Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats.

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1ß mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.

Gut Microbiota Is Not Involved in the Induction of Acute Phase Protein Expression Caused by Vitamin C Deficiency.

Using rats, we previously found that vitamin C deficiency increases serum levels of interleukin-6 (IL-6) and glucocorticoid, and changes the gene expression of acute phase proteins (APP) in the liver. However, it remains unclear how vitamin C deficiency causes these inflammation-like responses. In this study, we investigated the possibility that changes in gut microbiota are involved in the induction of APP gene expression by vitamin C deficiency. ODS rats that cannot genetically synthesize vitamin C were divided into 4 groups based on the presence or absence of vitamin C or antibiotics and were raised for 15 d. Neomycin, vancomycin, and ampicillin were used as antibiotics, and 300 mg L-ascorbic acid/kg was added to the AIN93G diet. Vitamin C deficiency affected neither the wet tissue weights nor relative abundance of bacteria in the cecal contents. Antibiotic administration increased wet weights of the cecum, cecal contents, and colon, changed the relative abundance of some bacteria in the cecal contents, and decreased serum IL-6 level. However, antibiotic administration had no effect on serum concentrations of corticosterone and α1-acid glycoprotein (AGP), vitamin C concentration in the liver, and mRNA levels of haptoglobin and AGP in the liver. Therefore, disturbance of gut microbiota did not attenuate the increase in glucocorticoid level and induction of APP gene expression due to vitamin C deficiency. This suggests that gut microbiota is not involved in the inflammation-like responses caused by vitamin C deficiency.

Ascorbic acid deficiency increases hepatic expression of acute phase proteins through the intestine-derived IL-6 and hepatic STAT3 pathway in ODS rats.

We have previously shown that ascorbic acid (AsA) deficiency elevates hepatic expression of acute phase proteins (APPs), inflammatory markers, in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. However, the precise mechanisms of this elevation are unknown. Signal transducer and activator of transcription 3 (STAT3) is one of the transcription factors inducing the expression of APPs and is activated by several cytokines including interleukin-6 (IL-6). The aim of this study was to determine whether AsA deficiency stimulates hepatic STAT3 activation and increases intestinal production of proinflammatory cytokines such as IL-6. Male ODS rats (6 weeks old) were fed either a basal diet containing 300 mg AsA/kg (control group) or an AsA-free diet (AsA-deficient group) for 18 days. AsA deficiency gradually and simultaneously elevated both mRNA levels of APPs (haptoglobin, α1-acid glycoprotein, C-reactive protein and α2-macroglobulin) and nuclear level of phosphorylated STAT3 (activated STAT3) in the liver. These results showed that the AsA-deficiency-induced expression of hepatic APPs is stimulated by proinflammatory cytokines activating STAT3. On day 14, AsA deficiency significantly elevated IL-6 mRNA level in the ileum and the concentration of IL-6 in portal blood. Furthermore, the portal concentration of IL-6 positively correlated with hepatic mRNA levels of STAT3-regulated genes. These findings suggest that IL-6, produced in the intestine as a result of AsA deficiency, is recruited to the liver via the portal vein and contributes to hepatic STAT3 activation and the elevated expression of APPs.

Effect of Docosahexaenoic Acid Ingestion on Temporal Change in Urinary Excretion of Mercapturic Acid in ODS Rats.

We hypothesized a suppressive mechanism for docosahexaenoic acid (22:6n-3; DHA)-induced tissue lipid peroxidation in which the degradation products, especially aldehydic compounds, are conjugated with glutathione through catalysis by glutathione S-transferases, and then excreted into urine as mercapturic acids. In the present study, ascorbic acid-requiring ODS rats were fed a diet containing DHA (3.6% of total energy) for 31 days. Lipid peroxides including degradation products and their scavengers in the liver and kidney were determined, and the temporal change in the urinary excretion of mercapturic acids was also measured. The activity of aldehyde dehydrogenase, which catalyzes the oxidation and detoxification of aldehydes, tended to be higher in the liver of DHA-fed rats. The levels of lipid peroxides as measured by thiobarbituric acid-reactive substances and aldehydic compounds were higher and that of alpha-tocopherol was lower in the liver, and the pattern of temporal changes in the urinary excretion of mercapturic acids was also different between the n-6 linoleic acid and DHA-fed rats. Accordingly, we presume from these results that after dietary DHA-induced lipid peroxidation, a proportion of the lipid peroxidation-derived aldehydic degradation products is excreted into urine as mercapturic acids.