trichinella spiralis C-type lectin elicited the protective immunity and enhanced gut epithelial barrier function.

C-type lectin (CTL) plays an important act in parasite adhesion, host's cell invasion and immune escape. Our previous studies showed that recombinant Trichinella spiralis C-type lectin (rTsCTL) mediated larval invasion of enteral mucosal epithelium. The aim of this study was to investigate protective immunity produced by vaccination with rTsCTL and its effect on gut epithelial barrier function in a mouse model. The ELISA results showed that subcutaneous vaccination of mice with rTsCTL elicited a systemic humoral response (high levels of serum IgG, IgG1/IgG2a and IgA) and significant gut mucosal sIgA responses. The levels of Th1/Th2 cytokines (IFN-γ/IL-4) secreted from spleen, mesenteric lymph nodes and Peyer's patches were distinctly increased at 6 weeks following vaccination (P < 0.05). At one week after challenge, the numbers of goblet cells and expression level of Muc2, Muc5ac and pro-inflammatory cytokines (TNF-α and IL-1β) in gut tissues of vaccinated mice were obviously decreased, while expression of anti-inflammatory cytokines (IL-4 and IL-10) was evidently increased, compared to the infected PBS group. It is interesting that expression levels of gut epithelial tight junctions (TJs; occludin, claudin-1 and E-cad) were prominently elevated and intestinal permeability was interestingly declined in vaccinated mice. The rTsCTL-vaccinated mice exhibited a 51.69 and 48.19% reduction of intestinal adult and muscle larva burdens, respectively. The female fecundity in rTsCTL vaccinated mice was reduced by 40.51%. These findings indicated that rTsCTL vaccination impeded larval invasion and improved gut epithelial integrity and barrier function, reduced worm burdens, and relieved gut and muscle inflammation. Vaccination of mice with rTsCTL elicited an obvious protective immunity against larval challenge, impeded larval invasion of gut mucosa, enhanced gut epithelial integrity and barrier function, reduced worm burdens; it also alleviated gut and muscle inflammation. TsCTL might be a novel candidate target molecule for anti-Trichinella vaccines.

Mechanoreceptor Piezo1 channel-mediated interleukin expression in conjunctival epithelial cells: Linking mechanical stress to ocular inflammation.

Mechanical stress on the ocular surface, such as from eye-rubbing, has been reported to lead to inflammation and various ocular conditions. We hypothesized that the mechanosensitive Piezo1 channel in the conjunctival epithelium contributes to the inflammatory response at the ocular surface after receiving mechanical stimuli. Human conjunctival epithelial cells (HConjECs) were treated with Yoda1, a Piezo1-specific agonist, and various allergens to measure cytokine expression levels using qRT-PCR. Piezo1 activation-induced intracellular signaling pathways were also investigated by Western blot. Mechanical stretching experiments were conducted to simulate Piezo1 activation in HConjECs. Specificity of Piezo1 was confirmed by PIEZO1 knockdown and GsMTx4. In in vivo studies, using immunohistochemistry, rats were administered Yoda1 eye drops to examine the inflammatory response in the conjunctiva and Piezo1-induced signaling activation. HConjECs expressed functional Piezo1 channel which was the dominant mechanoreceptor among putative channels and whose activation significantly increased IL-6 and IL-8 expression through the p38 MAPK-CREB pathway. Piezo1-induced [Ca2+]i elevation was crucial for the production of IL-6. The Yoda1-induced inflammatory responses were blocked by PIEZO1 knockdown. Mechanical stretching mimicked these effects, which were suppressed by GsMTx4. In vivo, Yoda1 administration led to increased phospho-p38 MAPK, phospho-CREB, and IL-6 in the rat conjunctival epithelium, with significant neutrophil infiltration. Mechanical stress-induced Piezo1 channel activation in conjunctival epithelial cells can cause ocular inflammation by upregulating pro-inflammatory cytokines via the p38 MAPK-CREB pathway and promoting neutrophil infiltration. These findings suggest that mechanical stimuli on ocular surface tissues are significant risk factors for ocular inflammation.

