RESUMEN
Impaired intestinal barrier function and gut microbiota dysbiosis are believed to be related to exacerbation of acute pancreatitis (AP). As a bacterial cell wall peptidoglycan component, diaminopimelic acid (DAP) is a specific ligand of NOD1 that regulates the NOD1/RIP2/NF-kB signaling pathway. Here, we investigated the role of DAP in the crosstalk between the gut microbiota and pancreas during the occurrence of AP. Upregulation of NOD1/RIP2/NF-kB and elevated serum DAP levels were found in severe AP (SAP) model rats. The accumulation of DAP in SAP patients corroborated its ability to serve as an indicator of disease severity. Subsequently, SAP rats were treated with oral administration of the traditional Chinese medicine Qingyi Keli (QYKL) as well as neomycin, which can widely eliminate DAP-containing bacteria. Both QYKL and neomycin intervention ameliorated intestinal and pancreatic damage and systemic inflammation in SAP rats. Through 16S rDNA sequencing, we found that QYKL could rehabilitate the gut microbiota structure and selectively inhibit the overgrowth of enteric bacteria, such as Helicobacter and Lactobacillus, in SAP rats without affecting some protective strains, including Romboutsia and Allobaculum. Interestingly, we demonstrated that the decrease in serum DAP was accompanied by suppression of the NOD1/RIP2/NF-kB signaling pathway in both the intestine and pancreas of the two intervention groups. Taken together, these results suggested that the gut microbiota-DAP-NOD1/RIP2 signaling pathway might play a critical role in the progression of AP and that SAP could be alleviated via intervention in the signaling pathway. Our work provides new potential early warning indicators of SAP and targets for intervention.
Asunto(s)
Microbioma Gastrointestinal , Pancreatitis , Enfermedad Aguda , Animales , Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Ácido Diaminopimélico/farmacología , Microbioma Gastrointestinal/fisiología , FN-kappa B/metabolismo , Neomicina , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD1/metabolismo , Ratas , Transducción de SeñalRESUMEN
The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/farmacología , Adyuvantes Inmunológicos/farmacología , Ácido Diaminopimélico/análogos & derivados , Microglía/efectos de los fármacos , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD2/agonistas , Fenotipo , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Forma de la Célula , Extensiones de la Superficie Celular/efectos de los fármacos , Células Cultivadas , Ácido Diaminopimélico/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/citología , Microglía/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The following aminopeptidase (AP) activities were found to be associated with the surface of mouse spleen cells: Leu-AP (138 pmol/10(5) cells X minute) and AP-B (16 pmol/10(5) cells X minute with Lys-beta-naphthylamide as substrate and 21 pmol/10(5) cells X minute with Arg-beta-naphthylamide substrate); AP-A activity was not detected by the assay system applied. The immunoactive peptide bestatin inhibited the Leu-AP, while AP-B activity decreased in the presence of both arphamenines A and B and bestatin. No effects on these enzymes were caused by amastatin (an AP-A inhibitor), FK-156, FK-565 and Bu-2743E; the latter peptide turned out to be not an inhibitor of cell surface associated microsomal Leu-AP but an inhibitor of cytosolic Leu-AP. The immunoactive peptides bestatin, arphamenines A and B, and amastatin increased [3H]thymidine incorporation into spleen cells containing lymphocytes and macrophages. These mitogenic actions were not observed when macrophages were removed from the cultures or the cells had been stimulated with ConA or LPS. The lactoyl- and heptanoyl peptides FK-156 and FK-565 caused a mitogenic action on lymphocytes independently of the presence of macrophages. The inhibitor of cytosolic Leu-AP did not change the incorporation into lymphocytes.