Experimental Cirrhosis Research Articles

Use of Mesenchymal Stem Cell Transplantation as a Treatment for Liver Cirrhosis in Animal Models.

Liver cirrhosis is one of the top 10 causes of death worldwide, and liver disease is in the top five in many developing countries. The treatment of liver cirrhosis at various stages necessitates the development of new organ transplantation techniques. One of these options is cell therapy, which has lately been used to treat a wide range of illnesses. The study used a rabbit model of liver cirrhosis to examine the efficiency of mesenchymal stem cell (MSC) transplantation. Subcutaneous injections of carbon tetrachloride (CCL4) were used to cause liver cirrhosis. When liver cirrhosis developed, autologous mesenchymal stem cells were infused into the portal vein. The histological image of the cirrhotic liver improved one month after MSC transplantation. This demonstrates that the intraportal delivery of autologous MSC to rabbits with experimental liver cirrhosis has good therapeutic outcomes.

Effect of Saccharomyces boulardii on Liver Diseases: A Systematic Review.

We aimed to systematize the results of published studies on the use of Saccharomyces boulardii (SB) for the treatment of various liver disorders (CRD42022378050). Searches were conducted using PubMed and Scopus on 1 August 2022. The PubMed search was updated on 15 June 2024. The review included sixteen studies: ten experimental animal studies (EASs) and six randomized controlled trials (RCTs). The CNCM I-745 strain was used in 68.8% of the included studies. SB reduced the severity of many manifestations of cirrhosis, and lowered the Child-Pugh scores in RCT. SB reduced the serum concentrations of TNF-α, IL-1β, IL-6, and IL-4 in animals with metabolic dysfunction-associated steatotic liver disease (MASLD); lowered the serum TNF-α and IL-6 levels in experimental cirrhosis in rats; and reduced the CRP levels in decompensated cirrhosis. The EAS of MASLD revealed that SB reduced liver steatosis and inflammation and lowered the liver expression of genes of TNF-α, IL-1β, interferon-γ, and IL-10. In studies on experimental cirrhosis and MASLD, SB reduced the liver expression of genes of TGF-β, α-SMA, and collagen as well as liver fibrosis. SB reduced the abundance of Escherichia (Proteobacteria), increased the abundance of Bacteroidetes in the gut microbiota, prevented an increase in intestinal barrier permeability, and reduced bacterial translocation and endotoxemia.

Hepatoprotective Effect of Cannabidiol on the Progression of Experimental Hepatic Cirrhosis in Rats.

Introduction: Liver cirrhosis is a condition characterized by the gradual replacement of normal liver tissue with scar tissue, ultimately leading to liver failure. This slow and progressive disease begins with a chronic inflammatory process induced by a noxious agent. In its advanced stages, the disease lacks effective therapies. Research has demonstrated the significant involvement of the endocannabinoid system in the pathogenesis of this disease. This study evaluated the hepatoprotective effect of cannabidiol (CBD) in the progression of experimental hepatic cirrhosis induced by thioacetamide (TAA) in rats. Methods: A randomized experimental design was employed using Holtzman rats. Hepatic cirrhosis was induced by intraperitoneal administration of TAA at a dose of 150 mg/kg for 6 weeks, with treatment initiated additionally. The groups were as follows: Group 1: TAA + vehicle; Group 2: TAA + CBD 2 mg/kg; Group 3: TAA + CBD 9 mg/kg; Group 4: TAA + CBD 18 mg/kg; Group 5: TAA + silymarin 50 mg/kg; and Group 6: Healthy control. Serum biochemical analysis (total bilirubin, direct bilirubin, ALT, AST, alkaline phosphatase, and albumin) and hepatic histopathological study were performed. The Knodell histological activity index (HAI) was determined, considering periportal necrosis, intralobular degeneration, portal inflammation, fibrosis, and focal necrosis. Results: All groups receiving TAA exhibited an elevation in AST levels; however, only those treated with CBD at doses of 2 mg/kg and 18 mg/kg did not experience significant changes compared to their baseline values (152.8 and 135.7 IU/L, respectively). Moreover, ALT levels in animals treated with CBD showed no significant variation compared to baseline. The HAI of hepatic tissue was notably lower in animals treated with CBD at doses of 9 and 18 mg/kg, scoring 3.0 and 3.25, respectively, in contrast to the TAA + vehicle group, which recorded a score of 7.00. Animals treated with CBD at 18 mg/kg showed a reduced degree of fibrosis and necrosis compared to those receiving TAA alone (p ≤ 0.05). Conclusion: Our findings demonstrate that cannabidiol exerts a hepatoprotective effect in the development of experimental hepatic cirrhosis induced in rats.

Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

Action of melatonin and physical exercise on the liver of cirrhotic rats: Study of oxidative stress and the inflammatory process.

Cirrhosis is characterized by structural and functional alterations of the liver. Melatonin (MLT) has antioxidant properties. Physical exercise (EX) can reverse muscle loss in cirrhotic patients. The objective was to evaluate the action of MLT and EX on the liver of rats subjected to the experimental model of bile duct ligation (BLD). 48 male Wistar rats were used, divided into groups: Control (CO), CO+MLT, CO+EX, CO+MLT+EX, BDL, BDL+MLT, BDL+EX, and BDL+MLT+EX. The treatments occurred from the 15th to the 28th day. The dose of MLT was 20 mg/kg via I.p (1x/day), and the EX was performed 10 min/day. Blood and liver were collected for analysis. The liver integrity enzymes AST, ALT, and ALP showed a significant reduction in the groups treated with MLT and EX. Histological analyses showed reorganization of the liver parenchyma, reduction of inflammatory infiltrate, and fibrotic nodules. Lipoperoxidation (LPO), the activity of antioxidant enzymes, and nitric oxide metabolites showed a significant reduction in the groups treated with MLT and EX. The expression of TNF-α and NF-kB decreased in the treated groups. Melatonin and physical exercise seem to be effective in restoring the parameters evaluated in this model of experimental cirrhosis.

Role of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. Chronic treatment with L-NAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.

Recombinant VEGF-C (Cys156Ser) improves mesenteric lymphatic drainage and gut immune surveillance in experimental cirrhosis

Lymphatic vessels (LVs) are crucial for maintaining abdominal fluid homoeostasis and immunity. In cirrhosis, mesenteric LVs (mLVs) are dilated and dysfunctional. Given the established role of vascular endothelial growth factor-C (VEGF-C) in improving LVs, we hypothesised that VEGF-C treatment could ameliorate the functions of mLVs in cirrhosis. In this study, we developed a nanoformulation comprising LV-specific growth factor, recombinant human VEGF-C (Cys156Ser) protein (E-VEGF-C) and delivered it orally in different models of rat cirrhosis to target mLVs. Cirrhotic rats were given nanoformulation without VEGF-C served as vehicles. Drainage of mLVs was analysed using tracer dye. Portal and systemic physiological assessments and computed tomography were performed to measure portal pressures and ascites. Gene expression and permeability of primary mesenteric lymphatic endothelial cells (LyECs) was studied. Immune cells in mesenteric lymph nodes (MLNs) were quantified by flow cytometry. Endogenous and exogenous gut bacterial translocation to MLNs was examined. In cirrhotic rats, mLVs were dilated and leaky with impaired drainage. Treatment with E-VEGF-C induced proliferation of mLVs, reduced their diameter, and improved functional drainage. Ascites and portal pressures were significantly reduced in E-VEGF-C rats compared with vehicle rats. In MLNs of E-VEGF-C animals, CD8+CD134+ T cells were increased, whereas CD25+ regulatory T cells were decreased. Both endogenous and exogenous bacterial translocation were limited to MLNs in E-VEGF-C rats with reduced levels of endotoxins in ascites and blood in comparison with those in vehicle rats. E-VEGF-C treatment upregulated the expression of vascular endothelial-cadherin in LyECs and functionally improved the permeability of these cells. E-VEGF-C treatment ameliorates mesenteric lymph drainage and portal pressure and strengthens cytotoxic T-cell immunity in MLNs in experimental cirrhosis. It may thus serve as a promising therapy to manage ascites and reduce pathogenic gut bacterial translocation in cirrhosis. A human recombinant pro-lymphangiogenic growth factor, VEGF-C, was encapsulated in nanolipocarriers (E-VEGF-C) and orally delivered in different models of rat liver cirrhosis to facilitate its gut lymphatic vessel uptake. E-VEGF-C administration significantly increased mesenteric lymphatic vesselproliferation and improved lymph drainage, attenuating abdominal ascites and portal pressures in the animal models. E-VEGF-C treatment limited bacterial translocation to MLNs only with reduced gut bacterial load and ascitic endotoxins. E-VEGF-C therapy thus holds the potential to manage ascites and portal pressure and reduce gut bacterial translocation in patients with cirrhosis.

