In this commentary, we discuss new findings indicating that microbiota transplantation has favorable impact on portal hypertension (PH) in the experimental model of cirrhosis induced by bile duct ligation (BDL) (Huang et al.; Clin Sci (Lond) (2021) 135(24): 2709-2728, doi: 10.1042/CS20210602). Sinusoidal PH is an ominous outcome of advanced chronic liver disease, characterized by increased intrahepatic vascular resistance (IHVR), splanchnic hyperemia, and the development of portosystemic collaterals. In the work of Huang et al., microbiota transplantation not only alleviated splanchnic hyperdynamic circulation by improving vascular responsiveness and decreasing mesenteric angiogenesis, but also reduced blood flow in portosystemic collaterals. Surprisingly, however, microbiota transplantation had no effect on intrahepatic vasoconstriction in this experimental model. We discuss these observations in the context of recent literature showing that manipulation of the gut microbiota (either by transplantation or through the use of probiotics) may improve IHVR, which is one of the earliest abnormalities in the pathogenesis of sinusoidal PH. Further research is needed to explore the specific molecular and cellular targets associated with the correction of dysbiosis in liver disease.

Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-alpha can stimulate muscle proteolysis; therefore, a potential relationship between TNF-alpha and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 (P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated (P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-alpha levels were increased and correlated positively with free and conjugated ubiquitin (P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-alpha might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.

Endotoxin has been proposed to participate in the development of hepatic encephalopathy. However, there is no published data concerning the effects of endotoxin neutralization on the degree of hepatic encephalopathy. The present study investigated the effect of chronic intraperitoneal injection of polymyxin B, a neutralizing antagonist of endotoxin, on hepatic encephalopathy in rats with thioacetamide (TAA)-induced fulminant hepatic failure.

Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of TAA (350 mg/kg/day) for 3 days. Two series of rats were designed to compare the effects of low dose (0.1 mg) or high dose (0.2 mg) intraperitoneal polymyxin B administration versus normal saline (NS) on hepatic encephalopathy. The injection was twice daily started from 2 days prior to TAA administration and lasted for 5 days. Severity of encephalopathy was assessed by the counts of motor activity in an Opto-Varimex animal activity meter. Plasma levels of endotoxin and tumor necrosis factor-alpha (an index of liver injury) were measured by Limulus assay and the ELISA method, respectively.

Neutralization of endotoxin by either low dose or high dose polymyxin B administration did not significantly alleviate the degree of hepatic encephalopathy, as represented by the counts of motor activities (P > 0.05). Plasma levels of endotoxin and tumor necrosis factor-alpha were comparable between rats treated with polymyxin B or NS (P > 0.05).

Our findings do not support the notion that endotoxin plays a major role in the pathogenesis of hepatic encephalopathy in rats with TAA-induced fulminant hepatic failure.

A 70-kDa heat shock protein (stress-inducible HSP70, HSP72) has been reported to be a cytoprotectant in a variety of organs. It has been reported that HSP72 protected non-cirrhotic rats against endotoxemia. However, its cytoprotective effect against endotoxemia in cirrhotic rats has not yet been studied. In this study, we investigated the cytoprotective effect of HSP72 on lipopolysaccharide (LPS)-induced liver injury in carbon tetrachloride (CCl(4))-induced cirrhotic rats.

Liver cirrhosis was produced by an 8-week intraperitoneal injection of CCl(4) in male Sprague-Dawley rats. Expression of HSP72 was investigated using western blot analysis. Cirrhotic rats were given an intraperitoneal injection of LPS (10 mg/kg) with or without hyperthermia (42.5 degrees C, 15 min) preconditioning. Liver injury was assessed biochemically (aspartate transaminase, alanine transaminase, bilirubin, lactate dehydrogenase, creatinine) and histologically. The plasma tumor necrosis factor (TNF)-alpha level was determined.

Hyperthermia preconditioning induced a 4-fold increase in HSP72 in the cirrhotic rat liver. Pre-induction of HSP72 prevented LPS-induced liver injury, as evaluated using serum biochemical parameters and histology with reduced TNF-alpha response.

These findings suggest that pre-induction of HSP72 may provide therapeutic strategies for Gram-negative sepsis-induced liver injury in liver cirrhosis.

Increased vascular nitric oxide (NO) production has been implicated in the pathogenesis of the hyperdynamic circulation in liver cirrhosis. This study investigated the expression of three isoforms of NO synthase (NOS) in rat cirrhotic livers. Cirrhosis was induced by chronic bile duct ligation (BDL). NOS enzyme activity was assessed by L-citrulline generation. Competitive RT-PCR was performed to detect the mRNA levels of NOS. In situ hybridization was done to localize NOS mRNA. Protein expression of NOS was evaluated by Western blotting and immunohistochemistry. The L-citrulline assay showed that constitutive NOS (cNOS) enzymatic activity was decreased, while inducible NOS (iNOS) activity was increased in BDL livers. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) mRNA were detected in BDL and sham rats, but with enhanced expression in BDL rats. eNOS protein was redistributed with less expression in sinusoidal endothelial cells, but the total levels in liver were not changed. nNOS was induced in hepatocytes of BDL rats, in contrast to only a weak signal observed around some blood vessels in sham livers. Intense mRNA and protein expression of iNOS was induced in livers of BDL rats and was localized in hepatocytes, with no or a negligible amount in control livers. In conclusion, iNOS was induced in cirrhotic liver with its activity increased. In contrast, cNOS activity was impaired, regardless of unchanged eNOS protein levels and enhanced nNOS expression. These results suggest that all three types of NOS have a role in cirrhosis, but their expression and regulation are different.

