Cannabidiol may prevent the development of congestive hepatopathy secondary to right ventricular hypertrophy associated with pulmonary hypertension in rats

Background Pulmonary hypertension (PH) can cause right ventricular (RV) failure and subsequent cardiohepatic syndrome referred to as congestive hepatopathy (CH). Passive blood stasis in the liver can affect inflammation, fibrosis, and ultimately cirrhosis. Cannabidiol (CBD) has many beneficial properties including anti-inflammatory and reduces RV systolic pressure and RV hypertrophy in monocrotaline (MCT)-induced PH in rats. Thus, it suggests that CBD may have the potential to limit CH development secondary to RV failure. The present study aimed to determine whether chronic administration of CBD can inhibit the CH secondary to RV hypertrophy associated with MCT-induced PH. Methods The experiments involved rats with and without MCT-induced PH. CBD (10 mg/kg) or its vehicle was administered once daily for 3 weeks after MCT injection (60 mg/kg). Results Monocrotaline administration increased the liver/body weight ratio. In histology examinations, we observed necrosis and vacuolar degeneration of hepatocytes as well as sinusoidal congestion. In biochemical studies, we observed increased levels of nuclear factor-κappa B (NF-κB), tumour necrosis factor-alpha (TNA-α), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6). CBD administration to PH rats reduced the liver/body weight ratio, improved the architecture of the liver, and inhibited the formation of necrosis. Cannabidiol also decreased the level of NF-κB, TNF-α, IL-1β and IL-6. Conclusions The studies show that CBD can protect the liver from CH probably through attenuating PH, protective effects on the RV, and possibly direct anti-inflammatory effects on liver tissue through regulation of the NF-κB pathway. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s43440-024-00579-4.


Introduction
Pulmonary hypertension (PH) is a rare disease diagnosed when the mean pressure in the pulmonary artery is higher than 20 mmHg [1].Under physiological conditions, the right ventricle (RV) is an integral part of the pulmonary circulation, which is characterized by high compliance and low pressure.The RV structure is optimized to adapt to changes in its preload, however, it is less effective in coping with increased afterload caused by high pressure in the pulmonary circulation.In PH, high pressure increases the RV afterload, which consequently contributes to the pathological RV remodeling and its systolic-diastolic failure [2].Right ventricular failure can lead to cardiohepatic syndrome referred to as congestive hepatopathy (CH).The primary pathomechanism of liver dysfunction is elevated RV pressure transmitted to the liver and portal vein, leading to passive blood stasis which causes the development of CH.It manifests as congestive fibrosis in response to hypoxia, hepatocyte necrosis, venous thrombosis, and inflammation, and can eventually result in cirrhosis [3][4][5][6].Liver damage caused by heart failure is accompanied by passive congestion and activation of Kupffer cells, with the development of inflammation and subsequent activation of multinucleated leukocytes and influx of cytokines and chemokines [7].The pathophysiology of cardiac-hepatic interactions is not fully understood and it is a subject of intense scientific interest, requiring a well-defined animal model [8].The animal model of monocrotaline (MCT)-induced PH is one of the most widely used animal models that allow for the development of severe PH, RV hypertrophy, and subsequent CH [6,9,10].
Cannabidiol (CBD) is a non-intoxicative phytocannabinoid with many beneficial properties including antioxidant, anti-inflammatory, and anti-fibrotic [11].It has been demonstrated that CBD, alleviates MCT-induced PH in rats [9,12] and Sugen + hypoxia-induced PH in mice by reducing RV systolic pressure (RVSP) [12].In previous publications, we also proved that CBD reduces pulmonary and RV hypertrophy, reduces perivascular and interstitial fibrosis in the RV as well as lowers plasma pro-B-type natriuretic peptide (NT-proBNP) levels in MCT-induced PH [9,10].Moreover, CBD reduced fibrosis in the left ventricle in a mouse model of autoimmune myocarditis [13].This evidence suggests that CBD may have the potential to limit organ changes developing secondary to heart failure (HF) including CH.
The pathomechanism of CH is complex and inflammation may be one of the mechanisms underlying this condition, since activation of hepatic stellate cells (HSCs), a key population of fibrogenic cells in the liver, is an important contributor to the process of liver fibrosis in CH.Studies indicate that the nuclear factor-κappa B (NF-κB) signaling pathway may play a role in liver fibrosis as a potent activator of HSCs [14].It has been proven that one of the mechanisms of CBD's anti-inflammatory effects involves the regulation of the NF-κB pathway and reduced production of pro-inflammatory mediators: tumour necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) [15].Recently, it has been suggested that therapeutic targeting of the NF-κB pathway may have beneficial effects in treating liver diseases [16,17].Cannabidiol also alleviates non-alcoholic steatohepatitis (NASH) in mice by inhibiting the NF-κB p65 nuclear translocation [17].In addition to targeting NF-κB, recent reports have shown that a promising therapeutic strategy for PH and its complications may be to inhibit IL-6.The use of a monoclonal antibody (IL-6R Ab) against the interleukin-6 receptor (IL-6R) was effective in reducing elevated pulmonary artery pressure and RVSP, and reduced RV remodeling and inflammation [18].It is known that one of the anti-inflammatory properties of CBD is the inhibition of IL-6 expression [19], which can directly improve the clinical condition of patients with PH, as well as prevent the development of RV failure and associated CH.
Therefore, this study aimed to determine whether chronic administration of CBD to rats can inhibit the CH secondary to RV hypertrophy associated with MCT-induced PH and whether inflammation is involved in the process.

