Rifaximin modulates TRH and TRH-like peptide expression throughout the brain and peripheral tissues of male rats

The TRH/TRH-R1 receptor signaling pathway within the neurons of the dorsal vagal complex is an important mediator of the brain-gut axis. Mental health and protection from a variety of neuropathologies, such as autism, Attention Deficit Hyperactivity Disorder, Alzheimer’s and Parkinson’s disease, major depression, migraine and epilepsy are influenced by the gut microbiome and is mediated by the vagus nerve. The antibiotic rifaximin (RF) does not cross the gut-blood barrier. It changes the composition of the gut microbiome resulting in therapeutic benefits for traveler’s diarrhea, hepatic encephalopathy, and prostatitis. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH2, where “X” can be any amino acid residue, have reproduction-enhancing, caloric-restriction-like, anti-aging, pancreatic-β cell-, cardiovascular-, and neuroprotective effects. TRH and TRH-like peptides occur not only throughout the CNS but also in peripheral tissues. To elucidate the involvement of TRH-like peptides in brain-gut-reproductive system interactions 16 male Sprague–Dawley rats, 203 ± 6 g, were divided into 4 groups (n = 4/group): the control (CON) group remained on ad libitum Purina rodent chow and water for 10 days until decapitation, acute (AC) group receiving 150 mg RF/kg powdered rodent chow for 24 h providing 150 mg RF/kg body weight for 200 g rats, chronic (CHR) animals receiving RF for 10 days; withdrawal (WD) rats receiving RF for 8 days and then normal chow for 2 days. Significant changes in the levels of TRH and TRH-like peptides occurred throughout the brain and peripheral tissues in response to RF. The number of significant changes in TRH and TRH-like peptide levels in brain resulting from RF treatment, in descending order were: medulla (16), piriform cortex (8), nucleus accumbens (7), frontal cortex (5), striatum (3), amygdala (3), entorhinal cortex (3), anterior (2), and posterior cingulate (2), hippocampus (1), hypothalamus (0) and cerebellum (0). The corresponding ranking for peripheral tissues were: prostate (6), adrenals (4), pancreas (3), liver (2), testis (1), heart (0). The sensitivity of TRH and TRH-like peptide expression to RF treatment, particularly in the medulla oblongata and prostate, is consistent with the participation of these peptides in the therapeutic effects of RF.

rodents [4,5]. The mechanisms underlying these effects include reduced levels of brain-derived neurotrophic factors in the cortex, hippocampus, and amygdala, and altered expression of genes encoding subunits of the glutamate and dopamine receptors [1]. Glutamatergic neurons are the most abundant excitatory class of nerves in the mammalian nervous system which requires co-release of the neuromodulatory thyrotropin releasing hormone (TRH) and TRH-like peptides to protect postsynaptic cells from the excitotoxic effects of excessive glutamate release [6][7][8][9]. The TRH/TRH-R1 receptor signaling pathway is an important mediator of brain-gut axis communication via the brain medulla oblongata and its associated TRH synthesizing neurons within the raphe pallidus, raphe obscura, and parapyramidal regions [10]. TRH and TRH-like peptides, with the structure pGlu-X-Pro-NH 2 where "X" can be any amino acid residue, have reproductive, antidepressant, anxiolytic, analeptic, anorexic, and anti-aging effects [11].
TRH and TRH-like peptides occur not only throughout the CNS but also peripheral tissues, with very high levels in the rat and human prostate [11]. This is particularly noteworthy given the vulnerability of humans to prostatitis and prostate cancer [12]. Recent studies have implicated bacterial infections as potential causes of prostate diseases. The antibiotic rifaximin (RF), which does not cross the gut-blood barrier, is a standard treatment for traveler's diarrhea and hepatic encephalopathy. Its therapeutic potential in the treatment of other brain and urogenital disorders is currently being evaluated [12].
RF has anti-depressant and anxiolytic effects in both humans and rodents which are mediated, at least in part, by its ability to modify the composition of the gut microbiota [13][14][15][16][17][18]. Inadequate and/or irregulate sleep and poor nutrition contribute to obesity, alterations in the microbiome and the expression of gut hormones, including leptin and ghrelin, which have a profound effect on both appetite [19][20][21][22][23] and TRH and TRH-like peptide release [24,25]. Leptin and ghrelin also have mood altering effects [24,25].
The present studies examine the effects of oral RF on TRH and TRH-like peptide levels in those brain regions, for example the medulla oblongata, and peripheral tissues which may play a role in the therapeutic effects of this gut-limited antibiotic [12].

