Effect of mild moxibustion on intestinal microbiota and NLRP6 inflammasome signaling in rats with post-inflammatory irritable bowel syndrome.

BACKGROUND: About one-third of refractory irritable bowel syndrome (IBS) cases are caused by gastrointestinal (GI) infection/inflammation, known as post-infectious/post-inflammatory IBS (PI-IBS). Although it is known that intestinal microbiota and host NOD-like receptor family pyrin domain containing 6 (NLRP6) inflammsome signaling are closely related to PI-IBS and moxibustion has a therapeutic effect on PI-IBS, whether moxibustion regulates the intestinal flora and host NLRP6 events in PI-IBS remains unclear. AIM: To examine the regulatory effect of moxibustion on intestinal microbiota and host NLRP6 inflammatory signaling in PI-IBS. METHODS: Sprague-Dawley rats were divided into a normal control group, a model control group, a mild moxibustion group, and a sham mild moxibustion group. PI-IBS rats in the mild moxibustion group were treated with moxibusiton at bilateral Tianshu (ST 25) and Zusanli (ST36) for 7 consecutive days for 10 min each time. The sham group rats were given the same treatment as the mild moxibustion group except the moxa stick was not ignited. Abdominal withdrawal reflex (AWR) score was measured to assess the visceral sensitivity, and colon histopathology and ultrastructure, colonic myeloperoxidase (MPO) activity, and serum C-reactive protein (CRP) level were measured to evaluate low-grade colonic inflammation in rats. The relative abundance of selected intestinal bacteria in rat feces was detected by 16S rDNA PCR and the NLRP6 inflammsome signaling in the colon was detected by immunofluorescence, qRT-PCR, and Western blot. RESULTS: The AWR score was significantly decreased and the low-grade intestinal inflammation reflected by serum CRP and colonic MPO levels was inhibited in the mild moxibustion group compared with the sham group. Mild moxibustion remarkably increased the relative DNA abundances of Lactobacillus, Bifidobacterium, and Faecalibacterium prausnitzii but decreased that of Escherichia coli in the gut of PI-IBS rats. Additionally, mild moxibustion induced mRNA and protein expression of intestine lectin 1 but inhibited the expression of IL-1ß, IL-18, and resistance-like molecule ß by promoting the NLRP6 and reducing the mRNA and protein expression of apoptosis-associated speck-like protein containing CARD (ASC) and cysteinyl-aspartate-specific proteinase 1 (Caspase-1). The relative DNA abundances of Lactobacillus, Bifidobacteria, Faecalibacterium prausnitzii, and Escherichia coli in each group were correlated with the mRNA and protein expression of NLRP6, ASC, and Caspase-1 in the colon. CONCLUSION: These findings indicated that mild moxibustion can relieve low-grade GI inflammation and alleviate visceral hypersensitivity in PI-IBS by regulating intestinal microbes and controlling NLRP6 inflammasome signaling.

Cellular and subcellular distribution of the type-2 vasopressin receptor in the kidney.

Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.