BISMUTH SUBSALICYLATE REVERSES HIGH FAT DIET-INDUCED INCREASE IN PORTAL VENOUS FLOW

doi:10.21203/rs.3.rs-4018738/v1

In animal studies, a high-fat diet (HFD) is associated with fatty liver disease and vascular complications characterized by hyperdynamic circulation. The underlying mechanism for these hemodynamic changes is not known. Hydrogen sulfide (H₂S) is a gaseous signaling molecule that plays several roles including that of a vasodilator in the peripheral vascular system. H₂S is produced by both mammalian cells and sulfate reducing bacteria (SRB). SRB in the gut are known to convert hydrogen, a by-product of bacterial fermentation, to H₂S. Bismuth subsalicylate (BSS) lowers the availability of luminal H₂S by binding directly to this gas in the gut lumen. In this study, we tested the hypothesis that a high fat diet may increase portal venous blood flow in a BSS-reversible fashion in rats (n = 8/group) fed one of 4 diets for 8 weeks: HFD, HFD + BSS, standard diet (SDD) and SDD + BSS. Portal venous blood flow was measured in laparotomized animals using a perivascular ultrasonic probe. To directly test the effect of H₂S, portal venous blood flow was also compared before (baseline) and after infusing NaHS, a donor of H₂S, into the jejunum. We found that baseline rates of portal venous blood flow were significantly greater in the HFD group [23.41 ± 0.4mL/min, p < 0.05] than SDD group [17.48 ± 0.2 mL/min], HFD + BSS [17.95 ± 0.3mL/min] or SDD + BSS [14.49 ± 0.2mL/min]. H₂S donor increased portal venous blood flow significantly more in the HFD group [6.1 ± 0.26mL/min, p < 0.05] when compared to SDD group [1.88 ± 0.18mL/min], HFD + BSS [2.00 ± 0.40mL/min] or SDD + BSS [3.45 ± 0.34mL/min). qPCR using primers targeting the dissimilatory sulfite reductase beta-subunit (dsrB) of SRB ([NiFe] hydrogenase gene fragment), the phyla Firmicutes and Bacteroidetes showed no difference between groups in the distal small intestine. The liver enzymes AST and ALT were significantly elevated in the HFD group than HFD + BSS, SDD and SDD + BSS (p = 0.044, 0.011, respectively). In conclusion, an increase in portal venous blood flow consistent with a hyperdynamic circulation was observed in HFD-fed animals with a further increase induced by infusion of NaHS. In HFD-fed animals, the increase in portal venous blood flow at baseline and the further increase following NaHS infusion were both reversed by H₂S-binding BSS administered to the gut supporting the conclusion that increased portal venous blood flow in HFD-fed animals was driven by gut bacteria-derived H₂S.

Hydrogen sulfide

portal circulation

bismuth subsalicylate

non-alcoholic fatty liver disease

In the past few decades, non-alcoholic liver disease (NAFLD) has become more prevalent in the US population¹; liver disease-related death is the fourth most common cause of death in the US² with NAFLD responsible for 48.9% of the 60,111 individuals who died from cirrhosis and 35.4% of 11,765 individuals who died from hepatocellular carcinoma in 2017³. Fatty liver disease has seen a steep rise due to, in part, a sedentary lifestyle and overconsumption of Western-diet that is high in calories and saturated fats^4,5. Animal models have demonstrated that a high fat diet (HFD) could lead to hepatic steatosis and inflammation⁴ and are used as a model to study sequelae of liver disease.

While NAFLD has been shown to be associated with increased portal venous blood flow without underlying cirrhosis⁶, the explanation is not well understood. Intrahepatic vascular vasoconstriction has been noted in fatty liver disease⁷. In addition, endothelial dysfunction was found in animal models of NAFLD^7–9. However, it is not known how those changes relate to increased portal venous blood flow. A role for nitric oxide (NO) has long been proposed¹⁰ as portal hypertension and hyperdynamic circulation in cirrhosis have been associated with increased nitric oxide production in the splanchnic circulation leading to vasodilation that is thought to increase the blood flow^11,12. However, NO cannot be the only explanation for increased portal venous blood flow in liver disease as mesenteric vasodilation in early stages of toxin-induced (carbon tetrachloride) cirrhosis is only partially corrected by chronic administration of an inhibitor of nitric oxide synthase in a rat of model of cirrhosis^13,14. It is likely, then, that other vascular modulators are involved in controlling splanchnic circulation in chronic liver disease.

