Gut Dysbiosis and Hemodynamic Changes as Links of the Pathogenesis of Complications of Cirrhosis


 Background. Hemodynamic changes (hyperdynamic circulation) and gut dysbiosis are observed in cirrhosis. It was suggested that gut dysbiosis contributes to the development of hyperdynamic circulation, which aggravates the course of cirrhosis. The aim is to test this hypothesis.Methods. The cross-sectional observational study included 47 patients with cirrhosis. Stool microbiome was assessed using 16S rRNA gene sequencing. Echocardiography with a simultaneous assessment of blood pressure and heart rate was performed. Hemodynamic parameters were calculated. Results. Hyperdynamic circulation was found in 34% of patients. Patients with hyperdynamic circulation had higher incidences of clinically significant ascites (p=0.018), overt hepatic encephalopathy (p=0.042), hypoalbuminemia (p=0.011), hypoprothrombinemia (p=0.019), systemic inflammation (p=0.002), and severe hyperbilirubinemia (p=0.042) than patients without hyperdynamic circulation. The abundance of Proteobacteria (p=0.012), Enterobacteriaceae (p=0.008), Bacilli (p=0.027), Streptococcaceae (p=0.044), Lactobacillaceae (p=0.034), Enterococcaceae (p=0.046), and Fusobacteria (p=0.026) increased and the abundance of Bacteroidetes (p=0.049) and Erysipelotrichia (p=0.029) decreased in the gut microbiome of patients with hyperdynamic circulation compared to patients without hyperdynamic circulation. The systemic vascular resistance value negatively correlated with the abundance of Proteobacteria (r=-0.423; p=0.003), Enterobacteriaceae (r=-0.417; p=0.004), and Fusobacteria (r=-0.401; p=0.005). Heart rate was negatively correlated with the abundance of Bacteroidetes (r=-0.453; p=0.001). The cardiac output value was positively correlated with the abundance of Proteobacteria (r=0.402; p=0.003), Enterobacteriaceae (r=0.424; p=0.003), Fusobacteria (r=0.281; p=0.049), and Bacilli (r=0.314; p=0.031), and negatively correlated with the abundance of Bacteroidetes (r=-0.313; p=0.032) and Erysipelotrichia (r=-0.329; p=0.024). Conclusion. Gut dysbiosis is associated with hyperdynamic circulation, which is associated with a number of complications of cirrhosis.


Introduction
Hemodynamics changes in cirrhosis were described half of a century ago and consist of arterial vasodilation (decreased systemic vascular resistance), hypotension, and increased cardiac output. This is de ned as hyperdynamic circulation [1][2][3] . The study of experimental models of cirrhosis led to the hypothesis that these changes arise as a response to subclinical systemic in ammation which in turn is a consequence of bacterial translocation, the penetration of bacteria and their components from the intestinal contents into ascitic uid, mesenteric lymph nodes, and portal and systemic blood ow [1][2][3][4][5][6][7] .

Patients
In this cross-sectional observational study, 100 consecutive patients with cirrhosis were admitted to the Department of Hepatology's Clinic for Internal Diseases, Gastroenterology and Hepatology at Sechenov University (Moscow, Russia) and screened for inclusion. The study procedures were explained to potential participants, and written informed consent was obtained before enrollment. The present study was approved by the Ethics Committee of Sechenov University in accordance with the Declaration of Helsinki.
The inclusion criteria were as follows: diagnosis of cirrhosis veri ed by histology or clinical, biochemical, and ultrasound ndings; and age between 18 and 70 years. The exclusion criteria were as follows: use of lactulose, lactitol, or other prebiotics, probiotics, antibiotics, or metformin in the past 6 weeks; alcohol consumption in the past 6 weeks; or in ammatory bowel disease, cancer, or any other serious disease. Of the original 100 patients screened for inclusion, 47 met the criteria and were enrolled in the study (Fig. 1).