Coagulation disorders commonly develop in patients with cirrhosis of the liver. They have also been reported in patients with non-cirrhotic portal fibrosis (NCPF) and extra-hepatic portal venous obstruction (EHPVO); the two conditions with portal hypertension and near-normal liver functions. The spectrum and prevalence of coagulation abnormalities and their association with the pathogenesis of these diseases and with hypersplenism was prospectively studied.

Eighteen EHPVO patients that included an equal number of NCPF patients and 20 healthy controls were prospectively studied. The coagulation parameters assessed included: international normalized ratio, partial thromboplastin time, and fibrinogen and fibrinogen degradation products. Platelet aggregation and malondialdehyde levels were measured.

Both EHPVO (83%) and NCPF (78%) patients had a significantly prolonged international normalized ratio and a decrease in fibrinogen and platelet aggregation. The EHPVO patients had a significant prolongation in partial thromboplastin time (67% patients), with increased levels of fibrinogen degradation product levels occurring in all patients; these were normal in NCPF patients. Platelet malondialdehyde levels were normal in both groups. Hypersplenism was present in four EHPVO and seven NCPF patients. It did not significantly influence the coagulation profile in either NCPF or EHPVO patients.

Coagulation anomalies are common and significant in both NCPF and EHPVO patients, suggestive of a mild disseminated intravascular coagulation disorder. These imbalances could be caused by chronic subclinical endotoxemia and cytokine activation after the initial portal thromboembolic event. The persistence of these abnormalities in adolescent patients indicates an ongoing coagulation derangement.

Nitric oxide (NO) has been implicated in the arterial vasodilation and associated vascular hyporesponsiveness to vasoconstrictors observed in liver cirrhosis. Bacteria, potent activators of NO and TNF-alpha synthesis, are found in the mesenteric lymph nodes (MLNs) of ascitic cirrhotic rats. Here, we investigated the impact of bacterial translocation (BT) to MLNs on TNF-alpha production, vascular NO release, and contractility in the mesenteric vasculature of ascitic cirrhotic rats. Vascular response to the alpha-adrenoagonist methoxamine, which is diminished in the superior mesenteric arterial beds of cirrhotic rats, is further blunted in the presence of BT. BT promoted vascular NO release in cirrhotic rats, an effect that depended on pressure-induced shear stress and was blocked by the NO inhibitor N(omega)-nitro-L-arginine. Removing the endothelium had the same effect. Endothelial NO synthase (eNOS), but not the inducible isoform (iNOS), was present in mesenteric vasculature of cirrhotic rats with and without BT, and its expression was enhanced compared with controls. TNF-alpha was induced in MLNs by BT and accumulated in parallel in the serum. This TNF-alpha production was associated with elevated levels of tetrahydrobiopterin (BH(4)), a TNF-alpha-stimulated cofactor and enhancer of eNOS-derived NO biosynthesis and NOS activity in mesenteric vasculature. These findings establish a link between BT to MLNs and increased TNF-alpha production and elevated BH(4) levels enhancing eNOS-derived NO overproduction, further impairing contractility in the cirrhotic mesenteric vasculature.

Excessive formation of nitric oxide may mediate the generalized vasorelaxation and hyporesponsiveness to vasoconstrictors observed in portal hypertensive states. Endotoxin, released from the bowel and detoxified by the liver, could stimulate inducible nitric oxide synthase directly or indirectly via the cytokine cascade. This study investigated the effect of chronic intraperitoneal injection of polymyxin B, a neutralizing antagonist of endotoxin, on the hemodynamics of partially portal vein-ligated (PVL) rats.

Concomitantly with endotoxin (600 EU) and dactinomycin (80 microg), polymyxin B (0.1 mg) or normal saline (N/S) was administered via an intraperitoneal route to male Sprague-Dawley rats. Twenty-four hours later, mean arterial pressure was determined. In PVL rats polymyxin B (0.1 mg in 5 cc N/S) or N/S was given intraperitoneally twice daily from 2 days prior to operation until 5 days (short-term) or 14 days (long-term) after the operation. Long-term polymyxin B- or N/S-treated sham-operated rats were included as controls. Hemodynamic studies with a thermodilution technique were performed at the end of treatment. Blood samples were collected from another series of PVL rats with long-term treatment to determine plasma levels of endotoxin and tumor necrosis factor-alpha. Plasma levels of endotoxin and tumor necrosis factor-alpha were measured by Limulus assay and the ELISA method, respectively.