THU-341 - Study of epigenetic control of LSECtin in hepatic antigen presenting cells during experimental cirrhosis

THU-341 - Study of epigenetic control of LSECtin in hepatic antigen presenting cells during experimental cirrhosis

THU-363 - Resection of mesenteric lymph nodes is associated with increased systemic immunosuppression in experimental cirrhosis following oral salmonella typhimurium challenge

THU-363 - Resection of mesenteric lymph nodes is associated with increased systemic immunosuppression in experimental cirrhosis following oral salmonella typhimurium challenge

THU-338 - ε-Lysine-melittin reduces pathological bacterial translocation and enhances gut immunity in experimental decompensated cirrhosis

THU-338 - ε-Lysine-melittin reduces pathological bacterial translocation and enhances gut immunity in experimental decompensated cirrhosis

Podoplanin-positive dilated lymphatic vessels in duodenum associates with three-month mortality in patients with cirrhosis.

Dilated and dysfunctional gut lymphatic vessels (LVs) have been reported in experimental cirrhosis. Here, we studied LVs in duodenal (D2)-biopsies of liver cirrhosis patients and investigated the prognostic role of a LV marker, podoplanin (PDPN), in predicting the mortality of patients with cirrhosis. A prospective, single-center cohort study was performed in liver cirrhosis patients (n = 31) and matched healthy controls (n = 9). D2-biopsies were obtained during endoscopy procedure, immunostained with PDPN, and scored based on 1) intensity and 2) density of positively-stained LVs per high power field. Gut and systemic inflammation were estimated by quantifying duodenal CD3+ intraepithelial lymphocytes (IELs), CD68+ macrophages, and serum TNF-α and IL-6 levels, respectively. Gut permeability and inflammation as assessed by quantifying gene expression of TJP1, OCLN, TNF-α, and IL-6 in D2-biopsies. Gene expression of LV markers, PDPN (8-fold), and LYVE1 (3-fold) was enhanced in D2-biopsies of cirrhosis patients compared to control (p < 0.0001). The mean PDPN score in decompensated cirrhosis patients (6.91 ± 1.26, p < 0.0001) was significantly increased as compared to those with compensated (3.25 ± 1.60). PDPN score positively and significantly correlated with the number of IELs (r = 0.33), serum TNF-α (r = 0.35), and IL-6 (r = 0.48) levels, while inversely correlated with TJP1 expression (r = -0.46, p < 0.05 each). In Cox regression, the PDPN score was a significant and independent 3-month-mortality predictor in patients (HR: 5.61; 1.08-29.109; p = 0.04). The area under the curve for the PDPN score was 84.2, and cutoff value for predicting mortality was ≥6.5 with 100% sensitivity and 75% specificity. Collectively, dilated LVs with high PDPN expression in D2-biopsies is a characteristic feature of patients with decompensated cirrhosis. PDPN score correlates with enhanced gut and systemic inflammation and also associates with 3-month mortality in cirrhosis.

Elevated bile acids are associated with left ventricular structural changes in biliary atresia.

In children with biliary atresia (BA), pathologic structural changes within the heart, which define cirrhotic cardiomyopathy, are associated with adverse perioperative outcomes. Despite their clinical relevance, little is known about the pathogenesis and triggers of pathologic remodeling. Bile acid excess causes cardiomyopathy in experimental cirrhosis, but its role in BA is poorly understood. Echocardiographic parameters of left ventricular (LV) geometry [LV mass (LVM), LVM indexed to height, left atrial volume indexed to BSA (LAVI), and LV internal diameter (LVID)] were correlated with circulating serum bile acid concentrations in 40 children (52% female) with BA listed for transplantation. A receiver-operating characteristic curve was generated to determine optimal threshold values of bile acids to detect pathologic changes in LV geometry using Youden index. Paraffin-embedded human heart tissue was separately analyzed by immunohistochemistry for the presence of bile acid-sensing Takeda G-protein-coupled membrane receptor type 5. In the cohort, 52% (21/40) of children had abnormal LV geometry; the optimal bile acid concentration to detect this abnormality with 70% sensitivity and 64% specificity was 152µmol/L (C-statistics=0.68). Children with bile acid concentrations >152µmol/L had ∼8-fold increased odds of detecting abnormalities in LVM, LVM index, left atrial volume index, and LV internal diameter. Serum bile acids positively correlated with LVM, LVM index, and LV internal diameter. Separately, Takeda G-protein-coupled membrane receptor type 5 protein was detected in myocardial vasculature and cardiomyocytes on immunohistochemistry. This association highlights the unique role of bile acids as one of the targetable potential triggers for myocardial structural changes in BA.