Another possible mediator of increased portal venous blood flow is hydrogen sulfide (H₂S). H₂S is a multi-functional signaling molecule produced endogenously by the vascular endothelial cells and smooth muscle cells and is known to be involved in regulating systemic blood pressure through its vasodilatory effects^15–19. However, exogenous H₂S is also produced as a metabolic product of sulfate reducing bacteria of the gut microbiome. The role of this exogenous bacteria-derived H₂S on portal venous blood flow is not known.

Bismuth subsalicylate (BSS) is a metal-based compound with a long history of medical use. This drug has previously been explored in the setting of gastrointestinal disorders as a treatment for microbial diseases such as Helicobacter Pylori infection of the stomach and bacteria gastroenteritis²⁰. BSS is known to bind very efficiently to gut luminal H₂S, effectively removing it from the gut lumen in humans^21,22 and in rats²³. We have also shown in mice that BSS administered into the gut could completely reverse the effects of bacteria-derived H₂S on intestinal transit²¹. In this study, we tested the hypothesis in rats that HFD may increase portal venous blood flow in a BSS-reversible fashion.

Experimental Overview

Animals were placed on high fat or standard diets with or without the addition of BSS. To further test the effect of H₂S on the portal circulation, NaHS, an H₂S donor, was administered into the jejunum with measurements taken before vs. after BSS. After an 8-week diet protocol, animals underwent non-survival surgery to measure portal venous blood flow and to harvest intestinal samples and blood for measuring liver enzymes.

Animals

All procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of New Mexico. All protocols conformed to the National Institutes of Health Guidelines for Animal Use in Research. Male Sprague-Dawley rats (100-125g, Harlan Laboratories, Indianapolis, IN) were housed (2 animals per cage) under conditions of 12-hour light/12-hour dark cycle with environmental temperature maintained at 20-23 °C and water available ad libidum. After initial acclimation, animals were placed into 1 of 4 groups (n=8 per group): high fat diet (HFD) (D12451, Research Diets, Inc, New Brunswick, NJ)^24–27, HFD plus Bismuth subsalicylate (HFD+BSS) (Research Diets, Inc, New Brunswick, NJ) (400mg Bismuth/100g chow) (26), standard chow (SDD), standard chow plus Bismuth (SDD+BSS). Animals were kept on the test diets for 8 weeks.

Surgical approach

At end of the 8-week diet protocol, all animals underwent non-survival surgery for measurement of portal venous blood flow at baseline. Animals were anesthetized under inhaled isoflurane (5% in oxygen for induction and 2% in oxygen for maintenance) and instrumented through a midline ventral incision. A perivascular ultrasonic probe (Transonic Flowprobe, Ithaca, NY) was placed around the portal vein to measure blood flow which was recorded as mL/min. Once flow was stabilized, blood flow was monitored for 5 minutes as the baseline data. Blood samples were then collected at the end of the baseline measurement (prior to NaHS infusion) via a tail vein for various liver enzyme measurements (aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin and alkaline phosphatase). Whole blood was spun for 7 minutes with the separated plasma analyzed for liver enzymes using the Catalyst One Chemistry Analyzer (Idexx Laboratories, Westbrooke, Maine).

NaHS Infusion

After baseline rates of portal venous blood flow were recorded, NaHS dissolved in saline (16.8mg/mL/kg) was injected into the jejunum as a bolus followed by continuous infusion (0.56 mg/mL/kg/min) using 25 gauge needle, 10 cm from small intestine origin using previously described methods²⁸ that allow for steady state infusion at concentrations that do not cause systemic toxicity. Control animals underwent same treatment using equi-volume saline only. Portal venous blood flow was recorded throughout the infusion. Measurements were recorded in 60-second time intervals for a total of 20-minutes. The final 60-second measurement was used as the after-infusion data that were compared with the baseline measurement.

