Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28d in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and β-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from -14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). Overall, these multitier changes revealed important features associated with the transition into lactation. Alterations in ruminal epithelium gene expression could be driven by nutrient intake-induced changes in microbes; microbial metabolism; and the systemic metabolic, hormonal, and immune changes. Understanding causes and mechanisms driving the interaction among ruminal bacteria and host immunometabolic responses merits further study.

Hepatic fibrosis or increased liver collagen contents drive functional abnormalities that, when extensive, may be life threatening. The purpose of this study was to assess the effects of the chronic stimulation or inhibition of nitric oxide synthesis in rats with hepatic fibrosis induced by permanent common bile duct ligation (3 weeks) and the role of expression of the different nitric oxide synthase isoforms. Bile duct ligation led to an important accumulation of collagen in the hepatic parenchyma, as shown both histologically and by the hydroxyproline contents of livers. Bilirubin and serum enzyme activities (measured as markers of cholestasis) increased several-fold after bile duct ligation. The area of fibrotic tissue, liver hydroxyproline content and serum markers of cholestasis were clearly related in obstructed rats. The absence of modifications in haemodynamic parameters excludes circulatory changes from being responsible for the development of liver alterations. In animals treated with NG-nitro-L-arginine methyl ester (L-NAME) the area of fibrosis was similar to that of untreated animals, the signs of cholestasis and cellular injury being more evident. In rats treated with L-arginine the area of fibrosis was almost three times larger than that found in bile duct ligated rats and in L-NAME-treated bile duct ligated rats, although the observed biochemical changes were similar to those seen in rats treated with L-NAME. Our results with inducible nitric oxide synthase, obtained by Western blots and immunohistochemistry, indicate a greater expression of the inducible enzyme in bile duct ligated and L-arginine-treated animals and a lower expression in the L-NAME and control groups. Constitutive nitric oxide synthase expression, obtained by Western blots, was very similar in all groups, except for the L-arginine-treated rats in which it was lower. These results suggest that nitric oxide production may be a key factor in the development of fibrosis in bile duct ligated rats. They also support the hypothesis of a dual role for nitric oxide; one beneficial, mediated by its circulatory effects, and the second negative, through its local toxic effects.

were only small nonsignificant changes in SO animals. ThereRecent work indicates that nitric oxide (NO) plays an imfore, these results indicate that the expression, activity and portant role in the systemic and renal alterations of cirrhosis.production of NO in kidneys, glomeruli, and mononuclear In the present study, we have evaluated whether the inducible lymphocyte cells is elevated in BDL rats, and this is partly NO synthase (iNOS) isoform participates in the enhanced because of a plasma-derived substance(s), which stimulates renal and systemic NO production of a rat model of cirrhosis.iNOS formation. The amelioration of the arterial hypotension In vitro and in vivo experiments were performed in rats suband the associated reduced excretory levels of these cirrhotic jected to chronic bile duct ligation (BDL) and in sham-operanimals by aminoguanidine further support the involvement ated (SO) animals. Plasma nitrite (3.1 { 0.1 mmol/L in SO of the inducible NO synthase isoform in the renal alterations and 6.6 { 0.2 mmol/L in BDL), glomerular nitrite production observed in BDL animals. (HEPATOLOGY 1997;26:268-276.) (6.4 { 0.1 vs. 9.8 { 0.1 nmol/24h/7,000 glomeruli, respectively), and mononuclear lymphocyte cells nitrite production During the last years, it has become increasingly clear that (0.3 { 0.04 vs. 0.6 { 0.12 nmol/10 6 cells, respectively) nitric oxide (NO) plays an important role as a mediator of were all significantly higher in BDL than in SO. Moreover, the systemic and renal alterations of liver cirrhosis. 1 Recent mononuclear lymphocytes and glomeruli from BDL rats studies have revealed that rats with experimentally induced showed an increased expression of macrophage-type iNOS, cirrhosis show an increased endothelium-dependent renal detected by Western blot. Kidneys from BDL animals also vasodilation, 2 as well as an increased renal sensitivity to NO showed an increased calcium-independent NO synthase activinhibition. [3][4][5] Moreover, NO inhibition results in a reduction ity, compared with those from SO rats. Constitutive endotheof the hyperdynamic circulation 3,6-7 and produces beneficial lial-type NO synthase expression in glomeruli or the activity effects on renal excretory function. 4,5,8 However, to the best of calcium-dependent NO synthase in whole kidney did not of our knowledge, no direct demonstration of increased renal show differences between BDL and SO rats. In cultured mesproduction of NO in experimental or clinical cirrhosis has angial cells from normal rats, the addition of plasma from been published. Moreover, the type of NO synthase (NOS) BDL but not of plasma from SO significantly stimulated (35%) isoforms involved and the mechanisms responsible for their nitrite production and increased the expression of macroactivation are not completely established. 9-13phage-type iNOS. In addition, administration of aminoguaIn the present study, we have evaluated whether the inducnidine (AG), a preferential iNOS inhibitor, elevated dose-deible NOS isoform participates in the enhanced renal a...

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