Pathomorphological changes in the organs of laboratory animals with experimental liver cirrhosis

Objective. Develop an experimental model of liver cirrhosis and describe pathomorphological changes in organs and tissues of laboratory animals.Materials and methods. Liver cirrhosis was simulated on 11 sexually mature Wistar male white rats. The control group consisted of 12 healthy laboratory animals. Acute toxic liver injury resulting in cirrhosis was induced by intraperitoneal injection of 50% tetrachloromethane (CCl4) solution on olive oil on the first day of the experiment in the dosage of 0.1 ml CCl4 + 0.4 ml olive oil per 100 g of the animal body weight, on the second day of the experiment 0.3 ml CCl4 + 0.2 ml olive oil per 100 g of the animal body weight. To synergize and potentiate the hepatotoxic effect of CCl4, animals had 10% ethanol solution freely available daily. The duration of the experiment was 65 days. Clinical and laboratory parameters were evaluated, histological evaluation of the preparations was performed. The obtained data were compared with the control group of animals.Results. Reproducibility of the model was 82% (9 animals). The indicator of lymphocyte blabbing index showed the development of liver cirrhosis in animals of the experimental group. Comprehensive morphological confirmation of the developed liver cirrhosis was obtained.Conclusion. The proposed methodology of liver cirrhosis formation correlates with pathomorphological changes in lymphocyte cell wall, changes in liver tissue and internal organs in human liver cirrhosis.

Age-Related Features of Ketanserin Effects in Experimental Liver Cirrhosis.

The effect of ketanserin on inflammation, liver fibrosis, and microviscosity of the plasma and mitochondrial membranes of hepatocytes was studied on young (3 months) and old (9 months) male Wistar rats with experimental liver cirrhosis. Ketanserin reduced inflammation, area of the connective tissue, and liver damage and improved serum biochemical parameters in rats of both age groups; in old rats, the effects were more pronounced than in young animals. In old rats, ketanserin reduced polarity of hepatocyte plasma and mitochondrial membranes in the area of protein-lipid contacts, which determined higher effectiveness of ketanserin during the treatment of liver cirrhosis in aged animals.

Conformationally Restricted Dipeptide-Based Nanoparticles for Delivery of siRNA in Experimental Liver Cirrhosis

Liver cirrhosis is a major health problem with multiple associated complications. The presently available drug delivery systems showed moderate site-specific delivery of antifibrotic molecules to the diseased liver; therefore, research on more effective and selective delivery systems in the context of liver cirrhosis remains a necessity in clinical investigation. The aim of the present study was to develop a peptide-based targeted nanocarrier to deliver an oligonucleotide to the hepatic sinusoidal and perivascular regions of the cirrhotic liver. We have synthesized and characterized a conformationally restricted targeted pentapeptide (RΔFRGD), which contains an unnatural amino acid, α,β-dehydrophenylalanine (ΔF). The RΔFRGD self-assembled into spherical nanoparticles (NPs) and was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Next, we investigated the delivery potential of the pentapeptide-based NPs to make a stable complex with a well-established small interference RNA and studied its site-specific delivery in experimental liver cirrhosis. We used siNR4A1 of the orphan nuclear receptor 4A1 (NR4A1), a well-known regulatory checkpoint for controlling liver fibrosis. Peptide NPs and their complex with siNR4A1 showed high biocompatibility against various mammalian cell lines. Hepatic tissue biodistribution analysis illustrated that targeted NPs predominantly accumulated in the cirrhotic liver compared to normal rats, specifically in sinusoidal and perivascular areas. A significant downregulation of the NR4A1 mRNA expression (−70%) andlower levels of the NR4A1/GAPDH ratio (−55%) were observed in the RΔFRGD-siNR4A1 nanocomplex-treated group in comparison to the RΔFRGD-vehicle group (RΔFRGD-Veh) at the gene and protein levels, respectively. In addition, in vivo inhibition of NR4A1 produced a significant aggravation in hepatic fibrosis compared with siRNA-vehicle-treated rats (+41% in the MT stain). The novel pentapeptide-based targeted delivery system can be further evaluated and validated for therapeutic purposes in various pathological conditions.