Quantitative PCR

Quantitative polymerase chain reaction (qPCR) was used to measure the density of the sulfate reducing bacteria, Firmicutes and Bacteroidetes contained in the mucosa-associated microbiome of the distal small intestinal mucosa. Tissue was homogenized with a Tissuelyser II (Qiagen, Germantown, MD). DNA was prepared using Qiagen DNeasy Blood and Tissue Kit following the manufacturer’s protocol. DNA quality was assessed with the DeNovix DS-11-FX+ Spectrophotometer (DeNovix, Inc., Wilmington, DE). DNA samples were diluted with Buffer AE (Qiagen) to ensure a total of 100ng of DNA was used per reaction. All reactions were prepared with Luna Universal qPCR Master Mix (New England Biolabs, Ipwich, MA). QuantStudio 7 (ThermoFisher, Waltham, MA) was used to perform qPCR. The universal 16s rRNA gene was used as an internal control in extracted genomic DNA using the Eub338/518 primer pair. Bacteria were measured as follows: Firmicutes using primer pair Firm934/1060; Bacteroidetes primer pair Cfb319/Eub518; and SRB using the DSRb primer pair DSRb-213f/DSRB-658r^29,30 (Table 4). Analysis was performed using the ΔΔCt method³¹ with the universal eukaryotic 18s rRNA gene for standardization. qPCR results are reported as fold-change (mean ± SEM) relative to the Standard Chow (SDD) group.

Statistical Analysis

Tables 1 and 3 summarize the measured variables (except for portal vein flow), reporting the mean and standard error (SE) as well as the P-value (F-test) and the minimum False Positive Risk (mFPR) for the corresponding 2-way ANOVA. The mFPR is a transformation of the Likelihood ratio³² which gives the smallest reasonable probability that “a result which is claimed to be statistically significant is actually a false positive”^33,34. Results with a large mFPR should be interpreted with caution, even if the P-value is small. When directly comparing averages of two groups (as we do often, in the results section), we use Dunnett’s t test to adjust for multiple comparisons³⁵.

The 25 minutes of portal vein flow rate data is represented as 1500 temporally correlated observations (300 baseline, 1200 for post NaHS Infusion). The baseline flow rate is taken as the average across the first five minutes. The “effective sample size” (ESS)³⁶ is computed for each subject, and the standard error for the group level mean is computed using the pooled ESS values for all subjects in the group. To estimate the post-infusion increase, we fit a linear model for flow rate based on group and pre/post infusion. The coefficients of this model give the estimated (average) post-infusion increase of flow rate. To account for autocorrelation and heteroskedasticity, standard errors for these estimates are calculated using the Newey-West method³⁷. Missing portal vein flow data is considered to be missing completely at random.

All analysis was conducted with the R programming language³⁸ and all figures were created with ggplot2³⁹.

Weight

Weight gain during the 8-week diet period (Table 1) was greatest in the HFD group (211.6 ± 7.7g), exhibiting between 5% and 16% more weight gain than the other groups, although not statistically significant (HFD+BSS: 183.0 ± 10.1, p = 0.113; SDD: 188.8 ± 5.8g, p = 0.040; SDD+BSS: 200.3 ± 15.0g, p = 0.609).

Portal Venous Blood Flow

Baseline portal venous blood flow rate (Table 2 & Fig. 1) was substantially greater in the HFD group (23.41 ± 0.4mL/min) than in the other groups (HFD+BSS: 17.95 ± 0.3mL/min; SDD: 17.48 ± 0.2 mL/min; SDD+BSS: 14.49 ± 0.2mL/min). Likewise, the increase in portal venous flow rate after NaHS infusion (Table 4 & Fig. 2) was also much greater in the HFD group (6.1 ± 0.26mL/min) than in the other groups (HFD+BSS: 2.00 ± 0.40mL/min; SDD: 1.88 ± 0.18mL/min; SDD+BSS: 3.45 ± 0.34mL/min).

Liver Enzymes

Based on plasma liver enzyme concentrations at the end of the experiment, the HFD group exhibited the highest average levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), plasma albumin and total bilirubin (TBil) (Table 1). The evidence supporting this relationship for AST (HFD+BSS: p = 0.036; SDD: p = 0.120; SDD+BSS: p = 0.044) (Fig. 3), and for ALT (HFD+BSS: p = 0.017; SDD: p = 0.036; SDD+BSS: p = 0.011) (Fig. 4) and for TBil (HFD+BSS: p = 0.051; SDD: p = 0.189; SDD+BSS: p = 0.085), but we found only limited evidence supporting the difference in albumin (HFD+BSS: p = 0.136; SDD: p = 0.359; SDD+BSS: p = 0.237). The results for the ratio of AST to ALT demonstrate similar results, with the HFD group exhibiting the largest ratio of all treatment groups (HFD: 2.8 ± 0.56; HFD+BSS: 1.5 ± 0.20, p = 0.060; SDD: 1.96 ± 0.38, p = 0.295; SDD+BSS: 1.29 ± 0.07, p = 0.079) (Fig. 5).

