BACKGROUND: Postmenopausal osteoporosis is a chronic metabolic bone disease caused by excessive osteoclast formation and function. Targeting osteoclast differentiation and activity can modulate bone resorption and alleviate osteoporosis. Cirsilineol, an active constituent of Vestita Wall, has shown numerous biological activities and has been used to treat many metabolic diseases. However, whether cirsilineol inhibits osteoclast activity and prevents postmenopausal osteoporosis still remain unknown.
METHODS: Primary bone marrow macrophages (BMMs) and RAW264.7 cells were used. Osteoclast activity was measured by TRAP staining, F-actin staining, and bone resorption assay after BMMs were treated with cirsilineol at concentrations of 0, 1, 2.5 and 5 µM. RT-PCR and western blotting were performed to evaluate the expression of osteoclast-related genes. In addition, female C57BL/6 mice underwent OVX surgery and were treated with cirsilineol (20 mg/kg) to demonstrate the effect of cirsilineol on osteoporosis.
RESULTS: Cirsilineol significantly inhibited receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclast differentiation in a concentration- and time-dependent manner, respectively. Additionally, cirsilineol inhibited F-actin ring formation, thus reducing the activation of bone resorption ability. Cirsilineol suppressed the expression of osteoclast-related genes and proteins via blocking nuclear factor (NF)-κb, ERK, and p38 signaling cascades. More importantly, cirsilineol treatment in mice with osteoporosis alleviated osteoclasts hyperactivation and bone mass loss caused by estrogen depletion.
CONCLUSIONS: In this study, the protective effect of cirsilineol on osteoporosis has been investigated for the first time. In conclusion, our findings prove the inhibitory effect of cirsilineol on osteoclast activity via NF-κb/ERK/p38 signaling pathways and strongapplication of cirsilineol can be proposed as a potential therapeutic strategy.

Introduction: IgA nephropathy (IgAN) is the most common disease leading to end-stage renal disease, and tubular fibrosis represents an important risk factor for disease progression. However, research on early molecular diagnostic indicators of tubular fibrosis and the mechanisms underlying disease progression is still lacking. Methods: The GSE93798 dataset was downloaded from the GEO database. DEGs were screened and analyzed for GO and KEGG enrichment in IgAN. The least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) algorithms were applied to screen for hub secretory genes. The expression and diagnostic efficacy of hub genes were confirmed by the GSE35487 dataset. ELISA was applied to detect the expression of APOC1 in serum. The expression and localization of hub genes in IgAN were verified by the expression of IHC and IF in human kidney tissues, and the correlation of expression with clinical data was verified in the Nephroseq database. Finally, cellular experiments clarified the role of hub genes in the signaling pathway. Results: A total of 339 DEGs were identified in IgAN, of which 237 were upregulated and 102 downregulated. The KEGG signaling pathway is enriched in the ECM-receptor interaction and AGE-RAGE signaling pathway. APOC1, ALB, CCL8, CXCL2, SRPX2, and TGFBI identified six hub secretory genes using the LASSO and SVM-RFE algorithms. In vivo and in vitro experiments demonstrated that APOC1 expression was elevated in IgAN. The serum concentration of APOC1 was 1.232 ± 0.1812 μg/ml in IgAN patients, whereas it was 0.3956 ± 0.1233 μg/ml in healthy individuals. APOC1 exhibited high diagnostic efficacy for IgAN (AUC of 99.091%, specificity of 95.455%, and sensitivity of 99.141%) in the GSE93798 dataset. APOC1 expression negatively correlated with eGFR (R 2 = 0.2285, p = 0.0385) and positively correlated with serum creatinine (R 2 = 0.41, p = 0.000567) in IgAN. APOC1 exacerbated renal fibrosis, possibly in part by activating the NF-κB pathway in IgAN. Conclusion: APOC1 was identified as the core secretory gene of IgAN, which was closely associated with blood creatinine and eGFR and had significant efficacy in the diagnosis of IgAN. Mechanistic studies revealed that the knockdown of APOC1 could improve IgAN renal fibrosis by inhibiting the NF pathway, which may be a potential therapeutic target for improving renal fibrosis in IgAN.