Pharmacokinetics and Therapeutic Potential of Teucrium polium against Liver Damage Associated Hepatotoxicity and Oxidative Injury in Rats: Computational, Biochemical and Histological Studies.

This study investigated the druggability, pharmacokinetics and ethyl acetate extract of Teucrium polium (EA T. polium) and the protective effect against carbon tetrachloride (CCl4) induced liver cirrhosis in rats. The total antioxidant capacity (TAC) and scavenging activity of the extract were examined. The in vivo protective study was based on the use of an animal model of CCl4-induced liver cirrhosis. Four groups of rats have been used: Group I: control rats; Group II: received CCl4 in olive oil (0.5 mL/kg); Group III: received the EA T. polium (25 mg/kg) of pretreatment for seven days by gavage then CCl4 in olive oil by gavage for 15 days. Group IV: received the EA of T. polium for seven days (25 mg/kg). EA T. polium was found to possess significant antioxidant capacity. CCl4 caused a hepatotoxicity associated increase in both levels of AST and ALT, which were reduced back to normal values following EA T. polium pretreatment. Hepatotoxicity associated structural modifications of liver tissues and increase in thiobarbituric acid reactive substances (TBARS), conjugated dienes (CD) and carbonyl proteins (CP), associated decreases in several assessed antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT). The in vivo findings on the protective effect of T. polium were supported by its druggability, its pharmacokinetic properties and molecular docking assays. These results confirm the modulatory antioxidant and hepatoprotective potential of T. polium in this experimental liver cirrhosis model. T. polium phytochemicals are good candidates for further pharmaceutical explorations and drug design.

OS153 - Vascular endothelial growth factor C mediated restoration of mesenteric lymphatic vessels permeability and drainage improves gut immunity surveillance in experimental cirrhosis

OS153 - Vascular endothelial growth factor C mediated restoration of mesenteric lymphatic vessels permeability and drainage improves gut immunity surveillance in experimental cirrhosis

Deletion of kif3a in CK19 positive cells leads to primary cilia loss, biliary cell proliferation and cystic liver lesions in TAA-treated mice.

Loss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood. Primary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3af/f;CK19CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays. At the end of 20weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro. TAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.

Decrease in ANG and VEGF mRNA levels during progressive angiogenesis of the liver venous system of Wistar rats in experimental cirrhosis

Decrease in ANG and VEGF mRNA levels during progressive angiogenesis of the liver venous system of Wistar rats in experimental cirrhosis

STUDY OF HEPATOPROTECTIVE ACTIVITY OF A COMBINED FLAMIN/SILYMARIN MIXTURE FROM HERBAL ORIGIN IN EXPERIMENTAL CIRRHOSIS.

The aim of this study is to investigate Flamin/Silymarin as a new herbal combined hepatoprotective drug in experimental liver cirrhosis of white rats. Flamin was obtained from the flowers of the reddish immortelle (Helichrysum rubicundum (C. Koch.)), аnd Silymarin from the seeds of the milk thistle (Silybum marianum) growing in Armenia.&#x0D; The histomorphological studies showed that in comparison with the two control groups under the influence of Flamin/Silymarin the destructive and dystrophic processes are much reduced in the liver parenchyma. The histostructure is mainly preserved: there are binucleated hepatocytes with nucleoli, the boundaries of the interlobular hepatic tracts are clearly formed. Compared with the group treated with Flamin, in animals treated with Flamin/Silymarin, there was a reduction of karyolysis and pyknosis in hepatocytes. Along with this, in some areas, there were small foci of fatty changes. It is assumed that with long-term therapy, the effectiveness of Flamin/Silymarin may be increased.&#x0D; Consequently, further in-depth study of the mixture will lead to the identification and introduction of a new herbal hepatoprotective for the prevention and treatment of liver cirrhosis.