Gene expression profiles in human dental pulp stem cells treated short-term with lipopolysaccharides before and after osteoinduction.

Dental pulp stem cells (DPSCs) are essential for reparative dentinogenesis following damage or infection. DPSCs surrounding the blood vessels in the central region of the dental pulp actively proliferate after tooth injury and differentiate into new odontoblast-like cells or odontoblasts to form reparative dentin. However, the signaling pathways involved in undifferentiated and osteodifferentiated DPSCs under inflammatory conditions remain unclear. This study aimed to compare the expression profiles of immortalized undifferentiated and osteo-differentiated human DPSCs (hDPSCs) treated with and without lipopolysaccharide (LPS) to elucidate the molecular regulatory mechanisms involved in inflammatory conditions. We investigated the differences between undifferentiated and osteodifferentiated hDPSCs in response to LPS. RNA-seq analyses of undifferentiated and osteodifferentiated hDPSCs were performed with and without LPS. Whole-transcriptome profiling revealed distinct differences in the expression patterns of LPS-treated undifferentiated and osteodifferentiated DPSCs. Death-associated protein kinase 1 levels downregulated in LPS-treated osteodifferentiated cells, inhibiting apoptosis and enhancing cell survival. After LPS treatment, osteodifferentiated DPSCs exhibited higher expression levels of various inflammatory cytokines and chemokines than undifferentiated DPSCs. This study provides valuable transcriptomic data as a critical resource for uncovering potential therapeutic targets to enhance cell survival and regulate inflammation within the dental pulp. By elucidating the key molecular mechanisms and identifying specific gene expression changes linked to inflammatory and immune responses, these findings provide significant insights into osteo-differentiated hDPSCs.

CCL4L2 participates in tendinopathy progression by promoting macrophage inflammatory responses: a single-cell analysis

BackgroundTendinopathy is very common in clinical practice, which is highly prevalent in athletes, sports enthusiasts and other people involved in high-load weight-bearing activities. Common types of tendinopathy include rotator cuff injury, Achilles tendinitis, tennis elbow and so on. Macrophages (Macs) are key immune cells in the pathogenesis of tendinopathy. In this study, CCL4L2+ M1-related signaling pathways were screened by combining single-cell RNA sequencing (scRNA-seq) to explore their significance in tendinopathy treatment.MethodsImmune cell populations were screened by Uniform Manifold Approximation and Projection (UMAP) downscaling, and Mac cell subsets were annotated using cell marker genes. The cellular communication mechanism between different cellular subsets such as Macs and tendon stem/progenitor cells (TSPCs) was demonstrated by cellular communication analysis. Based on cell marker genes of CCL4L2 + M1, Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed to compare the expression differences in M1 and M2 between the Disease and Healthy groups. Associations between CCL4L2+ M1 and TSPCs were inferred by cell-cell communication analysis. The effects of CCL4L2 on Mac polarization and TSPCs were verified by enzyme-linked immunosorbent assay (ELISA) and real-time fluorescence quantitative PCR (qPCR).ResultsThe proportions of TSPCs, endothelial cells (ECs), smooth muscle cells (SMCs), and immune cells were significantly elevated in the Disease group. The proportion of M1 cells in the Disease group was higher than that in the Healthy group, while the proportion of M2 cells was lower than that in the Healthy group. M1 differentially expressed genes (DEGs) were mainly enriched to disease-related and immunoinflammation-related signaling pathways. Signaling intensities between M1 and TSPCs in pathways related to immunoinflammation and ischemic injury were significantly increased in the Disease group. The proportion of CCL4L2 + M1 in the Disease group was significantly higher than in the Healthy group, and communications between CCL4L2 + M1 and TSPCs varied significantly. Compared with the Control group, the expression levels of inflammatory cytokines were higher in the CCL4L2 group, and the expression levels of tendon differentiation markers (Egr1, Mkx, Scx, Type 1 collagen, Tnmd) were significantly down-regulated.ConclusionThe present study analyzed the heterogeneous alterations in the Healthy and Disease groups by scRNA-seq data and found that there was a significant inflammatory infiltrate in the Disease group with markedly increased Mac activity, which was associated with activation of the CCL4L2 + M1-associated signaling pathways. CCL4L2 promotes M1 polarization and inhibits TSPC differentiation through activating M1-related inflammatory signaling pathways. These findings contribute to a more comprehensive understanding of tendon injury progression and provide potential targets for tendinopathy treatment.