Animals
"Young adult male Sprague-Dawley rats (n = 16, SPF, Envigo, Indianapolis, IN) were used for all experiments. These animals were group housed (2 animals per cage) on wood shavings with a red plastic tube for play and shelter. Standard Purina rodent chow #5001 and water were provided ad libitum during a standard one-week initial quarantine with 22 ± 2 °C and 50 ± 10% relative humidity; lights on: 6 am-6 pm. Cages, water and bedding were changed every 3 days. All animals were weighed on the day of receipt and on the morning of each experiment. Initial body weights did not differ between experimental groups. Animals were randomized prior to the start of rifaximin treatment. Research was approved by the VA Greater Los Angeles Healthcare System Animal Care and Use Committee (IACUC Protocol #030090-10) and conducted in compliance with the Animal Welfare Act and the federal statutes and regulations related to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and use of Laboratory Animals, Eighth Edition, NRC Publication, 2011. All efforts have been made to minimize the number of animals used and their suffering. Animal was handled for 10 min per day for one month and then transferred from the Veterinary Medical Unit to the laboratory 12 h before the start of experiments to minimize the stress of a novel environment" [11]. "The American Veterinary Medical Association has concluded that decapitation without prior sedation 'is conditionally acceptable if performed correctly, and it should be used in research settings when its use is required by the experimental design and approved by the Institutional Animal Care and Use Committee'" [26]. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) (https:// arriv eguid elines. org).
"Because of the 10-to 100-fold changes in TRH and TRH-like peptide levels in response to the estrus cycle. female rats were not included in the present study" [27].

Effect of acute, chronic and withdrawal treatment with rifaximin in normal rat chow on levels of TRH and TRH-like peptides in rat brain and peripheral tissues
Sixteen young adult male Sprague-Dawley rats, body weight (mean ± SD) 203 ± 6 g, 3.0% CV, were divided into 4 groups (n = 4/group). The control (CON) group remained on ad libitum standard Purina powdered rodent chow and water for 10 days until decapitation. The acute (AC) group received ad libitum powdered rodent chow and water for 9 days and then 1 g rifaximin (Sigma, St. Louis, MO)/500 g powdered rat chow for 24 h. Assuming 16.7 g chow consumption/day, this would provide 150 mg rifaximin/kg body weight for 200 g rats. The chronic (CHR) animals received RF in powdered chow for 10 days. The withdrawal (WD) rats received RF chow for 8 days and then normal chow for 2 days. The effect of RF withdrawal on TRH and TRH-like peptide levels when compared to the corresponding acute effects can reveal the relative contribution of changes in peptide biosynthesis (hours) to changes in peptide release (minutes) [28].

HPLC and RIA procedures, HPLC peak identification and quantitation
HPLC and RIA procedures, peak identification, and quantitation by co-chromatography with synthetic TRH and TRH-like peptides, relative potency analysis of multiple antibodies to TRH and TRH-like peptides, and mass spectrometry and have been previously reported in detail [11,28,[31][32][33].
Briefly, after boiling, tissues were dried, re-extracted with methanol, dried and defatted by water-ethyl ether partitioning. Dried samples were dissolved in 0.1%trifluroacetic acid (TFA) and loaded onto reverse phaseC18 Sep-Pak cartridges (Water, Milford, MA). TRH and TRH-like peptides were eluted with 50% methanol. Dried peptides were again dissolved in TFA, filtered and then fractionated by HPLC using a 4.6-150 mm Econosphere, 3 mm C18 reverse phase column (Dr. Maisch GmbH, Ammerbuch, Germany) and a 0.2%/min gradient of acetonitrile. The 0.5 ml fractions collected were dried completely and reconstituted with 0.10 ml of 0.02% NaN3 just before RIA (Fig. 1).
The antiserum used (8B9) cross-reacts with TRH and nine TRH-like peptides with a relative potency of displacement ranging from 2.31 (Lys-TRH) to 0.288 (Ser-TRH) relative to Tyr-TRH (Table 2), (see [28]). Two of the regularly observed peaks (2a, b) consist of a mixture of unidentified TRH-like peptides. Of the eight observed peptides three have so far been confirmed by mass spectrometry: TRH, Glu-TRH and Tyr-TRH [31]. Tissue samples from the 4 rats within each treatment group were pooled prior to HPLC to provide the minimum amount of immunoreactivity needed for reliable RIA measurements.
The mean recovery of TRH and TRH-like peptide immunoreactivity from all tissues studied was 84 ± 15% (mean ± SD). The within-assay and between-assay coefficient of variation for measuring 333 pg/ml TRH was 4.8% and 16.9%, respectively. All HPLC fractions obtained from a given brain region or peripheral tissue were analyzed in the same RIA. The minimum detectable dose for TRH was 5 pg/ml. The specific binding of [ 125 I]TRH (Bo/T) was 25%.

