Immunofluorescence Analysis Research Articles

Total tanshinones ameliorates cGAS-STING-mediated inflammatory and autoimmune diseases by affecting STING-IRF3 binding

BackgroundAn important signaling pathway connecting illness and natural immunity is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but aberrant activation of this pathway is associated with the development of autoimmune and inflammatory diseases. Hence, targeted inhibition of the activation of the cGAS-STING pathway is potentially valuable in the treatment of disease. The primary active component of Salvia miltiorrhiza is total tanshinone (TTN). Research has indicated that TTN possesses noteworthy anti-inflammatory properties. However, the protective mechanism of TTN against acute liver injury (ALI) and autoimmune diseases is unknown.MethodsA model of aberrant activation of the cGAS-STING pathway was established in various cells and treated with TTN, and the expression of cGAS-STING pathway-related proteins, type I interferon, interferon stimulated genes and inflammatory factors was assessed by western blotting, real-time qPCR. Immunofluorescence analysis of the effect of TTN on the entry of associated proteins into the nucleus following aberrant activation of the cGAS-STING pathway. The effect of TTN on STING oligomerisation was investigated using 2'-3'-cyclic GMP-AMP (2',3'-cGAMP) to induce STING oligomerisation. Western blotting was used to examine the impact of TTN on the interactions of STING, tank-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) after HA or Flag-labelled plasmids were transfected into HEK-293 T cells. A dimethylxanthenone-4-acetic acid (DMXAA) -induced activation model of the cGAS-STING pathway in mice was established to study the effect of TTN on aberrant activation of the cGAS-STING pathway in vivo. On the other hand, an animal model of lipopolysaccharide/D-galactosamine (LPS/D-GaIN)-induced ALI and an autoimmune disease model induced by trex1 knockout were established to study the effects of TTN on inflammatory and autoimmune diseases mediated by the cGAS-STING pathway in vivo.ResultsIn several models of aberrant activation of the cGAS-STING pathway, TTN significantly inhibited the phosphorylation of STING and IRF3, thereby suppressing the expression of type I interferon, interferon-stimulated genes and inflammatory factors. Additionally, TTN prevented P65 and IRF3 from entering the nucleus after the cGAS-STING signalling pathway was abnormally activated. Subsequent research indicated that TTN was not involved in the oligomerization of STING or the integration of STING-TBK1 and TBK1-IRF3. However, TTN was found to have a substantial effect on the binding process between STING and IRF3. On the other hand, DMXAA-induced STING activation and activation of downstream signalling in vivo are inhibited by TTN. Furthermore, TTN exhibits positive treatment effects on autoimmune diseases caused by deficiency of trex1 and LPS/D-GaIN-induced ALI.ConclusionOur research indicates that TTN effectively treats ALI and autoimmune illnesses mediated by the cGAS-STING pathway by inhibiting the abnormal activation of this pathway.Graphical

A New Renieramycin T Right-Half Analog as a Small Molecule Degrader of STAT3.

Constitutive activation of STAT3 contributes to tumor development and metastasis, making it a promising target for cancer therapy. (1R,4R,5S)-10-hydroxy-9-methoxy-8,11-dimethyl-3-(naphthalen-2-ylmethyl)-1,2,3,4,5,6-hexahydro-1,5-epiminobenzo[d]azocine-4-carbonitrile, DH_31, a new derivative of the marine natural product Renieramycin T, showed potent activity against H292 and H460 cells, with IC50 values of 5.54 ± 1.04 µM and 2.9 ± 0.58 µM, respectively. Structure-activity relationship (SAR) analysis suggests that adding a naphthalene ring with methyl linkers to ring C and a hydroxyl group to ring E enhances the cytotoxic effect of DH_31. At 1-2.5 µM, DH_31 significantly inhibited EMT phenotypes such as migration, and sensitized cells to anoikis. Consistent with the upregulation of ZO1 and the downregulation of Snail, Slug, N-cadherin, and Vimentin at both mRNA and protein levels, in silico prediction identified STAT3 as a target, validated by protein analysis showing that DH_31 significantly decreases STAT3 levels through ubiquitin-proteasomal degradation. Immunofluorescence and Western blot analysis confirmed that DH_31 significantly decreased STAT3 and EMT markers. Additionally, molecular docking suggests a covalent interaction between the cyano group of DH_31 and Cys-468 in the DNA-binding domain of STAT3 (binding affinity = -7.630 kcal/mol), leading to destabilization thereafter. In conclusion, DH_31, a novel RT derivative, demonstrates potential as a STAT3-targeting drug that significantly contribute to understanding of the development of new targeted therapy.

