Fructooligosaccharides (FOS) or dead <i>L</i>. <i>salivarius</i> feeding decreased the intestinal bacterial overgrowth in STZ-DM mice.

<p>(A) Relative abundance of bacteria across difference groups, as indicated by 16S rRNA gene sequencing. Values represent the mean abundance of Genus found at >1% relative abundance in at least one sample. (B) The collected mucosa from the terminal ileum were weighed and homogenized. Total bacteria counts were significantly increased in STZ-DM mice and FOS or dead <i>L</i>. <i>salivarius</i> feeding decreased them. (C) <i>E</i>. <i>coli</i> and <i>K</i>. <i>pneumoniae</i> of intestinal lumen were significantly increased in STZ-DM mice in comparison with those in SPF WT mice. FOS or dead <i>L</i>. <i>salivarius</i> feeding decreased the growth of pathogenic bacteria such as <i>E</i>. <i>coli</i> and <i>K</i>. <i>pneumoniae</i> in STZ mice. (D) The growth of <i>lactobacilli/bifidas</i> in intestinal lumen was significantly decreased in STZ-DM mice compared with SPF mice. FOS feeding significantly increased them in STZ-DM mice. STZ, streptozotocin; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *P<0.05, **P<0.01. n = 5/group.</p

L-NAME but not D-NAME decreased iNOS expression of the intestinal mucosa, hepatic bacteria clearance, and IL-1β as well as TNF-α expression of the Kupffer cells in STZ-DM mice.

<p>(A) STZ-DM mice demonstrated a significant increase of iNOS protein expression of the intestinal mucosa as compared with that in the WT group. L-NAME but not D-NAME supplementation decreased iNOS protein expression of the intestinal mucosa in STZ-DM mice. (B) L-NAME but not D-NAME feeding decreased the bacterial loads of liver in STZ-DM mice. STZ-DM mice were fed with an L-NAME (0.5 mg/ml) or D-NAME (0.5 mg/ml, as a control drug) in drinking water for one week. <i>K</i>. <i>pneumoniae</i> (1 × 10³ CFU, K2 serotype) were injected into the branch of superior mesenteric vein (SVC). The liver was collected, weighed and homogenized at 4 hr after the injection for bacterial culture. (C) STZ-DM mice demonstrated a significant increase of IL-1β and TNF-α expression of the Kupffer cells. L-NAME but not D-NAME supplementation markedly decreased diabetes-induced IL-1β and TNF-α mRNA expression of Kupffer cells in STZ-DM mice. STZ, streptozotocin; L-NAME, L-NG-Nitroarginine Methyl Ester; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *, <i>P</i>< 0.05; **, <i>P</i>< 0.01; ***, < 0.001. n = 6/group.</p

Increased <i>K</i>. <i>pneumoniae</i> translocation to liver and MLNs in STZ-DM mice.

<p>Mice were orally fed with nonpathogenic, pathogenic, or GFP-expressing <i>K</i>. <i>pneumoniae</i> for 2 weeks. The translocation of <i>K</i>. <i>pneumoniae</i> to liver and MLNs was examined. Non-pathogenic as well as <i>K</i>. <i>pneumoniae</i> translocation to liver and MLNs did not happen in the control mice. Translocation of pathogenic <i>K</i>. <i>pneumoniae</i> (K2) to liver and MLNs was significantly increased in STZ-DM mice as compared with the control group. Translocation of <i>K</i>. <i>pneumoniae</i> to liver was significantly increased by feeding pathogenic <i>K</i>. <i>pneumoniae</i> (K2) as compared with non-<i>K</i>. <i>pneumoniae</i> feeding and GFP-expressing <i>K</i>. <i>pneumonia</i>e feeding in STZ-DM mice. STZ, streptozotocin; DM, diabetes mellitus; GFP, green fluorescence protein; MLNs, mesenteric lymph nodes. *P<0.05, **P<0.01, ***P<0.001. n = 5/group.</p

Fructooligosaccharides (FOS) or dead <i>L</i>. <i>salivarius</i> feeding reduced enteric bacteria as well as <i>K</i>. <i>pneumoniae</i> translocation to liver and increased hepatic bacteria clearance in STZ-DM mice.

<p>(A) The level of serum ALT was significantly increased in STZ-DM mice. FOS and dead <i>L</i>. <i>salivarius</i> supplementation significantly decreased serum ALT levels in STZ-DM mice. (B) STZ-DM mice demonstrated a significant increase of <i>K</i>. <i>pneumoniae</i> loads of liver after injection of <i>K</i>. <i>pneumoniae</i> in the superior mesenteric vein as compared with that in the control group. FOS or dead <i>L</i>. <i>salivarius</i> feeding significantly decreased the bacterial loads of liver in STZ-DM mice. (C) FOS or dead <i>L</i>. <i>salivarius</i> feeding decreased diabetes-induced bacterial translocation to liver in STZ-DM mice. (D) FOS or dead <i>L</i>. <i>salivarius</i> feeding significantly decreased diabetes-induced pathogenic <i>K</i>. <i>pneumoniae</i> translocation to liver in STZ-DM mice. <i>K</i>. <i>pneumoniae</i> (5 × 10⁷ CFU in 500 μl of normal saline) was injected into the isolated intestinal segment. After 2 h, liver was collected, weighed, and homogenized in equal volume of sterile saline for culture. ALT, alanine transaminase; BT, bacterial translocation; STZ, streptozotocin; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *P<0.05, **P<0.01, ***P<0.001. n = 6/group.</p

STZDM-JNK1^-/- mice demonstrated a significant decrease of serum NO levels, IL-1β expression in Kupffer cells, and ICAM expression in the aorta compared with that in DM mice.