With the dosage of 0.1 mg polymyxin B, hypotension in rats subjected to endotoxin and dactinomycin administration could be corrected (polymyxin B vs. placebo: 130.0+/-7.7 vs. 108.8+/-6.7 mm Hg, p<0.05). However, long-term or short-term treatment with the same dosage of polymyxin B failed to ameliorate the hyperdynamic circulation of PVL rats. In addition, long-term treatment with polymyxin B did not change systemic and portal hemodynamics in sham-operated rats. Plasma levels of endotoxin and tumor necrosis factor-alpha were comparable in PVL rats treated with long-term polymyxin B or N/S (p>0.05).

Our findings do not support the role of endotoxin in the hyperdynamic circulation of PVL rats.

It has been suggested that excessive formation of nitric oxide (NO) is responsible for the hyperdynamic circulation observed in portal hypertension. Substance P is a neuropeptide partly cleared by the liver and causes vasodilatation through the activation of the endothelial NO pathway. However, there are no previously published data concerning the plasma level of substance P in cirrhotic rats and its relationship to NO.

Plasma concentrations of substance P and nitrate/nitrite (an index of NO production) were determined in control rats and cirrhotic rats with or without ascites using an enzyme-linked immununosorbent assay and a colorimetric assay, respectively. In addition, systemic and portal hemodynamics were evaluated by a thermodilution technique and catheterization.

Cirrhotic rats with and without ascites had a lower systemic vascular resistance (2.6 +/- 0.2 and 3.9 +/- 0.4 mmHg ml(-1) x min x 100 g body weight, respectively) and higher portal pressure (14.6 +/- 0.6 and 11.3 +/- 1.8 mmHg) than control rats (6.5 +/- 0.3 mmHg x ml(-1) x min x 100 g BW and 6.8 +/- 0.2 mmHg, respectively, P < 0.05), and cirrhotic rats with ascites had the lowest systemic vascular resistance. Plasma levels of nitrate/nitrite progressively increased in relation to the severity of liver dysfunction (control rats, 2.7 +/- 0.5 nmol/ml; cirrhotic rats without ascites, 5.6 +/- 1.3 nmol/ml; cirrhotic rats with ascites, 8.3 +/- 2.2 nmol/ml; P < 0.05). Cirrhotic rats with ascites displayed higher plasma values of substance P (57.7 +/- 5.9 pg/ml) than cirrhotic rats without ascites (37.9 +/- 3.1 pg/ml, P < 0.05) and control rats (30.1 +/- 1.0 pg/ml, P < 0.05). There was no significant difference in plasma substance P values between control rats and cirrhotic rats without ascites (P > 0.05). No correlation was found between plasma levels of substance P and nitrate/nitrite (r = 0.318, P > 0.05).

Excessive formation of NO may be responsible, at least partly, for the hemodynamic derangements in cirrhosis. Although substance P may not participate in the initiation of a hyperdynamic circulation in cirrhosis, it may contribute to the maintenance of the hyperdynamic circulation observed in cirrhotic rats with ascites.

To realize the roles of peripheral vasodilatation and atrial natriuretic peptide in the formation of cirrhotic ascites, the effects of long-term administration of octreotide on carbon tetrachloride-induced cirrhotic rats were evaluated.

Urine sodium excretion, hemodynamics, plasma atrial natriuretic peptide levels, renin activities and aldosterone concentrations were compared between cirrhotic and control rats (protocol 1); and between octreotide- (65 micrograms/kg, twice daily for 10 days, subcutaneously) and placebo-treated (5% dextrose) cirrhotic rats (protocol 2). In an in vitro experiment, right atrial tissue of cirrhotic rats was incubated with different concentrations of octreotide to evaluate the release of atrial natriuretic peptide (protocol 3).

Cirrhotic rats had significantly lower urine sodium excretion and systemic vascular resistance, and significantly higher plasma atrial natriuretic peptide levels, renin activities and aldosterone concentrations than control rats. Compared with placebo-treated cirrhotic rats, octreotide caused increased urine sodium excretion (-10 +/- 4% vs. 13 +/- 8% from baseline values, p < 0.05) and systemic vascular resistance (2.6 +/- 0.1 vs. 3.3 +/- 0.3 mmHg.min.100 g.ml-1, p < 0.05); and decreased plasma atrial natriuretic peptide levels (166.7 +/- 24.8 vs. 234.0 +/- 19.2 pg/ ml, p < 0.05), renin activities (2.45 +/- 0.49 vs. 4.36 +/- 0.53 ng.ml-1.h-1, p < 0.01) and aldosterone concentrations (290.2 +/- 40.0 vs. 483.3 +/- 82.6 pg/ml, p < 0.05). In the in vitro experiment, right atrial release of atrial natriuretic peptide of cirrhotic rats was not significantly changed when incubated with different concentrations of octreotide.

Octreotide ameliorates renal sodium retention and suppresses plasma levels of atrial natriuretic peptide of ascitic cirrhotic rats with a novel mechanism via, at least partly, the modification of peripheral vascular resistance.