Identification of IgE-mediated sensitization in children by enzyme immunoassay and immunofluorescence methods

The goal of research was to determine the frequency of detection and the structure of causally significant sensitization to the main groups of allergens in children with allergic diseases in Siberia when evaluating the results by enzyme immunoassay and immunofluorescence analysis. We researched 968 children with allergic rhinitis, bronchial asthma, urticaria, and/or allergic dermatitis undergoing treatment at the Consultative and Diagnostic Center for Allergology and Immunology, GDKB No. 2 named after V.P. Bisyarina of Omsk. Blood serum samples were diagnosed in the laboratory of clinical immunology. Also, the allergen-specific IgE concentrations were determined for 109 allergens, including food allergens (milk, egg, meat, fish, cereals, fruits, vegetables), household allergens (epidermal pets, dust and house dust mites, molds), and inhaled antigens (grass, trees). Analysis of 435 children was carried out by enzyme immunoassay, and 533 children by indirect immunofluorescence. The research revealed the predominance in children with allergic diseases in Siberia of IgE-mediated sensitization to household allergens, which were detected in 33.7% of the examined. IgE-mediated sensitization to inhalation and food allergens was 1.5 times less common. According to our research, the most common causative allergen among households was house dust (90%), dandruff and animal epithelium (86%), house dust mites (13.5%), and molds (1.4%). The most common causative allergen among pollen was tree allergens (47.8%), cereals (23.5%) and weeds (20.1%). The presence of multivalent sensitization with a predominant combination of household and respiratory allergens and a rarer combination of household and food allergens has been experimentally established.

DHA-enriched phosphatidylserine ameliorates cyclophosphamide-induced liver injury via regulating the gut-liver axis

ObjectiveThis study explores the therapeutic effects and mechanisms of DHA-enriched phosphatidylserine (DHA-PS) on liver injury induced by cyclophosphamide (CTX) in mice, focusing on the gut-liver axis. MethodsA mouse model was established by administering CTX (80 mg/kg) intraperitoneally for 5 days. DHA-PS (50 or 100 mg/kg) was administered for the next 7 days to assess its reparative impact on liver damage. ResultsThe findings revealed significant improvements in liver biochemical indices, inflammatory markers, and oxidative stress levels in the mice treated with DHA-PS. Through non-targeted metabolomics analysis, DHA-PS mitigated CTX-induced metabolic disruptions by modulating lipid, amino acid, and pyrimidine metabolism. Immunofluorescence analysis further confirmed that DHA-PS reduced the expression of liver-associated inflammatory proteins by inhibiting the TLR4/NF-κB pathway. Additionally, DHA-PS restored the intestinal barrier, evidenced by adjustments in the levels of intestinal lipopolysaccharide (LPS), secretory immunoglobulin A (sIgA), and tight junction proteins (Claudin-1, Occludin, and ZO-1). It also improved gut microbiota balance by enhancing microbial diversity, increasing beneficial bacteria, and altering community structures. ConclusionThese results suggest that DHA-PS could be a potential therapeutic agent or functional food for CTX-induced liver injury through its regulation of the gut-liver axis.

Enhanced interleukin-16-CD4 signaling in CD3 T cell mediates neuropathic pain via activating astrocytes in female mice

Immune cells and interleukins play a crucial role in female-specific pain signaling. Interleukin 16 (IL-16) is a cytokine primarily associated with CD4+ T cell function. While previous studies have demonstrated the important role of spinal CD4+ T cells in neuropathic pain, the specific contribution of IL-16 to neuropathic pain remains unclear. In this study, by using a spinal nerve ligation (SNL)-induced neuropathic pain mice model, we found that SNL induced an increase in IL-16 mRNA levels, which persisted for a longer duration in female mice compared to male mice. Immunofluorescence analysis further confirmed enhanced IL-16- and CD4-positive signals in the spinal dorsal horn following SNL surgery in female mice. Knockdown of spinal IL-16 by siRNA or inhibition of CD4 by FGF22-IN-1, a CD4 inhibitor, attenuated established mechanical and thermal pain hypersensitivity induced by SNL. Furthermore, female mice injected with IL-16 intrathecally exhibited significant spontaneous pain, mechanical and thermal hyperalgesia, all of which could be alleviated by FGF22-IN-1 or a CD3 antibody. Additionally, IL-16 induced astrocyte activation but not microglial activation in the spinal dorsal horn of female mice. Meanwhile, astrocyte activation could be suppressed by the CD3 antibody. These results provide compelling evidence that IL-16 promotes astrocyte activation via CD4 on CD3+ T cells, which is critical for maintaining neuropathic pain in female mice.