Quantitative PCR

We found no evidence of a relationship between diet and the Firmicutes/Bacteroidetes ratio (Table 3 & Fig. 6). To evaluate changes associated with SRB, dsrB was analyzed by qPCR. Again, we found no evidence for the relationship between experimental conditions and dsrB (Table 3 & Fig 7).

In this study, we demonstrated that a high fat diet increased portal venous blood flow and this increase was reversed by BSS. In addition, we showed that administration of an H₂S donor into the jejunum led to a further increase in portal venous blood flow in animals with HFD-induced weight gain and elevated AST, and this exaggerated blood flow was also restored to normal levels by BSS administered into the gut. Elevated AST and ALT in HFD group suggested liver injury potentially secondary to fatty liver disease.

A few studies have evaluated portal venous blood flow in terms of velocity (cm/min) in fatty liver disease indirectly using ultrasonic doppler study. These studies have reported a decrease in estimated portal vein velocity in the setting of hepatic disease^40–42. In contrast, we measured portal venous blood flow directly (rather than velocity) using a probe placed directly on the portal vein. Indirect assessment of portal venous blood flow by doppler ultrasonography has been shown to be less reliable than direct measurement^43,44. A further difference between our approach and that of previous published studies is that portal venous blood flow in our study was reported as flow (mL/min) which does not depend on cross-sectional area of the measured blood vessel⁴⁵.

BSS has several known mechanisms of action in the gastrointestinal tract including anti-bacterial and anti-inflammatory properties^21,46–49. However, there are no data on the effect of bismuth on mesenteric hemodynamics. While portal venous blood flow was increased in the HFD group, the portal venous blood flow in the HFD + BSS group was not different than the control group suggesting that the underlying change seen after 8 weeks of HFD was caused by a mechanism that is reversible by BSS. Such reversibility strongly argues away from structural or anatomic changes in the vasculature as the explanation for the abnormal blood flow. Nitric oxide is considered a key mediator that regulates sinusoidal and systemic circulation in the setting of portal hypertension with and without cirrhosis^6,50. This role is consistent with our previous report that H₂S modulates portal venous flow in a NO-dependent manner²⁸.

Recent animal studies have shown that non cirrhotic, NAFLD-induced changes to arterial circulation (prior to development of liver fibrosis) may be facilitated through another mediator, cyclooxygenase-2 (COX-2) (separate from the venous NO-mediated response in cirrhosis)⁶. Interestingly, bismuth subgallate, another bismuth salt, has been shown to suppress NO production and COX-2 activity, which acts as a vasoconstrictor on hepatic vasculature^51,52. Our result showing a further increase in portal venous blood flow with infusion of a H₂S donor provided additional support for this gaseous neurotransmitter as a key mediator of this vascular response. In addition, our data would suggest that abnormal portal venous blood flow could be a consequence of liver injury induced by even a short period of exposure to a HFD (8 weeks, too short for cirrhosis) and that this change in portal venous blood flow could be reversed by bismuth consistent with a key role for bacteria-derived H₂S and potentially, other vasoactive substances. Cirrhosis, as an end stage of liver disease, may not be needed for portal venous blood flow to be drastically changed (Fig. 1).

In addition to reversing elevated baseline portal venous flow with HFD, we found that BSS also reversed the augmented portal venous blood flow induced by acute administration of NaHS (Fig. 2). This immediate effect of BSS further suggests antibacterial actions are not responsible for the BSS prevention of HFD-induced increases in portal venous blood flow. Instead, the rapid action of BSS is better accounted by its ability to bind and inactivate H₂S.

The elevation of AST in the HFD group indicates underlying liver injury. Increased AST alone has been described as an indicator of liver injury even in the absence of changes in other liver enzymes⁵³. We found that the AST:ALT ratio in the HFD group was elevated compared to normal ranges in this strain of rats⁵⁴. An increased AST:ALT ratio has been previously used as a marker of NAFLD and as a predictor of fibrosis^55,56. Interestingly, AST:ALT ratio and the portal venous blood flow were normal in the HFD + BSS group suggesting that bismuth counteracts not only the vascular changes seen with early injury from a HFD but also the biochemical changes associated with liver injury induced by this diet. This is the first evidence of bismuth having a beneficial effect on hepatic injury. This benefit of bismuth may be due to mitigating harmful effects of H₂S. In a recent rat study examining H₂S as an environmental toxin, intraperitoneal NaHS increased the plasma concentration of liver enzymes, disrupted mitochondrial structure, induced apoptosis and injury of the liver via increased production of reactive oxygen species⁵⁷.