Gut microbiome analysis
The morning after admission, a stool sample was taken into a sterile disposable container and immediately frozen at -80°C [26] .
DNA from the stool was isolated using the MagNa Pure Compact Nucleic Acid Isolation Kit I (Roche, Basel, Switzerland) according to the manufacturer's instructions. Libraries for sequencing were prepared by two rounds of PCR ampli cation. In the rst round, speci c primers for the v3-v4 region of the 16S ribosomal RNA gene were used: 16S-F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG and 16S-R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.
After ampli cation, the PCR product was puri ed using AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Then, a second round of PCR was performed to attach speci c adapters and enable multiplexing of the samples. To begin, 5 µL of the rst PCR product was added to the reaction after ball cleaning with primers containing Illumina indices (Nextera XT Index v2 Primers; San Diego, CA, USA) and adapter sequences as well as 2× KAPA HiFi HotStart ReadyMix. The ampli cation products were also puri ed using AMPure XP beads (Beckman Coulter). The concentrations of the prepared libraries were then measured using a Qubit 2.0 uorimeter (London, UK) and quantitative PCR. The quality of the libraries was assessed using the Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The libraries were mixed in equal proportions and diluted to the required concentration to be run on a MiSeq (Illumina) device. Pair-end readings of 300 + 300 nucleotides were obtained. Reads were trimmed from the 3'-tail with Trimmomatic (Illumina) and then merged into a single amplicon with the MeFiT tool [27][28] . We did not perform operational taxonomic unit picking; instead, we classi ed amplicon sequences with the Ribosomal Database Project (RDP) classi er and RDP database [29] .