MCT and CBD treatment
The rats were randomly divided into 4 experimental groups as follows: (1) Control (CTR) rats received solvent for CBD (consisting of Tween The dose of CBD was determined using data, where 10 mg/ kg in a rat corresponds to an effective and safe dose of CBD used in humans [20,21].PH was confirmed in our previous paper [9] where the RVSP in the MCT group was 43.7 ± 3.9, in the CTR group 20.03 ± 0.9, in CTR + CBD 21.4 ± 0.9 and MCT + CBD 28.2 ± 0.7 [mmHg] (n = 10).Figure 1 shows the protocol of the experiments.

Tissue preparation
The animals underwent anesthesia (sodium pentobarbital (300 µmol/kg), Biowet, Puławy, Poland) a day after the last administration of CBD.The parts of the liver tissues were destined for histology studies (described below).The remains of liver tissue were weighed and then immediately frozen (flash frozen) with liquid nitrogen and stored at − 80 °C for Western blot, enzyme-linked immunosorbent assay (ELISA), and quantitative Real-Time PCR (qRT-PCR) studies.

Histology
Liver sections were fixed in 10% buffered formalin, cut into small pieces, put in a tissue cassette for the dehydration process, embedded in paraffin, sliced (4 μm), and stained with hematoxylin and eosin (H + E).Histopathological changes of each liver section (5 sections per each animal (n = 5-6)) were blindly evaluated in ≥ 10 randomly selected

Western blot
Liver samples were homogenized in a buffer Mammalian Protein Extraction Reagent (MPER, Thermo Scientific, Rockford, IL, USA) containing a protease inhibitor cocktail (Roche Diagnostics GmbH, Germany).The bicinchoninic acid method (Price Rapid Gold BCA, Protein Assay Kit, Thermo Scientific, Waltham, MA, USA) was used to determine the total protein concentration, and after calculation homogenates were properly reconstituted in Laemmli

Statistical analysis
Statistical analysis was performed using GraphPad Prism 9.3.0(GraphPad Software, San Diego, California, USA).The normality of the data was assessed for Gaussian distribution before conducting statistical tests.The data exhibited a normal distribution a two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test (when appropriate) was used.The results are presented as mean ± the mean standard error of the mean (SEM) of n animals.For non-parametric data (scores), a non-parametric Kruskal-Wallis test followed by post-hoc Dunn's (when appropriate) test was used and the data was expressed as medians and interquartile ranges.A significance level of pvalue < 0.05 was considered statistically significant.

Effects of MCT-induced PH and CBD on liver/total body weight ratio and selected liver function parameters in plasma
We observed the statistically significant effect of MCT (MCT effect: F 1,36 = 19.06,p < 0.001) and CBD (CBD effect: F 1,36 = 10.22,p = 0.003) on the liver/total body weight ratio in groups with the interaction between factors i.e., MCT x CBD (F 1,36 = 11.40,p = 0.002).Monocrotaline rats showed a higher liver/total body weight ratio of about 20% relative to CTR (Tukey multiple comparison test: q = 7.743, DF = 36, p < 0.001) rats suggesting CH.Chronic CBD administration to MCT rats reduced this ratio by 13% (Tukey multiple comparison test, q = 6.574,DF = 36, p < 0.001) (Table 2).Analysis of plasma ALT, AST, albumin, total bilirubin, GLDH, and GGT parameters showed no differences between CTR and MCT rats suggesting no damage to hepatocytes.Chronic administration of CBD did not modify these parameters in either the CTR + CBD or MCT + CBD groups (Table 2), for statistical significance, see Table 2.