The normal gut microbiota contains multiple genera that produce H₂S as a by-product of hydrogen generated from microbial fermentation. Collectively, these microbes are called sulfate reducing bacteria (SRB)^58,59. Identification of SRB is usually achieved by measuring the presence of enzymes in the bacterial dissimilatory bisulfate reductase pathway (dsrA and dsrB) via polymerase chain reaction (PCR)^58,60,61. In a healthy state, bacteria-derived H₂S is detoxified by several mechanisms in the colon including oxidation by colonocytes to thiosulfate⁶². Dysbiosis, alterations in the gut microbiome that lead to bacterial overgrowth in the small intestine, has previously been shown to increase the number of SRB in the small intestine^59,63,64. The overgrowth of SRB (SRB bloom) into more proximal sections of the intestine is especially challenging since these regions have fewer H₂S detoxifying mechanisms compared to the colon⁵⁸. The increased H₂S production resulting from overgrowth in the SRB population in the small intestine can lead to exposure to excessive amount of H₂S which, in turn, can damage the surrounding vasculature and lead to intestinal damage⁶⁴ not seen when H₂S production is confined to the healthy colon.

In the setting of small intestinal bacterial overgrowth, higher concentrations of H₂S in the proximal small intestine could be a factor leading to liver injury and disease progression. Excessive exposure to exogenous bacteria-derived H₂S in the setting of dysbiosis with concomitant liver disease has the potential of inducing toxic effects similar to that seen with accidental exposure to environmental H₂S. In that setting concentration-dependent responses to increasing concentrations of H₂S generated following meals with accompanying microbial fermentation could uniquely increase portal venous blood flow and contribute to portal hypertension¹⁷. Patients with liver disease and increased SRB in the proximal small intestine may thus have excessive exposure to H₂S resulting in hyperdynamic mesenteric circulation, portal hypertension and other known vascular complications of liver disease.

We did not see a change in the Firmicutes/Bacteroidetes ratio in our animal study. This specific criteria was chosen as an indirect measure of dysbiosis in obesity^65–67. Even though the HFD appears to induce early liver disease as seen by abnormal AST:ALT ratio, it did not cause detectable changes in these major bacterial phyla⁶⁸. In general, this study suggests the gut microbiome at the phylum level may be resistant to change by a HFD within the 8-week time frame of this study in contrast to the changes in AST and liver portal blood flow.

In addition, we found no significant changes in dsrB gene expression. This might be explained by the low number of SRB in the gut microbiome (< 0.1 percent), the diversity of SRB and reported difficulty in detecting alterations to the SRB population ⁶⁹. Specifically, primers targeting hydrogenase genes could only detect Desulfovibrio species⁷⁰. As the Desulfovibrio-like population has been estimated to make up only 30–40% of the SRB population⁷¹, future studies are needed to evaluating other SRB.

There are a few limitations in our study including no tissue histology to confirm fatty liver disease or no direct measures of H₂S concentrations in the liver. However, the adverse effects of HFD or H₂S on liver enzymes could occur without histologic changes⁵⁷.

The results of this study have significant clinical implications. As the portal vein delivers the nutrients from the small intestine to the liver, bacteria- derived H₂S coming from the small intestine could have an especially large impact on portal venous hemodynamics. Luminal hydrogen concentration is increased after delivery of fermentable substrates to the gut microbiome when microbial fermentation increases. In the presence of SRB, these postprandial surges in hydrogen availability could result in large increases in luminal H₂S concentration that should drive surges in portal venous blood flow. Importantly, the consequence of these postprandial changes in portal venous blood flow could be even worse in the setting of cirrhosis where portal hypertension is already a complication, and the vascular compliance is abnormal. In addition, the elevated portal venous flow was reversed with oral administration of BSS, suggesting BSS might be a useful therapy for the hyperdynamic complications of portal hypertension.