<p>(A) STZDM-JNK1^-/- mice demonstrated a significant decrease of serum NO levels compared with that in DM mice. (B) STZDM-JNK1^-/- mice demonstrated a significant decrease of IL-1β expression in Kupffer cells compared with that in DM mice. Kupffer cells were purified from liver. The total RNAs were extracted from Kupffer cells using the Miniprep Purification Kit (GeneMark). Real-time polymerase chain reaction was performed with the SYBR Green PCR Master Mix and ABI PRISM 7700 Sequence Detection Systems (Applied Biosystems, Foster City, CA). (C) STZ-DM mice demonstrated a significant increase of pJNK expression of intestinal mucosa as compared with that in WT mice. (D) STZDM-JNK1^-/- mice demonstrated a significant decrease of ICAM expression of aorta as compared with that in DM mice. ICAM, intercellular adhesion molecule; JNK, c-Jun NH2-terminal kinase; Fmo, flavin mono-oxygenase; DM, diabetes mellitus. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, < 0.001. n = 6/group.</p

Diabetes induced enteric dysbiosis and FOS feeding reversed it.

<p>Relative abundance of bacteria across difference groups, as indicated by 16S rRNA gene sequencing. Values represent the mean abundance of Genus found at >1% relative abundance in at least one sample. High-throughput 16S rRNA gene sequencing revealed that <i>Lactobacillus</i> was decreased in DM group compared with WT group and FOS feeding significantly increased <i>Lactobacillus</i> in the intestine in STZ-DM mice. STZ, streptozotocin; FOS, fructooligosaccharides; DM, diabetes mellitus. n = 3/group.</p

FOS feeding reversed diabetes-induced hepatic bacterial clearance impairment, IL-1β expression of the Kupffer, and iNOS expression of the intestinal mucosa in Ins2^Akita mice.

<p>(A) Ins2^Akita mice demonstrated a significant increase of <i>K</i>. <i>pneumoniae</i> translocation to liver and blood as compared with that in WT group. FOS supplementation significantly increased hepatic bacteria clearance and decreased K. pneumonia translocation to liver as well as blood in Ins2^Akita mice. <i>K</i>. <i>pneumoniae</i> (1 × 10³ CFU, K2 serotype) were injected into the branch of superior mesenteric vein (SVC). The liver was collected, weighed and homogenized in 1 ml of sterile saline at 4 hr after the injection. 100 μl of blood were taken from heart. Blood or aliquots of the homogenates were plated onto tryptic soy broth (TSB) agar plates (DIFCO). (B) Diabetes induced plasma NO levels in the portal vein as measured by Griess reagents and FOS feeding decreased them. (C) Diabetes significantly induced iNOS protein expression of the intestinal mucosa in Ins2^Akita mice and FOS feeding decreased it. (D) Diabetes induces a significant increase of IL-1β as well as IL6 expression of Kupffer cells in Ins2^Akita mice. FOS or dead <i>L</i>. <i>salivarius</i> supplementation markedly decreased it. (E) Diabetes induced iNOS mRNA expression of the intestinal mucosa in Ins2^Akita mice. FOS or dead <i>L</i>. <i>salivarius</i> supplementation decreased it. STZ, streptozotocin; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *, <i>P</i>< 0.05; **, <i>P</i>< 0.01; ***, < 0.001. n = 6/group.</p

The regulatory mechanism of enteric dysbiosis on Kupffer cell activation and <i>Klebsiella pneumonia</i> liver translocation in diabetes.

<p>L-NAME, L-NG-Nitroarginine Methyl Ester.</p

Inhibition of nitric oxide production reverses diabetes-induced Kupffer cell activation and <i>Klebsiella pneumonia</i> liver translocation

<div><p><i>Klebsiella pneumoniae</i> (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-<i>Ins</i>2^Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of <i>Enterococcus</i> as well as <i>E</i>.<i>coli</i> and decreased <i>lactobacilli/bifidas</i> growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1β and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead <i>L</i>. <i>salivarius</i> (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead <i>E</i>.<i>coli</i> (2×10⁸ CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1β and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4^-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead <i>L</i>. <i>salivarius</i> decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production.</p></div

FOS or dead <i>L</i>. <i>salivarius</i> feeding reversed diabetes-induced iNOS expression of the intestinal mucosa, plasma NO levels in the portal vein, and IL-1β as well as TNF-α expression of the Kupffer cells in STZ-DM mice.

<p>(A) STZ-DM demonstrated a significant increase of iNOS protein expression of the intestinal mucosa as examined by Western blotting and FOS or dead <i>L</i>. <i>salivarius</i> feeding significantly decreased it. (B) STZ-DM demonstrated a significant increase of plasma NO levels of the portal vein as measured by Griess reagents and FOS or dead <i>L</i>. <i>salivarius</i> feeding significantly decreased them. (C) STZ-DM demonstrated a significant increase of IL-1β and TNF-α expression of Kupffer cells and FOS feeding significantly decreased them. STZ, streptozotocin; DM, diabetes mellitus; FOS, fructooligosaccharides; dLac, dead <i>L</i>. <i>salivarius</i>. *, <i>P</i>< 0.05; **, <i>P</i>< 0.01; ***, < 0.001. n = 6/group.</p