OBJECTIVE: To observe the effect of "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique on the relative protein expression of the signaling pathway of nucleotide-binding oligomerization domain-containing protein 1 (NOD1)/ receptor-interacting protein 2 (RIP2)/nuclear factor kappa-B (NF-κB) and the expression of proinflammatory cytokines in the rats with primary dysmenorrhea (PD), so as to explore the underlying mechanism of this acupuncture technique for pain alleviation in PD. METHODS: Thirty female SD rats of SPF grade with normal estrous cycle were randomized into a blank group, a model group and an acupuncture group, 10 rats in each one. Using the intraperitoneal injection with estradiol benzoate combined with oxytocin, PD model was prepared in the model group and the acupuncture group. In the acupuncture group, during model preparation, the intervention with "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique was delivered simultaneously, 20 min each time, once daily for consecutive 10 days. On day 11, within 30 min after the intraperitoneal injection with oxytocin, the writhing reaction (latency, frequency and score) was recorded; the morphology of uterine tissue was observed with HE staining, the contents of prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), interleukin (IL)-1β, IL-18, cyclooxygenase-2 (COX-2), and tumor necrosis factor-α(TNF-α) in the serum were detected using ELISA method; the relative protein expression of NOD1, RIP2, NF-κB p65, phosphorylation-NF-κB p65 (p-NF-κB p65) was detected in the uterine tissue using Western blot method; and the mRNA expression of NOD1, RIP2 and NF-κB p65 was detected with the quantitative real-time PCR employed. RESULTS: Compared with the blank group, in the model group, the writhing latency was prolonged (P<0.01), the writhing frequency and score increased (P<0.01) in the rats; the endometrial epithelial cells showed massive degeneration and necrosis, with severe endometrial edema and widespread shedding, combined with neutrophil infiltration; the serum PGE₂ content was dropped (P<0.01), while those of PGF_2α, IL-1β, IL-18, COX-2, and TNF-α elevated (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65, and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue increased (P<0.01). In comparison with the model group, in the acupuncture group, the writhing latency was prolonged (P<0.01), the writhing frequency and score were reduced (P<0.01) in the rats; there was less degeneration and necrosis of endometrial epithelial cells, with mild endometrial edema and very little neutrophil infiltration; the serum PGE₂ content increased (P<0.01), while those of PGF_2α, IL-1β, IL-18, COX-2, and TNF-α decreased (P<0.01); the protein expression of NOD1, RIP2, NF-κB p65 and p-NF-κB p65 and the mRNA expression of NOD1, RIP2 and NF-κB p65 in uterine tissue were dropped (P<0.05, P<0.01). CONCLUSION "Zhibian" (BL54)-toward-"Shuidao" (ST28) needling technique can alleviate the pain symptom of PD rats, and its action mechanism may be related to inhibiting the active expression of NOD1/RIP2/NF-κB signaling pathway in the uterine tissue, thereby reducing the inflammatory response.
Animals ; Female ; Rats, Sprague-Dawley ; Rats ; Signal Transduction ; Dysmenorrhea/metabolism* ; NF-kappa B/metabolism* ; Acupuncture Points ; Humans ; Acupuncture Analgesia ; Nod1 Signaling Adaptor Protein/metabolism* ; Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism* ; Acupuncture Therapy