HIV infection is associated with compromised tumor microenvironment adaptive immune reactivity in Hodgkin lymphoma

AbstractThe impact of HIV infection on the tumor microenvironment (TME) of classic Hodgkin lymphoma (cHL), one of the most common comorbidities after HIV infection, is not well understood. Here, we have used multiplexed immunofluorescence and spatial transcriptomic analysis to dissect the impact of viral infections (Epstein-Barr virus [EBV] and HIV/EBV) on cHL TME. HIV−EBV+ cHL TME was characterized by higher cell densities of CD8high T cells coexpressing inhibitory receptors (PD-1 and TIGIT), macrophage subsets, and an in situ inflammatory molecular profile associated with increased expression of T-cell receptor (TCR) and B-cell receptor cell signaling pathways than HIV−EBV− cHL TME. Compared with HIV−EBV+, HIV+EBV+ cHL TME was characterized by significantly less CD8high T cells coexpressing PD-1 and TIGIT, a profile concomitant with significantly increased cell densities of CD155high neoplastic cells. Significant downregulation of in situ TCR signaling and upregulation of extracellular matrix reorganization pathways were found in HIV+EBV+ cHL TME, in line with an altered topological organization of CXCL13 and heparan sulfate, an extracellular matrix glycosaminoglycan. Our data reveal the complexity of the cellular and molecular composition of cHL TME in the presence of viral infections, with possible implications for combinatorial immunotherapies. Furthermore, the data suggest specific molecular targets and pathways for further investigation that could improve our understanding of possible mechanistic links between HIV and lymphomagenesis.

Schisandrin A Attenuates Diabetic Nephropathy via EGFR/AKT/GSK3β Signaling Pathway Based on Network Pharmacology and Experimental Validation.

Diabetic nephropathy (DN) is one of the common complications of diabetes and the main cause of end-stage renal disease (ESRD) in clinical practice. Schisandrin A (Sch A) has multiple pharmacological activities, including inhibiting fibrosis, reducing apoptosis and oxidative stress, and regulating immunity, but its pharmacological mechanism for the treatment of DN is still unclear. In vivo, streptozotocin (STZ) and a high-fat diet were used to induce type 2 diabetic rats, and Sch A was administered for 4 weeks. At the same time, protein-protein interaction (PPI) networks were established to analyze the overlapping genes of DN and Sch A. Subsequently, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses were performed to determine the hub pathway. In addition, molecular docking was used to preliminarily verify the affinity of hub proteins and Sch A. Further, H&E staining, Sirius red staining, immunohistochemistry, immunofluorescence, and western blot analysis were used to detect the location and expression of related proteins in DN. This study revealed the multi-target and multi-pathway characteristics of Sch A in the treatment of DN. First, Sch A could effectively improve glucose tolerance, reduce urine microprotein and urine creatinine levels, and alleviate renal pathological damage in DN rats. Second, EGFR was the hub gene screened in overlapping genes (43) of Sch A (100) and DN (2524). Finally, it was revealed that Sch A could inhibit the protein expression levels of EGFR and PTRF and reduced the expression of apoptosis-related proteins, and this effect was related to the modulation of the AKT/GSK-3β signaling pathway. In summary, Sch A has a protective effect in DN rats, EGFR may be a potential therapeutic target, throughout modulating AKT/GSK-3β pathway.