Immunoregulatory Effects of Codonopsis pilosula Polysaccharide Modified Selenium Nanoparticles on H22 Tumor-Bearing Mice.

Codonopsis pilosula polysaccharide (CPP) and rare element selenium (Se) have been proved to exert various biological activities, and our previous study demonstrated that selenium nanoparticles modified with CPP (CPP-SeNPs) possessed significantly enhanced tumor cytotoxicity in vitro. This study aimed to investigated the inhibitory effects of CPP-SeNPs complex on H22 solid tumors via immune enhancement. In this study, the H22 tumor-bearing mice model was constructed, and the potential mechanisms of CPP-SeNPs antitumor effects were further explored by evaluating cytokines expression levels, immune cells activities and tumor cells apoptotic indicators in each group. The results demonstrated that CPP-SeNPs effectively exerted dose-dependent protective effects on the immune organs of tumor-bearing mice in vivo, leading to increase in peripheral white blood cell counts and inhibition of solid tumor growth with inhibitory rate of 47.18% in high-dose group (1.5 mg/kg). Furthermore, CPP-SeNPs treatment significantly elevated the levels of TNF-α, IFN-γ, and IL-2 in mice sera, enhanced NK cell cytotoxicity, augmented macrophage phagocytosis capacity, as well as increased both the amounts and proliferation activity of lymphocyte subsets. CPP-SeNPs improved the immune system's ability to clear tumor cells by up-regulating Bax expression while down-regulating Bcl-2 expression within solid tumors, indicating the potential activation of mitochondrial apoptosis pathway. Therefore, CPP-SeNPs administration can effectively inhibit tumor growth by enhancing immune response in tumor-bearing mice, which might be relevant to the regulation of gut microbiota short-chain fatty acids metabolisms. These findings could provide theoretical support and data foundation for further development of CPP-SeNPs as functional food and drug adjuvants.

Production of recombinant protein of Tyr p 32 from Tyrophagus putrescentiae and identifying its immunoreactivity

The present study was aimed to produce the recombinant protein of Tyrophagus putrescentiae allergen component 32 (Tyr p 32) and to identify its immunoreactivity. The cDNA encoding Tyr p 32 was amplified from total RNA of T. putrescentiae and inserted into pET-28a (+) vector. The constructed plasmid pET-28a (+)-Tyr p 32 was transformed into BL21 (DE3) receptor cells. After being induced with IPTG, the recombinant protein was purified with Ni column, and then identified on SDS-PAGE and Western blotting. Serum of children with allergic asthma and(or) rhinitis was collected, IgE-ELISA and IgE-Western blotting were used to detect the binding rate of rTyr p 32 to human T.putrescentiae-positive serum. After human bronchial epithelial cells BEAS-2B being cultured with rTyr p 32, the expression levels of IL-6 and IL-8 cytokines was detected by ELISA and qRT-PCR, t-test was used for pairwise comparison between groups. The results showed that the cDNA length of Tyr p 32 was 885 bp. The sequence identity between Tyr p 32 and Der p 32, Der f 32 was 70.21% and 68.03%, respectively. According to SDS-PAGE and Western blotting, the molecular weight of rTyr p 32 was about 35 000 Da, which was consistent with the theoretical value. IgE-ELISA results showed that the positive rate of rTyr p 32 was 41.38% (12/29) against T. putrescentiae-positive serum. When BEAS-2B cells were cultured with rTyr p 32, the expression of IL-6 and IL-8 increased in the cell supernatant in a dose-dependent manner (t=-29.10,P=0.001 2;t=-33.69,P=0.000 9), which also significantly increased the mRNA expression levels of IL-6 and IL-8 (t=-9.15,P=0.011 7;t=-17.16,P=0.003 4). In conclusion, the recombinant protein rTyr p 32 was successfully prepared, which provides raw materials for component diagnosis and specific immunotherapy of allergic diseases.