Cancer stem cells (CSCs) are widely acknowledged as primary mediators to the initiation and progression of tumors. The association between microbial infection and cancer stemness has garnered considerable scholarly interest in recent years. Porphyromonas gingivalis (P. gingivalis) is increasingly considered to be closely related to the development of oral squamous cell carcinoma (OSCC). Nevertheless, the role of P. gingivalis in the stemness of OSCC cells remains uncertain. Herein, we showed that P. gingivalis was positively correlated with CSC markers expression in human OSCC specimens, promoted the stemness and tumorigenicity of OSCC cells, and enhanced tumor formation in nude mice. Mechanistically, P. gingivalis increased lipid synthesis in OSCC cells by upregulating the expression of stearoyl-CoA desaturase 1 (SCD1) expression, a key enzyme involved in lipid metabolism, which ultimately resulted in enhanced acquisition of stemness. Moreover, SCD1 suppression attenuated P. gingivalis-induced stemness of OSCC cells, including CSCs markers expression, sphere formation ability, chemoresistance, and tumor growth, in OSCC cells both in vitro and in vivo. Additionally, upregulation of SCD1 in P. gingivalis-infected OSCC cells was associated with the expression of KLF5, and that was modulated by P. gingivalis-activated NOD1 signaling. Taken together, these findings highlight the importance of SCD1-dependent lipid synthesis in P. gingivalis-induced stemness acquisition in OSCC cells, suggest that the NOD1/KLF5 axis may play a key role in regulating SCD1 expression and provide a molecular basis for targeting SCD1 as a new option for attenuating OSCC cells stemness.
Porphyromonas gingivalis/pathogenicity* ; Stearoyl-CoA Desaturase/metabolism* ; Humans ; Carcinoma, Squamous Cell/pathology* ; Mouth Neoplasms/metabolism* ; Animals ; Neoplastic Stem Cells/microbiology* ; Mice, Nude ; Mice ; Nod1 Signaling Adaptor Protein/metabolism* ; Kruppel-Like Transcription Factors/metabolism* ; Cell Line, Tumor

BACKGROUND/AIMS: The cytosolic host protein nucleotide binding oligomerization domain 1 (Nod1) has emerged as a key pathogen recognition molecule for innate immune responses in epithelial cells. The purpose of the study was to elucidate the mechanism by which Helicobacter pylori infection leads to transepithelial neutrophil migration in a Nod1-mediated manner. METHODS: Human epithelial cell lines AGS and Caco-2 were grown and infected with H. pylori. Interleukin (IL)-8 mRNA expression and IL-8 secretion were assessed, and nuclear factor kappaB (NF-kappaB) activation was determined. Stable transfections of AGS and Caco-2 cells with dominant negative Nod1 were generated. Neutrophil migration across the monolayer was quantified. RESULTS: Cytotoxin-associated gene pathogenicity island (cagPAI)(+) H. pylori infection upregulated IL-8 mRNA expression and IL-8 secretion in AGS and Caco-2 cells compared with controls. NF-kappaB activation, IL-8 mRNA expression and IL-8 secretion by cagPAI knockdown strains were reduced compared with those infected with the wild-type strain. NF-kappaB activation, IL-8 mRNA expression and IL-8 secretion in dominant-negative (DN)-Nod1 stably transfected cells were reduced compared with the controls. The transepithelial migration of neutrophils in DN-Nod1 stably transfected cells was reduced compared with that in controls. CONCLUSIONS: Signaling through Nod1 plays an essential role in neutrophil migration induced by the upregulated NF-kappaB activation and IL-8 expression in H. pylori-infected human epithelial cells.
Adult Stem Cells/physiology ; Caco-2 Cells ; Cell Line ; Epithelial Cells/*metabolism/microbiology ; Gene Expression ; Genomic Islands ; Helicobacter Infections/*genetics ; *Helicobacter pylori ; Humans ; Interleukin-8/genetics/secretion ; NF-kappa B/metabolism ; Neutrophils/*physiology ; Nod1 Signaling Adaptor Protein/*physiology ; RNA, Messenger/metabolism ; Signal Transduction ; Transendothelial and Transepithelial Migration/*physiology ; Up-Regulation