Systemic hemodynamic assessment
Echocardiography was performed at rest according to the guidelines of the American Society of Echocardiography [30][31][32][33] . The systolic and diastolic blood pressure and heart rate were measured using an automatic oscillometric sphygmomanometer (AND, Japan) simultaneously with the assessment of the stroke volume. Calculations of hemodynamic parameters are presented in Table 1 [30][31][32][33][34][35] . Stroke volume (Doppler velocity time integral)×(cross-sectional aorta area) [34] Mean arterial pressure ((systolic blood pressure) + 2×(diastolic blood pressure))/3 Cardiac output (stroke volume)×(heart rate) Systemic vascular resistance (mean arterial pressure)/(cardiac output) Systolic pulmonary artery pressure (right atrium pressure estimated from diameter of inferior vena cava and respiratory changes) + 4×(the peak velocity of the tricuspid valve regurgitant jet)2 [32][33] Mean pulmonary artery pressure 0.61×(systolic pulmonary artery pressure) + 2 mmHg [35] The criterion for portopulmonary hypertension was a combination of the presence of signs of portal hypertension and mean pulmonary artery pressure above 25 mm Hg [36] .
No generally accepted criteria for hyperdynamic circulation are available. Therefore, we diagnosed a patient with this disorder if their cardiac output was greater than the mean + 2 standard deviations (5.5 L/min) of healthy individuals examined in the same way during the check-up. The control group (n = 50) did not signi cantly differ from the patients with cirrhosis in terms of age and gender distribution. incidences of minimal ascites (grade 1 ascites according to the classi cation of the International Club of Ascites), mild hyperbilirubinemia, minimal hepatic encephalopathy, and esophageal varices, spleen size, main parameters of complete blood count, heart rate, mean blood pressure, left ventricular ejection fraction, and serum creatinine level. The difference between the groups of patients in mean pulmonary artery pressure, serum sodium and potassium levels almost reached the limit of signi cance (Table 2).    increased cardiac output was due to an increase in venous return to the heart, which led to an increase in end-diastolic volume. Heart rate and ejection fraction, which are other factors that could increase cardiac output, did not signi cantly differ between the groups of patients with and without hyperdynamic circulation, indicating their insigni cant in uence on its development. This is consistent with the under lling hypothesis, which considers vasodilation as a primary disorder, and uid retention and increased venous return to the heart with an increase in cardiac output as secondary changes [2][3][4] .
Notably, an increase in end-diastolic volume is usually characteristic of systolic heart failure, but it is not associated with a decrease in ejection fraction in patients with cirrhosis [9] . Moreover, the serum level of that biomarker of heart failure as N-terminal brain natriuretic peptide does not depend on ejection fraction, but is associated, on the contrary, with increased heart function in these patients [37] .
Complications of cirrhosis were differently associated with hyperdynamic circulation in our study. Some of them (hypoalbuminemia, hypoprothrombinemia, systemic in ammation, portopulmonary hypertension) were more often in patients with this disorder than in those without it. The presence of others (esophageal varices) was not associated with it. The association of hyperdynamic circulation with complications of cirrhosis from the third group (ascites, hyperbilirubinemia, hepatic encephalopathy) depended on their severity: it was absent in their mild and minimal forms, but their severe forms were associated with it. This may be considered as con rmation of the hypothesis that increased cardiac output aggravates the course of portal hypertension but is not its primary cause. Moreover, our study was cross-sectional and it is not entirely correct to judge causal relationships. The primary question here was whether decreased liver function led to the development of hyperdynamic circulation, whether hyperdynamic circulation worsened liver function, or whether they both exacerbated each other, leading to a vicious circle. Additional studies are required to determine the changes in liver function in patients with the same level of decreased liver function, depending on the presence or absence of hyperdynamic circulation. The incidence of hyperdynamic circulation development should be prospectively investigated and compared between patients with varying degrees of compensation for liver function in the other group of future studies.
Unfortunately, we could not measure the hepatic venous pressure gradient, which is considered to be the main quantitative characteristic of portal hypertension [38] .
Our study is the rst to assess the relationship between gut dysbiosis and hemodynamic changes in cirrhosis. Despite disagreements between the results of several previous studies, most indicated that the abundance of bacteria under the Proteobacteria phylum [10-18,21−23] , which contains active endotoxin, and Bacilli class [13][14][15][16][17][18][19][20][21][22][23] , which are capable of bacterial translocation, increase in the gut microbiome with cirrhosis. Thus, an increase in the abundance of these bacteria can be considered a biomarker of gut dysbiosis in cirrhosis. These bacteria are responsible for molecular (endotoxin) and cellular bacterial translocation in cirrhosis [39] .
In this study, the abundance of Bacilli and Proteobacteria increased in patients with hyperdynamic circulation and correlated with the values of the main markers of hyperdynamic circulation, namely systemic vascular resistance and cardiac output. This may support the hypothesis that bacterial translocation of these bacteria and their components leads to vasodilation and hyperdynamic circulation.
A similar relationship is also established for the minor taxon Fusobacteria, which also contain endotoxins.
Only one article [22] reported an increase in the content of these bacteria in the gut microbiome in cirrhosis.
This may be due to their low abundance in the gut microbiome, so these bacteria do not attract the attention of researchers.
An interesting nding was the decrease in Bacteroidetes abundance in patients with hyperdynamic circulation, considering these bacteria also have endotoxins. The abundance of these bacteria does not correlate with the degree of vasodilation but is associated with a decrease in heart rate, which can prevent the development of hyperdynamic circulation. The mechanism by which Bacteroidetes affect the heart rate is not clear. It seems that the presence of endotoxin is not an indicator of bacterial pathogenicity and its ability to translocate. It should be remembered that Bacteroidetes, together with bacteria under the Clostridia class, are the main taxa of normal human microbiota, and changes in their abundance in cirrhosis compared with healthy individuals are reported differently in different publications. Bacteroidetes abundance either increases [11,24] , decreases [10,19,22] , does not change [21] , or changes depending on the state of liver function [16] in cirrhosis. Bacteroidetes showed a protective effect against hyperdynamic circulation in our study.
The abundance of bene cial bacteria under the Clostridia class in the gut microbiome does not signi cantly differ between patients with and without hyperdynamic circulation and does not correlate with any of the hemodynamic parameters in cirrhosis.
An unexpected nding was the negative correlation between markers of hyperdynamic circulation and the abundance of Erysipelotrichia that are a minor class under the Firmicutes phylum. Among the 4 main classes under this phylum, it is the least studied and might be underestimated by researchers.
Changes in the gut microbiome in hemodynamic circulation mainly signi es a redistribution of the proportion of bacteria containing endotoxins, where Proteobacteria and Fusobacteria that have active endotoxins replace Bacteroidetes that have weak endotoxins [40] (Fig. 2).
Probiotics, which are living bacteria used for dysbiosis, showed their effects on hemodynamic parameters in cirrhosis in small uncontrolled studies, which require randomized controlled trials to con rm [41] .
Our study is the rst to con rm that gut dysbiosis is associated with hemodynamic changes in cirrhosis. We further showed that the presence of these changes is associated with a number of complications of cirrhosis. Thus, hemodynamic changes may be considered a pathogenetic link between gut dysbiosis and these complications of cirrhosis. However, this hypothesis requires veri cation in further prospective studies, the ideas of which we also proposed. All of these contribute to the strength of our study.
The limitation of our study is its small sample size, although this did not prevent us from obtaining signi cant results.
In conclusion, we have shown that gut dysbiosis is associated with hyperdynamic circulation, which in turn is associated with a number of complications of cirrhosis. Ethics approval and Consent to participate: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for being included in the study.
Consent for publication: Not applicable.
Availability of data and material: The data can be provided upon request to the corresponding author.
Code availability: Not applicable.  The composition of the gut microbiome in the patients with and without hyperdynamic circulation