TIGIT expression in renal cell carcinoma infiltrating T cells is variable and inversely correlated with PD-1 and LAG3

PurposeImmune checkpoint inhibitors have revolutionized the treatment of renal cell carcinoma (RCC), but many patients do not respond to therapy and the majority develop resistant disease over time. Thus, there is increasing need for alternative immunomodulating agents. The co-inhibitory molecule T-cell immunoglobulin and ITIM domain (TIGIT) may play a role in resistance to approved immune checkpoint inhibitors and is being investigated as a potential therapeutic target. The purpose of this study was to quantify TIGIT positivity in tumor-infiltrating T cells in RCC.MethodsWe employed tissue microarrays containing specimens from primary RCC tumors, adjacent normal renal tissue, and RCC metastases to quantify TIGIT within tumor-infiltrating CD3+ T cells using quantitative immunofluorescent analysis. We also compared these results to TIGIT+ CD3+ levels in four other tumor types (melanoma, non-small cell lung, cervical, and head and neck cancers).ResultsWe did not observe significant differences in TIGIT positivity between primary RCC tumors and patient-matched metastatic samples. We found that the degree of TIGIT positivity in RCC is comparable to that in lung cancer but lower than that in melanoma, cervical, and head and neck cancers. Correlation analysis comparing TIGIT positivity to previously published, patient-matched spatial proteomic data by our group revealed a negative association between TIGIT and the checkpoint proteins PD-1 and LAG3.ConclusionOur findings support careful evaluation of TIGIT expression on T cells in primary or metastatic RCC specimens for patients who may be treated with TIGIT-targeting antibodies, as increased TIGIT positivity might be associated with a greater likelihood of response to therapy.

Hyperoxia exposure induces ferroptosis and apoptosis by downregulating PLAGL2 and repressing HIF-1α/VEGF signaling pathway in newborn alveolar typeII epithelial cell

ABSTRACT Backgroud Bronchopulmonary dysplasia (BPD) is one of the most important complications plaguing neonates and can lead to a variety of sequelae. the ability of the HIF-1α/VEGF signaling pathway to promote angiogenesis has an important role in neonatal lung development. Method Newborn rats were exposed to 85% oxygen. The effects of hyperoxia exposure on Pleomorphic Adenoma Gene like-2 (PLAGL2) and the HIF-1α/VEGF pathway in rats lung tissue were assessed through immunofluorescence and Western Blot analysis. In cell experiments, PLAGL2 was upregulated, and the effects of hyperoxia and PLAGL2 on cell viability were evaluated using scratch assays, CCK-8 assays, and EDU staining. The role of upregulated PLAGL2 in the HIF-1α/VEGF pathway was determined by Western Blot and RT–PCR. Apoptosis and ferroptosis effects were determined through flow cytometry and viability assays. Results Compared with the control group, the expression levels of PLAGL2, HIF-1α, VEGF, and SPC in lung tissues after 3, 7, and 14 days of hyperoxia exposure were all decreased. Furthermore, hyperoxia also inhibited the proliferation and motility of type II alveolar epithelial cells (AECII) and induced apoptosis in AECII. Upregulation of PLAGL2 restored the proliferation and motility of AECII and suppressed cell apoptosis and ferroptosis, while the HIF-1α/VEGF signaling pathway was also revived. Conclusions We confirmed the positive role of PLAGL2 and HIF-1α/VEGF signaling pathway in promoting BPD in hyperoxia conditions, and provided a promising therapeutic targets.

Fuchs endothelial corneal dystrophy: aspects of pathogenesis

Fuchs endothelial corneal dystrophy (FECD) – multigenic bilateral disorder, characterized by dysfunction of corneal endothelium cells (CECs) that eventually results in loss of transparency. Purpose: To evaluate the pathogenesis of FECD with histological and immunohistochemical methods, immunofluorescence, scanning electron microscopy (SEM). Methods: Endothelium–Descemet membrane (EDM) complexes, corneal buttons were obtained from 76 patients (85 eyes) with FECD during keratoplasty and divided into 1A, 2A, 3A groups, 15 EDMs (donor tisuue) – 1B, 2B, 3B groups (control). Morphological (hematoxylin/eosin staining) and immunohistochemical (primary antibodies to cytokeratin AE1/AE3 and vimentin) studies (1A, 1B groups), phase-contrast microscopy and immunofluorescence analysis of tight junction protein ZO-1 (2A, 2B groups), SEM (3A, 3B groups) were performed. Results: FECD is characterized by decline of CECs, cell and nuclear enlargement. Nuclear-cytoplasmic ratio of CECs is relative to control. CECs expression ZO-1 is decreased in the area of guttae ingrowth. Coexpression of cytokeratin AE1/AE3 and vimentin is found. Morphological and immunohistochemical studies show DM’ thickening, stromal and epithelial edema and local fibrosis, vimentin (stromal cells) and cytokeratin AE1/AE3 (epithelium) expression in FECD. Conclusion: Pathogenesis of FECD include CECs’ epithelial-mesenchymal transition, followed by guttae formation – the excrescences, which destroy cytosketon and lead to progressive loss of CECs. Changes in permeability of endothelium cause edema and fibrosis of stroma and epithelial layer.