OBJECTIVE: To explore role of Nods (nucleotide-binding oligomerization domain Nod Like receptors) kind of pattern recognition receptors (PRR) in patients with allergic rhinitis. METHOD: The mRNA and protein of Nod1, Nod2 of Nalp3 were analyzed in the turbinate mucosa of patients with allergic rhinitis, nasal septum deviation (NSD) nasal mucosa of patients and nasal polyp mucosa with Real-Time RT-PCR, Western blot and immunohistochemistry respectively, and Nod1 expression changes was explored in PBMC with wad explored Western-blot and then the level of IL-4, IL-6, IL-10, IFN-γ were detected in serum of AR after desensitization treatment. RESULT: These Nods like receptors, mainly found in nasal mucosa epithelial cells, glandular epithelium and inflammatory cells (e. g. plasma cells, eosinophils), were expressed in the nasal mucosa tissues. In AR group, Nod1 (mRNA and protein) expression were lower than NSD group (P<0.05), Nalp3 expression were higher than (P<0.05), while, there was no significant difference of Nod2 (mRNA and protein) between groups. After 6 months desensitization therapy, the change of Nod1 in PBMC was negatively correlated with the change of IL-10 in the peripheral blood, r=-0.88, P<0.05; while, change of Nod1 was positively correlated, with the change of IL-6, r=0.57, P>0.05. CONCLUSION Nod1, Nod2 and Nalp3 expression were seen in the two groups,and the Nod1 expression in allergic rhinitis group was lower than other two groups, while, the Nalp3 was higher than other two groups. It showed Nod1, Nalp3 may be involved in the pathogenesis of allergic rhinitis. Expression of Nod1 in PBMC reduced after sublingual desensitization treatment. Besides, the change of Nod1 was negatively correlated with the change of IL-10 in PBMC. So,it seemed that Nod1 may regulate IL-10 changes and be involved in sublingual desensitization therapy.
Carrier Proteins ; metabolism ; Humans ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Interleukin-6 ; blood ; Leukocytes, Mononuclear ; metabolism ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nasal Mucosa ; metabolism ; Nasal Polyps ; metabolism ; Nod1 Signaling Adaptor Protein ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism ; Receptors, Pattern Recognition ; metabolism ; Rhinitis, Allergic ; metabolism ; Turbinates ; metabolism

OBJECTIVETo investigate the potential role of nucleotide-binding oligomerization domain 1 (NOD1), a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes.

METHODSAdipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB (NF-κB) activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon fatty acids treatment were analyzed.

RESULTSOleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake.

CONCLUSIONNOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

Adipocytes ; drug effects ; metabolism ; Animals ; Fatty Acids ; pharmacology ; Insulin Resistance ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; physiology ; Nod1 Signaling Adaptor Protein ; physiology ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; physiology

OBJECTIVE: To investigate the expression and role of a new pattern-recognition receptors (PRR), nucleotide binding oligomerization domain (Nod) like receptors (NLRs), in the patients with nasal polyps and nasal septum normal control group. METHOD: The expressions of Nod1, Nod2 and Nalp3 mRNA and protein were explored with real-time RT-PCR, Western-Blot and immunohistochemistry respectively. RESULT: The protein levels of Nod1, Nod2 and Nalp3 were expressed in nasal polyp and the control, but the expression of Nod1 and Nalp3 in nasal polyps were higher than those in control. No significant difference of Nod2 was seen between the two groups. And then, there was no significant difference of Nod1, Nod2, Nalp3 mRNA between two groups with Real-time RT-PCR. CONCLUSION The expression of Nod1 and Nalp3 are increased in nasal polyp tissues and maybe a etiological factors in the formation of nasal polyps.
Adult ; Carrier Proteins ; metabolism ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; NLR Family, Pyrin Domain-Containing 3 Protein ; Nasal Polyps ; metabolism ; pathology ; Nod1 Signaling Adaptor Protein ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism ; Receptors, Pattern Recognition ; metabolism