Co-culture system of breast cancer and normal cells to investigate inflammation: using doxorubicin encapsulated in adipose-derived exosomes.

Doxorubicin (DOX) chemotherapy for breast cancer is an effective treatment option, but it also has disadvantages. Exosomes (EXOs) have safely and successfully transported DOX and reduced its adverse effects; however, its use is still being explored. In this study, a co-culture system of malignant and non-malignant breast cells was used to generate an in vitro model reflecting the in vivo cellular microenvironment, and the effects of this treatment were investigated by examining inflammatory genes. Extracellular matrices (EXOs) were extracted from mesenchymal stem cells derived from human adipose tissue by ultracentrifugation. Later, Western blotting, dynamic light scattering (DLS) and transmission electron microscopy methods were used to examine the properties of the EXO. DOX was encapsulated in the EXOs by sonication and the loading rate was measured by spectrophotometry. In the current study, a co-culture system was used to investigate the cytotoxic effects of free DOX and DOX encapsulated in EXOs (EXO-DOX) on various breast cell lines, including MCF-7, MCF-10A, MDA-MB-231, and A-MSC. Additionally, the expression levels of inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) were examined. Methylthiazolyldiphenyl-tetrazolium bromide assay demonstrated that free DOX showed the highest cytotoxicity against MCF-10A cells, followed by MCF-7 cells. Conversely, EXO-DOX indicated a greater effect on MCF-7 cells and had a lower IC50 compared to MDA-MB-231 cells. Free DOX significantly downregulated the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), particularly in MCF-7 and MCF-10A cells, while concurrently upregulating IL-10 expression. EXO-DOX induced a more significant alteration in cytokine expression than the control and free DOX treatment groups. The co-culture system revealed a synergistic effect of free DOX on cancer cells while simultaneously mitigating the toxic effects of DOX on normal cells. This study suggests that EXO-DOX has promising potential as a targeted drug delivery system that could potentially improve therapeutic efficacy and minimize off-target toxicity.

Xuebijing Exerts Protective Effects on Myocardial Cells by Upregulating TRIM16 and Inhibiting Oxidative Stress and Apoptosis.