Characterization of c-Jun N-terminal kinase (JNK) gene reveals involvement of immune defense against Vibrio splendidus infection in Apostichopus japonicus

The c-Jun N-terminal kinase (JNK) constitutes an evolutionarily conserved family of serine/threonine protein kinases, pivotal in regulating various physiological processes in vertebrates, encompassing apoptosis and antibacterial immunity. Nevertheless, the involvement of JNK in the innate immune response remains largely unexplored in pathogen-induced echinoderms. We isolated and characterized the JNK gene from Apostichopus japonicus (AjJNK) in our investigation. The full-length cDNA sequences of AjJNK spanned 1806 bp, comprising a 1299 bp open reading frame (ORF) encoding 432 amino acids, a 274 bp 5′-untranslated region (UTR), and a 233 bp 3′-UTR. Structural analysis revealed the presence of a classical S_TKc domain (37–335 amino acids) within AjJNK and contains several putative immune-related transcription factor-binding sites, including Elk-1, NF-κB, AP-1, and STAT5. Spatial expression analysis indicated ubiquitous expression of AjJNK across all examined tissues, with the highest expression noted in coelomocytes. The mRNA, protein, and phosphorylation levels of AjJNK were obviously induced in coelomocytes upon V. splendidus challenge and lipopolysaccharide stimulation. Immunofluorescence analysis demonstrated predominant cytoplasmic localization of AjJNK in coelomocytes with subsequent nuclear translocation following the V. splendidus challenge in vivo. Moreover, siRNA-mediated knockdown of AjJNK led to a significant increase in intracellular bacterial load, as well as elevated levels of Ajcaspase 3 and coelomocyte apoptosis post V. splendidus infection. Furthermore, the phosphorylation levels of AjJNK inhibited by its specific inhibitor SP600125 and also significantly suppressed the expression of Ajcaspase 3 and coelomocyte apoptosis during pathogen infection. Collectively, these data underscored the pivotal role of AjJNK in immune defense, specifically in the regulation of coelomocyte apoptosis in V. splendidus-challenged A. japonicus.

Selective autophagy receptor p62/SQSTM1 inhibits TBK1-IRF7 innate immune pathway in triploid hybrid fish

The production of type I interferon is tightly regulated to prevent excessive immune activation. However, the role of selective autophagy receptor SQSTM1 in this regulation in teleost remains unknown. In this study, we cloned the triploid fish SQSTM1 (3nSQSTM1), which comprises 1371 nucleotides, encoding 457 amino acids. qRT-PCR data revealed that the transcript levels of SQSTM1 in triploid fish were increased both in vivo and in vitro following spring viraemia of carp virus (SVCV) infection. Immunofluorescence analysis confirmed that 3nSQSTM1 was mainly distributed in the cytoplasm. Luciferase reporter assay results showed that 3nSQSTM1 significantly blocked the activation of interferon promoters induced by 3nMDA5, 3nMAVS, 3nTBK1, and 3nIRF7. Co-immunoprecipitation assays further confirmed that 3nSQSTM1 could interact with both 3nTBK1 and 3nIRF7. Moreover, upon co-transfection, 3nSQSTM1 significantly inhibited the antiviral activity mediated by TBK1 and IRF7. Mechanistically, 3nSQSTM1 decreased the TBK1 phosphorylation and its interaction with 3nIRF7, thereby suppressing the subsequent antiviral response. Notably, we discovered that 3nSQSTM1 also interacted with SVCV N and P proteins, and these viral proteins may exploit 3nSQSTM1 to further limit the host's antiviral innate immune responses. In conclusion, our study demonstrates that 3nSQSTM1 plays a pivotal role in negatively regulating the interferon signaling pathway by targeting 3nTBK1 and 3nIRF7.

Effect of low-intensity pulsed ultrasound on the mineralization of force-treated cementoblasts and orthodontically induced inflammatory root resorption via the Lamin A/C-Yes associated protein axis.