Statistical analysis
"Statistical methods for comparing peak areas were made with the aid of Statview (Abacus Concepts, Inc., Berkeley, CA), a statistical software package for the Macintosh computer. All multi-group comparisons were carried out by one way analysis of variance using post hoc Scheffe contrast with the control group" [30].
"The mean within-group coefficient of variation (CV) (SD/mean, CV-within group) for each tissue and TRH/ TRH-like peptide combination, across four photoperiod intervals, has been previously reported (circadian rhythm experiment) for untreated Sprague-Dawley male rats" [30]. Mean within-group CVs in brain ranged from 4.5% for TRH levels in AY to 43% for Phe-TRH in HY, and from 12% for Val-TRH in testis to 41% for Trp-TRH in EP for peripheral tissues. These CVs were then used to estimate the level of significance, by on way ANOVA, of changes in the pooled mean values (see [34]) of TRH and TRHlike peptide levels following acute (AC), chronic (CHR) and withdrawal (WD) ingestion of RF" [30]. Pooling of at least 4 tissue extracts was required to provide sufficient signal-to-noise in the RIA for many brain regions and to keep the total number of HPLC fractions to be analyzed reasonable: 4 treatment groups × 19 tissues × 100 HPLC fractions/tissue pool = 7600 RIA samples for the present study. Without pooling the total number of HPLC fractions would have been 4 × 7600 = 30,400.

Body weights
Mean body weights for all animals at the time of decapitation (9 weeks) was 269 ± 12 g, 4.5% CV. Mean animal weights for each RF treatment group did not differ significantly with the untreated controls by one way ANOVA.

Serum hormone levels following oral rifaximin
Serum glucose levels for the CHR group were significantly lowers than the WD group (p < 0.05). All other serum hormone levels did not differ significantly between experimental groups by one way ANOVA ( Table 1).

HPLC results in brain and peripheral tissues
Significant, 25-to 37-fold, increases in Tyr-TRH and 93-94% decreases in Val-TRH levels in medulla oblongata (all treatment groups, Table 2) and increases in TRH (13-fold, WD group) and Val-TRH (fivefold, WD group) and 99% decreases in Tyr-TRH concentrations in ventral prostate and a 36-fold increase in liver Tyr-TRH (WD group) ( Table 3) were observed following rifaximin administration. Fig. 1 Representative profiles of TRH and TRH-like peptide responses in male rats to RF treatment. The response patterns in A and D are consistent with rapid and sustained increase in peptide release (reduced peptide level). The profile in B suggests rapid and sustained decrease in peptide release during and after RF exposure. C could be explained by RF stimulation of sustained peptide release which is compensated by increased peptide synthesis. Withdrawal of RF reduced peptide release but the compensatory increase in peptide synthesis results in a rebound increase in peptide content. The persistence of changes in TRH and TRH-like peptide levels in the WD group after RF has completely cleared from the GI tract is consistent with lingering effects of an altered microbiome Table 1 Effect of oral rifaximin on serum hormone levels of male rats There were no significant changes by one way ANOVA versus the corresponding control group. All results are mean ± SD * p < 0.05 by one-way ANOVA versus the WD group  All results are mean ± SD * p < 0.05; **p < 0.01; ***p < 0.002 by one way ANOVA using post hoc Scheffe contrasts versus the control group  Table 3 Effect of acute (AC), chronic (CHR) and withdrawal (WD) treatments with rifaximin on TRH and TRH-like peptide levels in peripheral tissues (pg) All results are mean ± SD * p < 0.05; **p < 0.01 by one way ANOVA using post hoc Scheffe contrasts versus the control group The number of significant changes in TRH and TRHlike peptide levels in brain resulting from RF treatment (In parentheses), in descending order were: MED (16), PIR (8), NA (7), FCX (5), STR (3), AY (3), ENT (3), ACNG (2), PCNG (2), HC (1), HY (0) and CBL (0) as seen in Table 2. The corresponding ranking for peripheral tissues were: PR (6), AD (4), PAN (3), L (2), T (1), H (0), (see Table 3). The pooled EP controls were lost during extraction so results for this tissue could not be analyzed.