OBJECTIVE: To investigate the expression of NOD1 and NOD2 in the nasal mucosa of healthy individuals and allergic rhinitis(AR), and explored the regulation of glucocorticoids on them. METHOD: RT-PCR and immunohistochemistry were used to detect the expression of NOD1 and NOD2 in nasal mucosa from healthy control and AR. Nasal explant culture was used to explore the effect of glucocorticoids on NOD1 and NOD2 expression. RESULT: NOD1 and NOD2 mRNA expression level was significantly increased in AR compared with control. Immunohistochemical staining demonstrated that NOD1 and NOD2 were mainly expressed by epithelial cells and some unknown cells in lamina propria and there were significantly more positive staining cells were observed in AR tissue when compared with control. Glucocorticoids down-regulated NOD1 and NOD2 expression in AR. CONCLUSION NOD1 and NOD2 as two PRRs may take part in the pathogenesis of AR, glucocorticoids may play a therapeutical role on allergic rhinitis through down-regulated the expression of NOD1 and NOD2.
Adult ; Case-Control Studies ; Female ; Gene Expression Regulation ; Glucocorticoids ; pharmacology ; Humans ; Male ; Middle Aged ; Nasal Mucosa ; drug effects ; metabolism ; Nod1 Signaling Adaptor Protein ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism ; Rhinitis, Allergic ; Rhinitis, Allergic, Perennial ; metabolism ; Young Adult

OBJECTIVETo investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine ÷ cytokines and insulin-dependent glucose uptake in human differentiated adipocytes.

METHODSAdipose tissues were obtained from patients undergoing liposuction. Stromal vascular cells were extracted and differentiated into adipocytes.A specific ligand for NOD1, was administered to human adipocytes in culture. Nuclear factor-κB transcriptional activity and proinflammatory chemokine ÷ cytokines production were determined by reporter plasmid assay and enzyme-linked immunosorbent assay, respectively.Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[³H] glucose uptake assay. Furthermore, chemokine ÷ cytokine secretion and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon stimulation of NOD1 ligand were analyzed.

RESULTSNuclear factor-κB transcriptional activity and monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and IL-8 secretion in human adipocytes were markedly increased stimulated with NOD1 ligand (all P<0.01).Insulin-induced glucose uptake was decreased upon the activation of NOD1 (P<0.05).NOD1 gene silencing by siRNA reduced NOD1 ligand-induced MCP-1,IL-6, and IL-8 release and increased insulin-induced glucose uptake (all P<0.05).

CONCLUSIONNOD1 activation in adipocytes might be implicated in the onset of insulin resistance.

Adipocytes ; metabolism ; Adult ; Cells, Cultured ; Cytokines ; biosynthesis ; Female ; Glucose ; metabolism ; Humans ; Inflammation ; etiology ; Insulin Resistance ; Ligands ; Middle Aged ; NF-kappa B ; physiology ; Nod1 Signaling Adaptor Protein ; physiology

OBJECTIVETo explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells (BMDCs) and T lymphocytes in vitro.

METHODSAfter BMDCs stimulated by E.coli LLO/OVA, their Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) receptor signalling pathway were examined by superarray hybridization; and the priming effect of the vaccine activated BMDCs on CD4(+)T and CD8(+)T was determined by [3H]thymidine uptake and ELISA, the tumor cytotoxic effect of activated CD8(+)T cells was determined by cytotoxic assay.

RESULTSAfter BMDCs were activated by E. coli LLO/OVA via TLR4, NOD1 receptor and NF-κB signalling pathway, the expression of their surface molecules including MHC class I, MHC class II, CD40, CD80 and CD86 significantly up-regulated; the secretion of IL-12 and IFN-γ increased also. The mature BMDCs stimulated the allergic CD4(+)T and CD8(+)T cells proliferation and their IL-2 and IFN-γ secretion, and the activated CD8(+)T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.

CONCLUSIONE.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.

Animals ; Antigens, Neoplasm ; genetics ; pharmacology ; Bacterial Toxins ; genetics ; pharmacology ; Cancer Vaccines ; genetics ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; immunology ; Coculture Techniques ; Cytokines ; immunology ; secretion ; Dendritic Cells ; cytology ; drug effects ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Female ; Flow Cytometry ; Heat-Shock Proteins ; genetics ; pharmacology ; Hemolysin Proteins ; genetics ; pharmacology ; Immunity, Innate ; drug effects ; Mice ; Mice, Inbred C57BL ; Nod1 Signaling Adaptor Protein ; genetics ; physiology ; Ovalbumin ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Toll-Like Receptor 4 ; genetics ; physiology