ELISA method
The concentrations of TNF-α and IL-1β (ab100785 and ab255730; Abcam, Cambridge, UK) were determined through enzyme-linked immunosorbent assay (ELISA), following the guidelines provided by the manufacturer.

qRT-PCR
The liver was homogenized in TRIsure™ (BIO-38,032, Bioline GmbH, Luckenwalde, Germany) and RNA was isolated following the guidelines provided by the manufacturer.RNA was reverse-transcribed into cDNA using SensiFAST™ cDNA Synthesis Kit (BIO-65,053, Bioline, London, UK).Quantitative Real-Time PCR was performed in a thermocycler (Thermocycler CFX96 Real-Time System, Bio-Rad) using a SensiFAST™ SYBR Kit (BIO-98,005, Bioline).The sequence of the primers and parameters of qRT-PCR are presented in Table 1 (provided by Genomed, Warsaw, Poland).Relative expression was calculated using the delta-delta Cycle Threshold (Ct) method and normalized to GAPDH [23].
CBD) or its vehicle was administrated once a day for 21 days.Data are presented as mean ± SEM; n = 5-6 animals per group.Two-way ANOVA, followed by Tukey's multiple comparisons test.A significance level is set at p < 0.05.In total bilirubin and gamma-glutamyltransferase results are under the detection threshold thus the statistic comparison is impossible.*** p < 0.001 CTR vs. MCT, ### p < 0.001 MCT vs. MCT + CBD.Abbreviations: ALT: alanine transaminase; AST: aspartate transaminase; CTR: control; GGT: gamma-glutamyltransferase; GLDH: glutamate dehydrogenase; MCT: monocrotaline; NS: nonspecific  in the MCT group and a decreasing tendency after CBD administration to MCT rats (MCT + CBD) (Fig. 3G).