Orthodontic treatment commonly results in orthodontically induced inflammatory root resorption (OIIRR). This condition arises from excessive orthodontic force, which triggerslocal inflammatory responses and impedes cementoblasts' mineralization capacity. Low-intensity pulsed ultrasound (LIPUS) shows potential in reducing OIIRR. However, the precise mechanisms through which LIPUS reduces OIIRR remain unclear. This study aimed to explore the effects and mechanisms of LIPUS on the mineralization of force-treated cementoblasts and its impact on OIIRR. We established a rat OIIRR model and locally administered LIPUS stimulation for 7 and 14 days. We analyzed root resorption volume, osteoclast differentiation, and the expression of osteocalcin and yes-associated protein 1 (YAP1) using micro-computed tomography (micro-CT), hematoxylin and eosin, tartrate-resistant acid phosphatase, immunofluorescence and immunohistochemistry staining. Invitro, we applied compressive force and LIPUS to the immortalized mouse cementoblasts (OCCM30). We assessed mineralization using alkaline phosphatase (ALP) staining, alizarin red staining, real-time quantitative polymerase chain reaction, Western blotting and immunofluorescence staining. In rats, LIPUS reduced OIIRR, as evidenced by micro-CT analysis and histological staining. Invitro, LIPUS enhanced mineralization of force-treated OCCM30 cells, as indicated by ALP and alizarin red staining, upregulated mRNA expression of mineralization-related genes, and increased protein expression of mineralization markers. Mechanistically, LIPUS activated YAP1 signaling via the cytoskeleton-Lamin A/C pathway, supported by immunofluorescence and Western blot analysis. This study demonstrates that LIPUS promotes mineralization in force-treated cementoblasts and reduces OIIRR by activating YAP1 through the cytoskeletal-Lamin A/C signaling pathway. These findings provide fresh insights into how LIPUS benefits orthodontic treatment and suggest potential strategies for preventing and treating OIIRR.

Characterization of an RR-2 cuticle protein DcCP8 and its potential application based on SPc nanoparticle-wrapped dsRNA in Diaphorina citri.

The insect cuticle consists of chitin fibers and a protein matrix, which plays an important role in protecting the body from invasion of various pathogens and prevents water loss. Periodic synthesis and degradation of the cuticle is required for the growth and development of insects. Key genes involved in cuticle formation have long been considered a potential target for pest control. In this study, a member of the RR-2 subfamily of cuticular protein 8 (DcCP8) was identified from the Diaphorina citri genome database. Immunofluorescence analysis suggested that DcCP8 was mainly located in the Diaphorina citri exocuticle and can be induced to up-regulate 12 h following 20-hydroxyecdysone (20E) treatment. Silencing of DcCP8 by RNA interference (RNAi) significantly disrupted the metamorphosis to the adult stage, and improved the permeability of the cuticle. Transmission electron microscopy (TEM) analysis revealed that the synthesis of the exocuticle was impressed after silencing of DcCP8. Furthermore, the recombinant DcCP8 protein exhibited chitin-binding properties in vitro, down-regulation of DcCP8 significantly inhibited expression levels of chitin metabolism-related genes. Additionally, a sprayable RNAi method targeting DcCP8 based on star polycation (SPc) nanoparticles-wrapped double-stranded RNA (dsRNA) significantly increased Diaphorina citri mortality. Transcriptome sequencing further confirmed that genes associated with the endocytic pathway and immune response were up-regulated in Diaphorina citri after SPc treatment. The current study indicated that DcCP8 is critical for the formation of Diaphorina citri exocuticles, and lays a foundation for Diaphorina citri control based on large-scale dsRNA nanoparticles. © 2024 Society of Chemical Industry.