BACKGROUND: NK cell is one of innate immune cells and can protect the body from cancer-initiating cells. It has been reported that GPR116 receptor is involved in inflammation and tumors. However, the effect of GPR116 receptor on the NK cells remains largely unclear.
RESULTS: We discovered that GPR116-/- mice could efficiently eliminate pancreatic cancer through enhancing the proportion and function of NK cells in tumor. Moreover, the expression of GPR116 receptor was decreased upon the activation of the NK cells. Besides, GPR116-/- NK cells showed higher cytotoxicity and antitumor activity in vitro and in vivo by producing more GzmB and IFNγ than wild-type (WT) NK cells. Mechanistically, GPR116 receptor regulated the function of NK cells via Gαq/HIF1α/NF-κB signaling pathway. Furthermore, downregulation of GPR116 receptor promoted the antitumor activity of NKG2D-CAR-NK92 cells against pancreatic cancer both in vitro and in vivo.
CONCLUSIONS: Our data indicated that GPR116 receptor had a negatively effect on NK cell function and downregulation of GPR116 receptor in NKG2D-CAR-NK92 cells could enhance the antitumor activity, which provides a new idea to enhance the antitumor efficiency of CAR NK cell therapy.

OBJECTIVE: Limonin has received significant attention due to its multiple biological effects, intervertebral disc degeneration (IDD) is also of interest due to the high prevalence of this disease. In this study, we determined the effects of limonin on IDD and the underlying mechanism of action to find novel ways to treat IDD.
METHODS: An IL-1β-induced cell inflammation model and a lumbar instability model inducing IDD were established to assess the progression of IDD with or without limonin treatment. We further evaluated MAPK/NF-κB and necroptosis pathways and alterations in the extracellular matrix specific within the disc.
RESULTS: Limonin suppresses inflammation in the nucleus pulposus in vitro by reducing the production of pro-inflammatory markers such as iNOS and COX-2. Limonin reduced the activation of the MAPK/NF-κB signalling pathway and the RIP1/RIP3/MLKL necroptosis pathway in the NP cells. Moreover, limonin delays the IDD progression in the lumbar instability model.
CONCLUSIONS: Limonin could potentially delay IDD by inhibiting NP cell necroptosis and modulating peripheral matrix proteins within the intervertebral disc and is a potential pharmacological research direction for the therapy in patients with IDD.

OBJECTIVE: As one of the major causes of low back pain, intervertebral disc degeneration (IDD) has caused a huge problem for humans. Increasing evidence indicates that NLRP3 inflammasome-mediated pyroptosis of NP cells displays an important role in the progression of IDD. Maltol (MA) is a flavoring agent extracted from red ginseng. Due to its anti-inflammatory and antioxidant effects, MA has been widely considered by researchers. Therefore, we hypothesized that MA may be a potential IVD protective agent by regulating NP cells and their surrounding microenvironment.
METHODS: In vitro, qRT-PCR, and Western blot were used to explore the effect of MA on the transcription and protein expression of the anabolic protein (ADAMTS5, MMP3, MMP9) catabolic protein (Aggrecan), and pro-inflammatory factor (iNOS COX-2). Next, the effects of MA on PI3K/AKT/NF-κB pathway and pyroptosis pathway were analyzed by Western blot and immunofluorescence. Molecular docking was used to investigate the relationship between PI3K and MA. Moreover, ELISA was also used to detect the effects of MA on inflammatory factors (TNF-α, PGE2, IL-1β, and IL-18). In vivo, the effects of MA on the vertebral structure of IDD mice were studied by HE and SO staining and the effects of MA on ECM and PI3K/AKT/NF-κB and pyroptosis pathway of IDD mice were studied by immunohistochemical staining.
RESULTS: MA can ameliorate intervertebral disc degeneration in vivo and in vitro. Specifically, the molecular docking results showed that the binding degree of MA and PI3K was significant. Second, in vitro studies showed that MA inhibited the degradation of ECM and inflammatory response by inhibiting the PI3K/AKT/NF-κB pathway and the pyroptosis mediated by NLRP3 inflammasome, which increased the expression of anabolic proteins, decreased the expression of catabolic proteins, and decreased the secretion of inflammatory mediators such as IL-18 and IL-1β. In addition, according to the study results of the mouse lumbar instability model, MA also improved the tissue disorder and degradation of the intervertebral disc, reduced the loss of proteoglycan and glycosaminoglycan, and inhibited intervertebral disc inflammation, indicating that MA has a protective effect on the intervertebral disc to intervertebral disc in mice.
CONCLUSIONS: Our results suggest that MA slowed IDD development through the PI3K/AKT/NF-κB signaling pathway and NLRP3 inflammasome-mediated pyroptosis, indicating that MA appeared to be a viable medication for IDD treatment.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although β-catenin signaling is reported to be associated with inflammatory responses and BFT is known to cleave E-cadherin linked with β-catenin, little is known about the β-catenin-mediated regulation of inflammation in ETBF infection. This study was conducted to investigate the role of β-catenin as a cellular signaling intermediate in the induction of proinflammatory responses to stimulation of intestinal epithelial cells with BFT. Expression of β-catenin in intestinal epithelial cells was reduced relatively early after stimulation with BFT and then recovered to normal levels relatively late after stimulation. In contrast, phosphorylation of β-catenin in BFT-exposed cells occurred at high levels early in stimulation and decreased as time passed. Concurrently, late after stimulation the nuclear levels of β-catenin were relatively higher than those early after stimulation. Suppression of β-catenin resulted in increased NF-κB activity and interleukin-8 (IL-8) expression in BFT-stimulated cells. However, suppression or enhancement of β-catenin expression neither altered the phosphorylated IκB kinase α/β complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3β was associated with increased β-catenin expression and attenuated NF-κB activity and IL-8 expression in BFT-exposed cells. These findings suggest the negative regulation of NF-κB-mediated inflammatory responses by β-catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection.