Discussion
In this study, MCT administration increased the liver/body weight ratio, necrosis and vacuolar degeneration of hepatocytes, as well as sinusoidal congestion.In biochemical studies, we also observed increased levels of NF-κB, TNAα, IL-1β, and IL-6.CBD administration to PH rats reduced the liver/body weight ratio and inhibited the formation of necrosis.Cannabidiol also decreased the level of NF-κB, TNF-α, IL-1β and IL-6.
In our experiments, despite the lack of effect of MCT at 60 mg/kg on plasma parameters, the ratio of liver/body weight increased 21 days after MCT administration.It may indicate the onset of CH development in the MCT group due to passive blood stasis in the liver caused by RV failure rather than toxic MCT-induced liver damage.Our previous papers support these speculations as using the same rats, we have demonstrated PH with RV failure as evidenced by increased RVSP, elevated Fulton index (the RV weight to the left ventricle (LV) plus septum (RV/LV + S) ratio) [9], as well as cardiomyocyte enlargement and RV fibrosis [10].Dai et al. [3] also did not show an increase in ALT, AST, GGT, and total bilirubin in the serum of patients with CH.Similarly, a single oral administration of MCT at doses of 40, and 80 mg/kg did not modify the activity of liver enzymes AST and ALT in rat plasma, only 240 mg/kg increased the activity of the aforementioned enzymes [24].Otaka et al. [25] demonstrated that a single administration of MCT at a dose of 600 mg/kg to mice resulted in higher ALT activity, peaking at 48 h after injection and returning to normal after 120 h.The above reports and the fact that the liver has a high regenerative potential suggest that only high doses of MCT cause toxic damage to the liver and regeneration occurs after 120 h [25].
We observed influence of MCT (MCT effect: F 1, 16 = 15.55,p = 0.01) but no CBD (CBD effect: F 1, 16 = 0.8653, p = 0.366) on the expression of TGF-β1 in groups with no interaction between factors F 1, 16 = 0.1104, p = 0.774.Tukey multiple comparison test showed differences between the CTR + CBD vs. MCT (q = 4.873, DF = 16, p = 0.016) and CTR + CBD vs. MCT + CBD (q = 4.275, DF = 16, p = 0.037) groups, however, in the context of our study, significances in these groups were not considered and are not shown in Fig. 3.The expression of Col I (Fig. 3G) was at a similar level in each group (no MCT effect: F 1,15 = 1.934, p = 0.185, no CBD effect: F 1,15 = 3.385, p = 0.086, no interaction between factors: F 1,15 = 0.5934, p = 0.453) but we noticed an increasing tendency in the expression of this parameter severe on the 37th day of the study [6].The limitation of our study is that it lasted only 21 days.Prolonged exposure of the liver to passive congestion could have induced fibrotic changes and would have allowed us to assess the effect of CBD on more advanced changes typical of CH.However, prolonging the study was associated with increased mortality in rats due to severe PH.In the future, we plan to study liver function in a different PH model, the use of which may allow us to extend the experiment.
Accordingly, we are the first to prove that CBD can alleviate CH secondary to RV hypertrophy associated with PH.We suggest that one of the likely mechanisms of hepatoprotective effects involves the improvement of cardiac function by chronic administration of CBD.In our previous papers, we already proved that CBD reduces perivascular and interstitial fibrosis in the RV, as well as decreases cardiomyocyte width [10] which was associated with lower RV systolic pressure [9] and may have contributed to the inhibition of the development of mild sinusoidal congestion.
In PH rats, histological examination showed the beginning of the development of sinusoidal congestion and vacuolization of hepatocyte cytoplasm.We did not observe advanced fibrosis, only an upward trend in TGF-β1 and prevention of liver enlargement in the MCT + CBD group compared to MCT rats, presumably through the beneficial effect on RV function that we have already demonstrated in our previous studies [9,10].Thus, we confirmed the results of other authors where chronic (90 days) oral administration of CBD at doses of 0.66 and 1.33 mg/kg to healthy rats did not modify the most biochemical parameters (e.g., AST, ALT) and did not modify liver weight or liver/body weight ratio [26].It correlated with no abnormalities in hepatic morphology in the CTR and CTR + CBD groups (Fig. 2A,  B).
The histological images confirm the development of mild sinusoidal congestion 21 days after MCT injection (Fig. 2C, E).Based on the pathological changes in the liver described in the results section we suggest that during passive venous congestion, hepatic cells became ischemic with the initiation of metabolic changes leading to accumulation of sodium and water in the cell.Gewehr et al. [6] showed that a single administration of 60 mg/kg MCT to rats causes CH secondary to PH-related RV hypertrophy.After 15, 30, and 37 days after MCT administration, histological changes were found in the liver of these animals.After 15 days, mild sinusoidal hyperemia was observed, which intensified to and monocrotaline (MCT)-induced pulmonary hypertension rats.All data are presented as mean ± SEM, n = 6; a two-way ANOVA followed by Tukey's post-hoc test lungs of rats with MCT-induced PH [31].In addition, CBD in the livers of mice with NASH reduced the translocation of NF-κBp65 which leads to a decrease in the expression of TNF-α and IL-1β.The same authors suggest that inactivation of NF-κB in macrophages by CBD may be a key mechanism for its anti-inflammatory effects in the liver [17].Moreover, CBD administration inhibited macrophage recruitment and suppressed the activation of the NF-κB pathway in the livers of mice with NASH thus confirming the hepatoprotective properties of CBD [32].We suggest that CBD, through its direct effect on inhibiting the NF-κB pathway, may indirectly contribute to inhibiting the activation of HSCs and thus prevent liver fibrosis, the next stage of CH.Other researchers have shown that CBD, by inducing the selective death of activated HSCs, may find use as a therapeutic agent for liver fibrosis [33].Effects of CBD on other liver cell types have also been reported.CBD inhibited NF-κB activation and TNF-α production in response to endotoxin in mouse Kupffer cells and reduced the expression of adhesion molecules and multinucleated cell adhesion to human sinusoidal endothelial cells [34].
Since there have been reports that the activation of CB 1 -Rs has a fibrogenesis-promoting effect on the liver, and activation of CB 2 -Rs has a hepatoprotective effect [35], we tested whether PH and administration of CBD affect the expression of classical cannabinoid receptors.We noted no effect of MCT or CBD on the expression of these receptors.This is not surprising, since CBD has a low affinity for cannabinoid receptors [21] and its hepatoprotective effect probably includes other mechanisms.