Discussion
Acute, chronic and withdrawal treatment with RF resulted in significant decreases in TRH, Val-TRH, Phe-TRH and Trp-TRH and marked increases in Tyr-TRH levels in the MED ( Table 2). These changes result from alterations in the biosynthesis and release of these tripeptides. The rapidity of these responses is consistent with increased TRH, Val-TRH, Phe-TRH and Trp-TRH and decreased Tyr-TRH release, respectively [32]. These remarkable changes in peptide levels within the MED is consistent with current knowledge regarding the role of TRH (and TRH-like peptides) as mediators of brain-gut communication via the vagus nerve [10,36]. The antidepressant activity of Tyr-TRH [31] and analeptic effect of Val-TRH [37] correspond with actions of TRH [11]. TRH and TRH-like peptide biosynthesis occurs within large dense core vesicles (LDCV) of glutamatergic neurons [32,33]. They are co-released with glutamate and act to moderate the effects of this excitotoxic neurotransmitter [7,8]. Neuropeptides, such as TRH, which are co-released with classical neurotransmitters are now considered primary mediators of brain circuit connectivity with a longer duration of action [38].
Dysbiosis of the microbiome has been implicated in prostatitis and prostate cancer [12,39]. Rifaximin, an antibiotic which does not cross the blood-gut barrier, is currently being evaluated as a treatment for these pathologies [40]. It is noteworthy that among the peripheral tissues analyzed, PR had the highest number of significant changes in TRH and TRH-like peptide levels (Table 3) in response to RF treatment. PR has very high levels of TRH and TRH-like peptides which are subject to marked circadian rhythmicity [11]. TRH stimulates the adenylyl cyclase in basal cell membranes of the rat ventral prostate [41].
Withdrawal of RF increased TRH and Val-TRH levels in prostate (Table 3), which is consistent with RF stimulation of both biosynthesis and release of these peptides. Because the reduction in the peptide release rate is rapid but the changes in RF-stimulated peptide synthesis is slow with RF withdrawal, a rebound in the levels of these peptides is observed. Acute RF treatment increased Phe-TRH levels consistent with a rapid inhibition of release for this peptide in response to RF followed by a compensatory decrease in biosynthesis returning CHR Phe-TRH levels to CON values. AC, CHR and WD treatment with RF all decreased Tyr-TRH levels significantly in prostate (Table 3). These observations may reveal a rapid and sustained increase in Tyr-TRH release in response to RF treatment which is not accompanied by marked changes in the processing of Tyr-TRH progenitor peptides.
Withdrawal of RF resulted in significant decreases in the levels of all TRH and TRH-like peptides measured in the adrenals (Table 3). This is consistent with an acute decrease in biosynthesis and/or increase in release of these peptides [32]. RF has modest, transient, and beneficial effects on stress-related changes in the gut microbiome, inflammation, permeability and hyperalgesia as well as central responses to social stress [15,17,18,42]. Manipulation of the gut microbiome can have significant effects on cortisol levels in urine [1]. The influence of the microbiota on the function of the HPA axis which regulates corticosterone levels was first demonstrated by Sudo et al. [43]. Germ-free mice have reduced levels of brainderived neurotrophic factors in the cortex, hippocampus, and amygdala, and altered expression of glutamate and dopamine receptors in specific regions of the brain [44].
Administration of the probiotic bacterium Lactobacillus rhamnosus strain JB-1 ™ to mice significantly altered the expression of the gene coding for a GABA receptor in multiple regions of the brain, including the amygdala, hippocampus, and cortex. This effect was abolished by vagotomy [45].
The vagus nerve is the principal neuronal link between internal organs and the brain and has now been shown to be integral to the regulation of an array of autonomic functions, such as breathing, heart functions, pancreatic and liver regulation of metabolism, modulation of immune and inflammatory responses via the spleen, mood, and even consciousness [46,47].
The TRH-degrading serum enzyme is a product of liver [48]. It rapidly metabolizes TRH and most TRHlike peptides in the circulation except Glu-TRH [49]. Rifaximin is used for the prevention of recurrent overt hepatic encephalopathy [50]. Significant increases in Val-TRH and Tyr-TRH levels were observed in liver in response to RF treatment (Table 3). Functional brain MRI studies of the responses of patients with cirrhosis to rifaximin treatment reveal higher activation in various brain regions including the frontal cortex, hippocampus, anterior and posterior cingulate [51]. RF is also utilized for the treatment of irritable bowel disease, diverticular disease, and small bowel bacterial overgrowth [16,17,50]. RF modulates inflammatory cytokines and intestinal permeability [52]. Medullary TRH and gastric vagal efferent and afferent circuits play a crucial role in the modulation of gastric integrity [53].

Conclusions
The marked responsivity of TRH and TRH-like peptide expression to RF-induced alterations in gut microbiota of normal rats is consistent with the participation of these peptides in vagally-mediated brain-gut signaling. The observed effects persist after RF, which does not cross the blood-gut barrier, has cleared the GI tract. We expect future studies will extend this concept to antidepressant, anxiolytic, anti-obesity, GI-, liver-and prostate-protective effects of rifaximin [54].