This study utilized transcriptomic sequencing combined with cellular and animal models to explore the potential mechanisms of Xuebijing in treating sepsis-induced myocardial dysfunction, also known as sepsis-induced myocardial injury. We investigated potential targets and regulatory mechanisms of XBJ injection using network pharmacology and RNA sequencing. The effects of XBJ on oxidative stress and apoptosis levels in human cardiac myocytes (AC16) and C57BL/6 mice exposed to lipopolysaccharide (LPS) were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA), fluorescent probe, Fluorescent Quantitative Polymerase Chain Reaction (qPCR), Western Blot, Transmission Electron Microscopy, oxidative stress-related indicators detection kit, flow cytometry, and Immunohistochemistry (IHC). First, it was verified that XBJ can reduce the deformation of AC16 cardiomyocytes induced by LPS and the production and secretion of ROS (P <0.01). The transcriptome sequencing results showed that the TRIM16 gene was significantly increased after XBJ treatment, and the data of KEGG and GO analyses demonstrated that XBJ could inhibit the pathway expression of oxidative stress damage in AC16 cells, and PCR verified that XBJ could indeed increase the expression level of TRIM16 gene in AC16 cells (P <0.01). Basic animal and cell experiments showed that LPS could inhibit the expression of TRIM16 and NRF2 in cardiomyocytes (P <0.05) and promote the expression of Keap1 (P <0.01), while XBJ could significantly upregulate the expression levels of TRIM16 and NRF2 (P <0.01) and inhibit the expression of Keap1 (P <0.01), thereby affecting the expression levels of downstream proinflammatory cytokines and alleviating LPS-induced oxidative stress damage. In addition, XBJ also inhibited the expression of the pro-apoptotic proteins Bax and c-caspase3 (P <0.01), promoted the expression of the anti-apoptotic protein Bcl2 (P <0.01), and reduced LPS-induced apoptosis by upregulating TRIM16. Our comprehensive data demonstrated that TRIM16 is a key gene in the therapeutic action of Xuebijing in sepsis-induced myocardial dysfunction, protecting myocardial cells from injury through antioxidative stress and anti-apoptotic mechanisms.

D609 Suppresses Antituberculosis Response by Regulating Dendritic Cells Antigen Presentation.

To elucidate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in the antituberculosis (anti-TB) immune response mediated by dendritic cells (DCs). In vivo, C57BL/6J mice infected with the Mycobacterium tuberculosis strain H37Rv. Before infection, the mice were pretreated with the PC-PLC inhibitor D609. Bacillary loads in lung and spleen tissues were quantified through colony-forming unit (CFU) assays. Hematoxylin and eosin (H&E) staining was performed to assess inflammatory infiltration and tissue damage. Levels of inflammatory mediators in peripheral venous blood were quantified using enzyme-linked immunosorbent assays (ELISAs). Flow cytometry was employed to determine the proportions of conventional DCs (cDCs) and their subsets, cDC1 and cDC2, within lung, spleen, and lymph node tissues. In vitro, mouse bone marrow-derived dendritic cells (BMDCs) pretreated with D609. The expression levels of chemokines and pro-inflammatory cytokines were assessed via quantitative polymerase chain reaction (qPCR) and ELISA. BMDCs were loaded with H37Rv expressing red fluorescent protein (RFP-H37Rv) or DQ-OVA, and flow cytometry was utilized to analyze the impact of D609 on antigen phagocytosis and processing. Furthermore, flow cytometry was employed to evaluate the effect of D609 pretreatment on the expression levels of costimulatory molecules on BMDCs. The capacity of D609-treated BMDCs to activate and proliferate T cells, as well as to induce interferon-gamma (IFN-γ) secretion, was assessed through a DC-T cell coculture system. In vivo analysis revealed that mice pretreated with D609 exhibited a marked increase in tissue bacterial load, enhanced inflammatory infiltration, and a reduction in pro-inflammatory mediator expression in peripheral venous blood. There was a notable decrease in the number of cDCs in lung and lymph node tissues, with a pronounced reduction in cDC1 in the lungs and cDC2 in the lymph nodes. In vitro studies demonstrated that D609 pretreated BMDCs displayed a significant decline in inflammatory mediator production, antigen phagocytosis, and antigen processing capabilities, potentially due to altered expression of costimulatory molecules. Coculture experiments indicated that D609 pretreated BMDCs showed a substantial reduction in their ability to stimulate T cell activation, proliferation, and IFN-γ secretion. Our findings suggest that PC-PLC plays a critical role in the functionality of DCs, including the production of chemokines and pro-inflammatory cytokines, migration to lymph nodes, and antigen presentation to T cells, which collectively contribute to T cell activation and effective clearance of Mycobacterium tuberculosis. Further investigation into the regulatory mechanisms of PC-PLC in DCs may uncover novel therapeutic targets for the development of advanced anti-TB treatments.

Zuyangping formula promotes skin wound healing in diabetic rats.