Conclusions
In conclusion, we confirmed that the NF-κB pathway may be involved in the development of CH, especially at an early stage.Furthermore, the studies presented show that CBD can protect the liver from CH probably through attenuating PH [9], protective effects on the RV [10], and possibly direct anti-inflammatory effects on liver tissue through regulation of the NF-κB pathway (Fig. 5).In addition, like other authors, we confirm that CBD did not cause any adverse changes in the liver of healthy rats, demonstrating its high safety potential.
Col I parameters, which are among the markers of remodeling and fibrosis.Interestingly, before the development of fibrotic lesions, we observed in PH rats an activation of the NF-κB pathway, which regulates the secretion of the mediators TNF-α, IL-1β and IL-6, and in histological images we observed an influx of inflammatory cells in the area of the central vein.There is still a lot of ambiguity about the mechanisms of CH development which is due to the lack of an adequate experimental model to determine the molecular mechanisms contributing to the development of CH [5].As mentioned, inflammation in the course of CH which has been overlooked for many years, has recently become the focus of research.In liver damage caused by heart failure, concomitant passive congestion is accompanied by activation of Kupffer cells, with subsequent activation of multinucleated leukocytes and influx of cytokines and chemokines [7].Hamberger et al. [14] observed that the livers of rats with PH induced by Sugen + hypoxia developed congestive liver failure secondary to HF, and higher expression of T lymphocytes, macrophages, and increased expression of TNF-α were found.In patients with CH, moderate hepatitis with mild liver cell damage has been observed [3].Hamberger et al. [14] showed that activation of HSCs, part of the main fibrogenic cell population in the liver, contributes to hepatic fibrosis in CH.Importantly, the NF-κB signaling pathway is a potent activator of HSC and thus may be the mechanism that initiates fibrosis in CH [27].It is known that fibrotic changes occur in advanced CH [7].One of the central regulators of fibrosis that leads to deposition of extracellular matrix and production of, for example, collagen I in many liver diseases of various etiologies is TGF-β1 [28].Overexpression of TGF-β1 leads to activation and migration of HSCs, which as mentioned above are responsible for fibrotic changes in CH [5].Known factors leading to increased TGF-β1 expression are IL-1β and also NF-κB [29].In our study, we observed an increase in both NF-κB and IL-1β in the livers of rats after MCT administration, as well as a trend toward an increase in TGF-β1, suggesting that rats on day 21 after MCT administration had already developed inflammation in the liver, but fibrosis processes had not yet been initiated, which is also confirmed by histological images (Fig. 2C).
Cannabidiol in our study decreased the level of NF-κB, TNF-α, IL-6, and IL-1β parameters, suggesting that it may inhibit the NF-κB pathway and thus regulate inflammation in the liver.NF-κB is assumed to be a central regulator of inflammation that responds to a wide variety of immune receptors, and its dysregulated activation has been linked to many inflammatory diseases.It is reported that targeting the NF-κB pathway may find application in the treatment of inflammatory diseases [30].Previously, we confirmed that CBD can regulate the activity of the NF-κB pathway in the use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.

Fig. 2
Fig. 2 Representative liver sections stained with hematoxylin and eosin control (CTR) rats (A), CTR rats treated with cannabidiol (CBD) (B), rats with monocrotaline (MCT)-induced pulmonary hypertension (C), MCT rats treated with CBD (D).The black arrows indicate congestion, the blue arrow indicates the area of necrosis, the green arrow indicates vacuolization and the red arrows indicate the influx of inflammatory cells around the central vein.Magnification ×200

Fig. 4
Fig.4 The influence of cannabidiol (CBD) on the protein levels of cannabinoid type 1 receptor (CB 1 ) (A), cannabinoid type 2 receptor (CB 2 ) (B) determined by Western blot in livers from control (CTR) Experiments received approval from the Local Ethical Committee for Animal Experiments at the University of Warmia and Mazury in Olsztyn, Poland (project code 88/2018, granted on November 27, 2018) and were conducted following the ARRIVE guidelines and European Directive (2010/63/EU).Rats were obtained from the Experimental Medicine Centre (Medical University of Białystok, Białystok, Poland).Animals were kept in plastic cages (two rats per cage) under a 12 h/12 h light/dark cycle and had ad libidum access to standard food pellets and water.The experiments were performed on 40 Wistar rats: male, 5-8 weeks old, with body weights: of 150-250 g.