Engineered Immunomodulatory Nanoparticles Inhibit Root Resorption and Ankylosis

IntroductionExternal root resorption following avulsion injury is a complex process wherein differentiation of macrophages (Mϕ) to multinucleated osteoclasts is temporally regulated by resident periodontal fibroblasts (PDLF). The current study aims to assess the effect of engineered bioactive chitosan nanoparticles (CSNP), sustained released dexamethasone conjugated CSNP (CS-DEX) and CSNP functionalized with photosensitizer Rose Bengal (CSRB) for application in root resorption using an in-vitro PDLF-Mϕ direct coculture model and in-vivo delayed reimplantation model. MethodsPDLF-Mϕ direct coculture system was exposed to lipopolysaccharide (LPS), macrophage colony stimulating factor, receptor activator of nuclear factor kappa β ligand with or without CSNP/CS-DEX for 7 days. Clastic differentiation was assessed by tartrate resistant acid phosphatase (TRAP) staining on day 7. On day 2 and 7, immunofluorescence analysis was conducted to assess the expression of Mϕ polarization markers (CD80, CD206), multinucleation markers (NFATc1, STAT6) in Mϕ and matricellular protein periostin in PDLF and cytokine profiling in cell culture supernatants. Delayed replantation model with extraoral air dry/LPS exposure for 1h followed by root surface treatment with CS-DEX/CSRB was used in Wistar rats. After 21 days, rats were euthanized for histologic and immunofluorescence analysis. Statistical analysis one-way ANOVA with Tukey's multiple comparisons was used to analyze the data (P < .05). ResultsCS-DEX significantly reduced TRAP+ multinucleated cells and CSNP treatment showed no TRAP+ cells. Immunofluorescence analysis showed that CSNP/CS-DEX reduced CD80, NFATc1 and STAT6 expression and increased periostin as expressed by fluorescence intensity. CSNP/CS-DEX significantly reduced TNFα, MMP9 and increased IL10, TGFβ1. Osteoprotegerin was upregulated only by CSNP. Root surface treatment in delayed replantation model showed that CS-DEX and CSRB substantially reduced the degree of resorption and ankylosis. Further, CD80, CD206, and MMP2 expression in groups with root surface treatment with CS-DEX and CSRB was lower than airdry/LPS group and similar to healthy control and NFATc1, STAT6, and MMP9 expressions were lower than healthy control. ConclusionThe engineered nanosized immunomodulatory bioactive materials chitosan nanoparticles functionalized with photosensitizer and dexamethasone effectively reduced the clastic differentiation of Mϕ in in-vitro coculture and minimized the resorption and ankylosis in a delayed reimplantation model. These biomaterials have the potential to serve as root modification agents, promoting favorable healing outcomes in cases of dental avulsion.

RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7days and sustained for 28days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

Overcoming ABCB1 mediated multidrug resistance in castration resistant prostate cancer

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. PCa that relapses after hormonal therapies, referred to as castration resistant PCa (CRPC), often presents with metastases (mCRPC) that are the major cause of mortality. The few available therapies for mCRPC patients include taxanes docetaxel (DTX) and cabazitaxel (CBZ). However, development of resistance limits their clinical use. Mechanistically, resistance arises through upregulation of multidrug resistance (MDR) proteins such as MDR1/ABCB1, making ABCB1 an attractive therapeutic target. Yet, ABCB1 inhibitors failed to be clinically useful due to low specificity and toxicity issues. To study taxanes resistance, we produced CBZ resistant C4-2B cells (RC4-2B) and documented resistance to both CBZ and DTX in cell culture and in 3D prostaspheres settings. RNAseq identified increased expression of ABCB1 in RC4-2B, that was confirmed by immunoblotting and immunofluorescent analysis. ABCB1-specific inhibitor elacridar reversed CBZ and DTX resistance in RC4-2B cells, confirming ABCB1-mediated resistance mechanism. In a cell-based screen using a curated library of cytotoxic drugs, we found that DNA damaging compounds Camptothecin (CPT) and Cytarabine (Ara-C) overcame resistance as seen by similar cytotoxicity in parental C4-2B and resistant RC4-2B. Further, these compounds were cytotoxic to multiple PC cells resistant to taxanes with high ABCB1 expression and, therefore, can be used to conquer the acquired resistance to taxanes in PCa. Finally, inhibition of cyclin-dependent kinases 4/6 (CDK4/6) with small molecule inhibitors (CDK4/6i) potentiated cytotoxic effect of CPT or Ara-C in both parental and resistant cells. Overall, our findings indicate that DNA damaging agents CPT and Ara-C alone or in combination with CDK4/6i can be suggested as a new treatment regimen in CRPC patients, including those that are resistant to taxanes.

Unveiling the Mechanism of Liangxue Siwu Decoction in Treating Rosacea Through Network Pharmacology and in-vitro Experimental Validation.