Myeloid differentiation factor 88 (MyD88) is a pivotal adapter protein that involved in interleukin-1 receptor/toll-like receptor (IL-1R/TLR) signal transduction, which could spur downstream cascades and eventually drawn into innate immune response. MyD88 has been extensively studied in vertebrates, however, the information ascribe to MyD88 in invertebrates is still very scarce especially its function annotation remains extremely obscure. At here, three novel MyD88 isoforms termed McMyD88a, McMyD88b and McMyD88c were firstly cloned from thick shell mussel Mytilus coruscus. McMyD88a, McMyD88b and McMyD88c shared domain topology containing the Death domain (DD) and TIR domain (TIR) with its counterparts in mammals. All three McMyD88s were ubiquitously expressed in examined tissues in thick shell mussel, with the higher expression levels in immune-related tissues such as haemocytes, gills and digestive glands. Upon Vibrio alginolyticus, polyinosine-polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) challenge, McMyD88a, McMyD88b and McMyD88c transcripts were significantly induced in haemocytes despite of differential expression levels and responsive time points. Overexpression of McMyD88a, McMyD88b and McMyD88c showed a dose-dependent induction to NF-κB or ISRE in mammalian cell lines. Taken together, these results suggested that McMyD88a, McMyD88b and McMyD88c are members of MyD88 family and play potential roles in innate immune response to pathogenic invasions in thick shell mussel. Moreover, these results suggested indirectly the existence of a MyD88-dependent signaling pathway in thick shell mussel, and provide insight into the immunoregulatory effect in molluscs.