Author Contribution

A.B and H.L and N.K wrote the main manuscript text. C.C and K.R. prepared figures and tables. All authors Reviewed the manuscript.

Acknowledgment:

Dr. Lin’s work is supported by the Winkler Bacterial Overgrowth Research Fund.

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Table 1 – Weight and liver test results at end of the 8-week diet study. Mean and standard error (in parentheses) for each variable across four groups. 2-way ANOVA results include P-value (F-test) and minimum False Positive Risk. Weight at end of study in grams. Weight gain in grams. Aspartate aminotransferase (AST) in U/L Alanine aminotransferase (ALT) in U/L. Total bilirubin (Tbil) in mg/dL. Albumin in g/dL. Error bars denote the Newey-West standard error.

	HFD	HFD+BSS	SDD	SDD+BSS	P-val	mFPR
Weight (wk 8)	369.7 (7.8)	344.4 (8.7)	344.9 (5.6)	374.8 (11.9)	0.028	0.007
Weight Gain	211.6 (7.7)	183.0 (10.1)	188.8 (5.8)	200.3 (15.0)	0.113	0.040
AST	211.9 (53.6)	83.6 (17.2)	111.3 (28.1)	60.3 (2.5)	0.037	0.014
ALT	72.9 (5.2)	52.6 (4.0)	54.9 (5.9)	46.8 (0.9)	0.010	0.002
Bilirubin	0.4 (0.12)	0.1 (0.02)	0.2 (0.04)	0.1 (0.00)	0.065	0.030
Albumin	2.6 (0.06)	2.3 (0.13)	2.4 (0.14)	2.3 (0.06)	0.192	0.072

Table 2- Baseline portal vein flow rates (5 min average, after stabilization) in mL/min across treatment groups and the average increase in flow after NaHS infusion. SE for baseline flow rate is computed using “effective sample sizes” and the SE for the increase in Flow is estimated using the Newey-West method. Measurements recorded using invasive Doppler flow probe on portal vein at 20 minutes of infusion.

	Baseline Flow (mL/min)	Flow Increase (NaHS)	P-value
HFD	23.4 (0.4)	6.1 (0.3)	<0.000001
HFD+BSS	18.0 (0.3)	2.0 (0.4)	0.000001
SDD	17.5 (0.2)	1.9 (0.2)	<0.000001
SDD+BSS	14.5 (0.2)	3.5 (0.3)	<0.000001

Table 3 – Quantitative PCR test results at end of the 8-week diet study of mucosa-associated bacterial density from distal small bowel evaluating Firmicutes/Bacteroidetes ratio as a measure of dysbiosis associated obesity and dsrB gene attributed to dissimilatory reductase. Normalized to universal eukaryotic 18S rRNA. Mean and standard error for each variable across four groups. 2-way ANOVA results include P-value (F-test) and minimum False Positive Risk. The distribution of the F/B ratio for each group is represented as a gray boxplot (and with circular points). The square point denotes the mean and the error bar denotes the standard error.

	HFD	HFD+BSS	SDD	SDD+BSS	P-val	mFPR
Firmicutes/Bacteroidetes	0.95 (0.32)	0.91 (0.23)	0.89 (0.30)	0.66 (0.05)	0.955	0.528
dsrB	0.81 (0.26)	1.07 (0.28)	1.03 (0.25)	0.85 (0.31)	0.876	0.477

Table 4 –Primer pairs used for quantitative PCR; references listed for each primer pair.

Target Group	Primer		Reference
All bacteria	Eub338	5’-ACTCCTACGGGAGGCAGCAG	²⁹
	Eub518	5’-ATTACCGCGGCTGCTGG	²⁹
Firmicutes	Firm934	5’-GGAGYATGTGGTTTAATTCGAAGCA	⁷²
	Firm1060	5’-AGCTGACGACAACCATGCAC	⁷²
Bacteroidetes	Cfb319	5’-GTACTGAGACACGGACCA	²⁹
	Eub518	5’-ATTACCGCGGCTGCTGG	²⁹
dsrB	dsrB213f	5’-CTGTTGTTTGGAGATGAGCCC	³⁰
	dsrB-658r	5’-ATTCCACTTCCTTCTCCCATA	³⁰

No competing interests reported.

BISMUTH SUBSALICYLATE REVERSES HIGH FAT DIET-INDUCED INCREASE IN PORTAL VENOUS FLOW

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Acknowledgment:

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