To evaluate the effects of Zuyangping (, ZYP) formula on wound healing in diabetic rats, as well as the molecular mechanisms involved. The main compounds in ZYP formula were identified by the Liquid chromatography-tandem mass spectrometry. Sprague-Dawley rats, injected with streptozotocin (STZ) to establish diabetes model, then, formed a defective skin trauma in the back, and each group was treated with corresponding drugs once a day. Granulation was taken from each time node for histological analysis. The Western blotting was used to measured protein expression of advanced glycation end products receptor (RAGE) and hypoxia-inducible factor-1α (HIF-1α) axis-related proteins. The relative expression levels of inflammatory cytokines and growth factors were measured by the enzyme-linked immunosorbent assay method. The main ingredients were identified in the ZYP formula. Histological analysis showed that the ZYP formula could inhibit the expression of inflammation, promote angiogenesis and collagen deposition. In addition, the ZYP formula could regulate the expression of RAGE and HIF1-α axis-related proteins, thus promoting the wound healing in diabetic rats. The ZYP formula could accelerate wound healing in diabetic rats.

Molecular mechanism through which Tripterygium hypoglaucum (Lévl.) Hutch alleviates psoriasis

Tripterygium hypoglaucum (Lévl.) Hutch rhizome (THH) is mainly used in the clinical setting for the treatment of autoimmune diseases such as rheumatoid arthritis. In total, four active compounds were isolated from THH methanol extract （THH-MeOH）and identified. The HPLC results showed that the proportions of the active ingredients in THH-MeOH (i.e., celastrol, triptolide, and 3-O-acetyloleanolic acid) were 0.79‰, 0.46‰, and 0.76‰, respectively. THH-MeOH attenuated the M5-induced hyperproliferation of HaCaT cells, decreased the mRNA expression levels of inflammatory cytokines, and inhibited the phosphorylation of IκBα, NF-κB p65, MAPK, and STAT3/JAK2. Furthermore, THH-MeOH significantly reduced PASI scores in mice; reduced the level of Ki67 expression in skin tissues; decreased the expression of inflammatory cytokines in the skin lesions and serum; and ameliorated the IMQ-induced imbalance in the RORγt/Foxp3 ratio. The extract can attenuate psoriasis-like lesions by inhibiting cellular hyperproliferation, ameliorating inflammatory reactions, and modulating immune responses. Our work provides a theoretical basis to support the use of THH for treating psoriasis.

Tectorigenin Reduces Dabie bandavirus-Induced Cytokine Storm by Regulating Toll-Like Receptor 7/Extracellular Signal-Regulated Kinase Pathway.

Severe fever with thrombocytopenia syndrome (SFTS) is a severe emerging infectious disease caused by Dabie bandavirus (DBV). Tectorigenin has been demonstrated to exert anti-inflammatory effect. Here, we aimed to investigate the effects of tectorigenin on DBV-induced cytokine storm. Effects of tectorigenin on cytokines in DBV-infected THP-1 cells and plasma samples of Type I interferon receptor (IFNAR)-/- mice infected with DBV were detected. The changes in body weight and survival time of mice were recorded. The liver, spleen, kidney, and lymph node tissues were collected for hematoxylin-eosin staining. We demonstrated that tectorigenin reduced the expression levels of inflammatory cytokines in both DBV-infected THP-1 cells and plasma samples of IFNAR-/- mice infected with DBV. Tectorigenin attenuated DBV-induced histopathological changes in mice. Mechanistically, tectorigenin attenuated DBV-induced phosphorylation of inhibitor of kappa-B kinase alpha/beta (IKKα/β) of the nuclear factor-κB (NF-κB) signaling pathway, extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway and might function by downregulation of Toll-like receptor. The result of this study suggested that tectorigenin exerted anti-inflammatory effects invivo and invitro and could serve as a novel potential therapeutic strategy for SFTS.