Simulated-microgravity (SMG) promotes cell-apoptosis. We demonstrated that SMG inhibited cell proliferation/metastasis via FAK/RhoA-regulated mTORC1 pathway. Since mTORC1, NF-κB, and ERK1/2 signaling are important in cell apoptosis, we examined whether SMG-enhanced apoptosis is regulated via these signals controlled by FAK/RhoA in BL6-10 melanoma cells under clinostat-modelled SMG-condition. We show that SMG promotes cell-apoptosis, alters cytoskeleton, reduces focal adhesions (FAs), and suppresses FAK/RhoA signaling. SMG down-regulates expression of mTORC1-related Raptor, pS6K, pEIF4E, pNF-κB, and pNF-κB-regulated Bcl2, and induces relocalization of pNF-κB from the nucleus to the cytoplasm. In addition, SMG also inhibits expression of nuclear envelope proteins (NEPs) lamin-A, emerin, sun1, and nesprin-3, which control nuclear positioning, and suppresses nuclear positioning-regulated pERK1/2 signaling. Moreover, rapamycin, the mTORC1 inhibitor, also enhances apoptosis in cells under 1 g condition via suppressing the mTORC1/NF-κB pathway. Furthermore, the FAK/RhoA activator, toxin cytotoxic necrotizing factor-1 (CNF1), reduces cell apoptosis, restores the cytoskeleton, FAs, NEPs, and nuclear positioning, and converts all of the above SMG-induced changes in molecular signaling in cells under SMG. Therefore, our data demonstrate that SMG reduces FAs and alters the cytoskeleton and nuclear positioning, leading to enhanced cell apoptosis via suppressing the FAK/RhoA-regulated mTORC1/NF-κB and ERK1/2 pathways. The FAK/RhoA regulatory network may, thus, become a new target for the development of novel therapeutics for humans under spaceflight conditions with stressed physiological challenges, and for other human diseases.

The imidazoquinoline compound R848 is a specific agonist of toll-like receptor (TLR) 7/TLR8 that has been used as an immunostimulant in humans against viral diseases. Although R848-induced immune response has been reported in teleost fish, the relevant mechanism is not clear. In this study, we investigated the antiviral potential and the signaling pathway of R848 in a model of Japanese flounder (Paralichthys olivaceus). We found that R848 was able to inhibit the replication of megalocytivirus, stimulated the proliferation of peripheral blood leukocytes (PBL), enhanced the expression of immune genes, and reduced apoptosis of PBL. When endosomal acidification was blocked by chloroquine (CQ), R848-mediated antiviral activity and immune response were significantly reduced. Likewise, inhibition of Myd88 activation markedly impaired the pro-proliferation and anti-apoptosis effect of R848. Cellular study showed that cultured founder cells treated with R848 exhibited augmented NF-κB activity, which, however, was dramatically reduced in the presence of CQ and Myd88 inhibitor. Furthermore, when NF-κB was inactivated, the effect of R848 on cell proliferation and apoptosis was significantly decreased. Taken together, these results indicate that R848 is an immunostimulant with antiviral property in a teleost species, and that the immune response of R848 is mediated by, most likely, TLR7/TLR8 signaling pathway, in which Myd88 and NK-κB play an essential role.

Tumor necrosis factor-induced protein 3 (TNFAIP3; also known as A20) negatively regulates NF-κB and MAPK signals to control inflammatory responses. TNFAIP3 also protects against TNF-induced cell death. Intestinal epithelial cell (IEC) expression of TNFAIP3 improves barrier function and tight junction integrity and prevents dextran sulfate sodium (DSS)-induced IEC death and colitis. We therefore investigated the effects of TNFAIP3 expression in IEC on immune homeostasis in the intestines of immune-compromised mice. Villin-TNFAIP3 (v-TNFAIP3) transgenic mice were interbred with IL-10(-/-) mice (v-TNFAIP3 × IL-10(-/-)) and incidence, onset, and severity of colitis was assessed. v-TNFAIP3 × IL-10(-/-) mice displayed severe, early onset, and highly penetrant colitis that was not observed in IL-10(-/-) or v-TNFAIP3 mice. V-TNFAIP3 mice displayed altered expression of mucosal cytokines, increased numbers of mucosal regulatory T cells, and altered expression of mucosal antimicrobial peptides (AMPs). Microbial colonization of the inner mucus layer of v-TNFAIP3 mice was observed, along with alterations in the microbiome, but this was not sufficient to induce colitis in v-TNFAIP3 mice. The relative sterility of the inner mucus layer observed in wild-type and IL-10(-/-) mice was lost in v-TNFAIP3 × IL-10(-/-) mice. Thus IEC-derived factors, induced by signals that are inhibited by TNFAIP3, suppress the onset of inflammatory bowel disease in IL-10(-/-) mice. Our results indicate that IEC expression of TNFAIP3 alters AMP expression and allows microbial colonization of the inner mucus layer, which activates an IL-10-dependent anti-inflammatory process that is necessary to prevent colitis.