Rosacea, a recurring dermatological disorder, demands effective therapeutic approaches. Traditional Chinese medicine, particularly Liangxue Siwu Decoction (LXSWD), has shown promise in managing inflammatory skin diseases, such as rosacea. This study focuses on uncovering LXSWD's specific effects on the inflammatory symptoms of rosacea. Our research investigates LXSWD's therapeutic effectiveness in rosacea treatment and delves into its underlying mechanisms. Network pharmacology was utilized to identify LXSWD's key components and their targets in rosacea management, which were then validated by molecular docking. An in vivo rosacea-like model in LL-37-induced mice was developed, subdividing them into control, model, and LXSWD groups. The LXSWD group received oral administration (25.0 g/kg/day) for six days before model induction. Post-treatment evaluations included skin tissue analyses to verify our network pharmacology predictions. Key active ingredients in LXSWD, such as quercetin, kaempferol, and luteolin, were identified alongside central target proteins like TNF and MMPs. Our molecular docking study confirmed the interactions between these ingredients and targets. Analyses through GO and KEGG pathways indicated LXSWD's role in mitigating inflammation, particularly influencing the TNF and IL-17 pathways. LXSWD treatment in vivo markedly alleviated LL-37-induced symptoms in mice, showing a marked reduction in inflammatory cytokines (p < 0.05) and modulation of crucial genes (p < 0.05). These results, supported by immunofluorescence analysis and Western blot, underline the modulatory effects of LXSWD on MMPs, offering significant protection against rosacea's inflammation alterations (p < 0.05). Integrating network pharmacology, molecular docking, and in vivo experiments, this study elucidates LXSWD's potential mechanisms in rosacea treatment. It offers a novel theoretical framework for its clinical use in managing rosacea.

A Tensioned Human Skin Explant Model Used for Preliminary Assessment of Chemexfoliant-Stimulated Bioeffects

A tensioned exvivo full-thickness human skin explant platform was used to assess the bioeffects arising from application of several commercial chemexfoliation agents. Although such treatments are well-established, and improved understanding of the underlying mechanistic processes continues to emerge, research into the optimum treatments for specific skin types/conditions is still needed for enhanced efficacy while minimizing recovery time. The 3 commercial chemexfoliation agents employed all contained trichloroacetic acid at well-defined concentrations (6, 10, and 20%) and were applied to the explants' stratum corneum. Subsequently, measurements of dermal remodeling factors (COL1A1, ELN, HAS2, HAS3, and procollagen type I) and inflammatory marker (IL-1b) were undertaken using qPCR and immunofluorescent analyses. Statistical analysis of these data facilitated the establishment of benchmarking biological responses to these trichloroacetic acid-containing agents against untreated controls. The performance of an innovative trichloroacetic acid-free chemexfoliation agent was then measured and, upon comparison with the previous benchmarking data, indicated that dermal remodeling factors could be upregulated in fashion comparable with that of the trichloroacetic acid-containing agents but with significant suppression of inflammatory response. Our measurements thus underscore the promise of the tensioned explant over prolonged study periods and also that potentially valuable insights to guide preclinical strategies may be forthcoming from the protocol developed.

OTULIN's influence on neuroinflammation and pain modulation in trigeminal neuralgia.

Trigeminal neuralgia (TN), marked by chronic pain from neural damage, is closely associated with inflammation. The role of OTULIN, a key regulator in inflammation and autophagy, is not fully understood in TN. The regulatory mechanism of OTULIN, a key protein involved in modulating inflammatory responses and autophagy processes, remains incompletely elucidated, particularly in the context of TN and neuroinflammation. An infraorbital nerve ligation-induced rat model of TN was used. OTULIN's expression was modulated using adenovirus vectors and short hairpin RNA. The impact on pain and inflammatory responses was assessed via quantitative real-time polymerase chain reaction, western blot, immunofluorescence, and transcriptomic analysis. Enhanced OTULIN expression significantly increased head withdrawal thresholds and reduced pain sensitivity and neuroinflammatory markers in the model. Conversely, silencing OTULIN exacerbated pain and inflammation. Transcriptomic data revealed OTULINs influence on both inflammatory and autophagy pathways, specifically in suppressing NLR family pyrin domain containing 3 (NLRP3) inflammasome and promoting autophagy. Invitro experiments demonstrated OTULIN's inhibition of inflammatory markers in microglia and neurons. OTULIN is crucial in modulating TN, reducing neuropathic pain and neuroinflammation by activating the autophagy pathway and inhibiting the NLRP3 inflammasome.