Dr. Naglaa M. El-Lakkany Theodor Bilharz Research Institute Antifibrotic effects of gallic acid on activation/proliferation of cultured hepatic stellate cells and in thioacetamide-induced liver fibrogenesis in rats. Dr. Naglaa M. El-Lakkany Professor and Head of Pharmacology Dept Theodor Bilharz Research Institute

Background

What Is Liver Fibrosis? Liver fibrosis is a wound healing process to chronic liver injury. Formation of scar tissue is a normal body response to injury, but in fibrosis this healing process goes away. In a healthy liver, the synthesis and breakdown of matrix tissue are in balance. Fibrosis occurs when excessive scar tissue builds up faster than it can be broken down.

Liver fibrosis is an outcome of many chronic liver diseases. Liver fibrosis and its end-stage of cirrhosis are major causes of morbidity and mortality worldwide (31 million DALYS or 2% of all deaths worldwide 2010-2013). Preventing or inhibiting the progression of fibrosis to cirrhosis may reduce the decline in health-related quality of life.

Hepatic stellate cells (HSCs) are the major cell type involved in development of liver fibrosis.

Is Liver Fibrosis Reversible? During fibrosis the balance between the production and the degradation of matrix constituents is affected, resulting in increased deposition of ECM in fibrotic livers. MMPs or TIMP ECM synthesis. MMPs or TIMP ECM degradation. Fibrosis regression Apoptosis of activated HSCs

Treatment Strategy ( A ) Cure the Primary Disease ( B ) ( G ) Reduction of Inflammation and Immune Response ( G ) Increased Degradation of ECM ( C ) Inhibition of HSCs Activation Treatment Strategy ( F ) Stimulation of HSCs Apoptosis ( D ) Neutralizing Fibrogenic and Proinflammatory Response of HSCs ( E ) Inhibition of Collagen Production

Despite significant advances in the understanding of the basic mechanisms underlying fibrosis, “silver bullet” antifibrotic drug remains elusive. The efficiency of current synthetic agents in treating chronic liver disease is not satisfactory and they have undesirable side effects. Recently, edible fruits and natural products have attracted substantial interest because they contain several antioxidants and bioactive phytocompounds that may act as possible remedial agents.

Gallic acid (GA), 3, 4, 5-trihydroxybenzoic acid, a natural phenolic acid found abundantly in tea, grapes, pomegranate, berries and other fruits. GA provides desirable health benefits beyond basic nutrition, including antioxidant, anti-inflammatory and anticancer properties. However, little has been documented concerning the therapeutic role of GA on liver fibrosis. GA

Aim of the work Recently, apoptosis of activated HSCs has been considered as a promising strategy for hepatic fibrosis treatment. Accordingly, this study investigates the ameliorative effects and potential mechanisms of GA on TAA-induced hepatic fibrogenesis in vivo and on HSCs in vitro.

Materials & Methods

Isolation and identification of GA from pomegranate peel extract Gallic acid Identification of Gallic acid by HPLC.

In vitro study Cell culture Hepatic stellate cells T6 (HSCs), a generous gift from Prof. S.L. Friedman (Mount Sinai School of Medicine, New York, NY), were cultured. Primary hepatocytes were freshly isolated from male Sprague–Dawley rat (300-350 g) by a two-step portal collagenase perfusion of the liver. Hepatocytes cell viability was assessed by trypan blue dye exclusion.

In vitro study HSCs proliferation, viability and cytotoxicity assay. Confluent HSCs-T6 (5×103 cells/ml) were cultured. Different concentrations of GA (0, 25, 50, 100, 200, 400 and 500 μg/mL) or vehicle (DMSO) were added and incubated for 24 and 48 h. The anti-proliferative activity of GA was determined using SRB staining.

In vitro study Safety of GA on hepatocytes was assessed by MTT cell viability/cytotoxicity assay on freshly isolated rat hepatocytes.

Experimental groups 32 male rats Normal Control TAA (200 mg/kg, IP twice a week for 12 weeks) TAA + Silymarin (oral at a daily dose of 50 mg/kg) TAA + GA (oral at a daily dose of 50 mg/kg)

Parameters of assessments 1- Liver function Serum ALT and AST. 2- Oxidative stress markers Hepatic GSH and MDA. 3- Liver fibrotic markers Hepatic TGF-B, PDGF-BB, TIMP-1, HP. 4-Histopathology and immunohistochemical assays (α-SMA, Caspase-3, PCNA) in tissues and cells.

Results

Effect of various concentrations of GA on HSCs and hepatocytes viability IC50= 48 µg/ml Effect of various concentrations of GA (25–400 μg/ml) on HSCs (A) and hepatocytes (B). GA selectively induces HSCs cytotoxicity.

Effect of various concentrations of GA on TGF- production in culture medium. HSCs were treated with GA (12.5, 25, 50 µg/ml) for 24 h. ap < 0.05 as compared with the control group, bp < 0.05 as compared with the corresponding 12.5 µg/ml concentration, cp <0.05 as compared with the corresponding 25 µg/ml concentration.

Effect of GA (IC50) on -SMA and caspase-3 expression in activated HSCs B C D A HSCs was cultured in 6-well plates and treated with IC50 of GA for 48h; (A) untreated HSCs cells showing an increase in α-SMA positive cytoplasmic stain (arrow); (B) HSCs treated with GA showing a decrease in α-SMA expression (positive cytoplasmic stain); (C) untreated HSCs cells showing few positively stained caspase-3 cells; (D) HSCs treated with GA showing an increase in caspase-3 expression (positive cytoplasmic stain).

Effect of GA on liver enzymes ALT and AST in TAA-intoxicated rats ALT, alanine amiotransaminase; AST; aspartate aminotranamiase; Data presented as mean of 8 rats ± SEM. ap <0.05 vs. normal control group, bp <0.05 vs. TAA intoxicated group.

Effect of GA on oxidative stress markers GSH and MDA in TAA-intoxicated rats GSH, reduced glutathione; MDA, malondialdehyde Data presented as mean of 8 rats ± SEM. ap <0.05 vs. normal control group, bp <0.05 vs. TAA intoxicated group, cp <0.05 vs. silymarin group.

Effect of GA on liver fibrosis marker s TGF-1 and PDGF-BB in TAA-intoxicated rats Profibrotic cytokines (PDGF-BB and TGF-β1) were determined in the liver homogenates. TAA, thioacetamide; SY, silymarin; GA, galic acid. ap < 0.05 vs. normal control group, bp < 0.05 vs. TAA intoxicated group, cp < 0.05 vs. silymarin group.

Effect of GA on liver fibrosis markers TIMP-1 and HP content in TAA-intoxicated rats TIMP-1 and HP levels were determined in the liver homogenates. TAA, thioacetamide; SY, silymarin; GA, gallic acid. ap < 0.05 vs. normal control group, bp < 0.05 vs. TAA intoxicated group, cp < 0.05 vs. silymarin group. Hydroxyproline is an amino acid unique to all collagens and its level reflects the amount of collagen present and therefore can be used to determine the extent of fibrosis.

Effect of GA on histopathology of liver Silymarin Normal TAA A E B F C G D H H & E Masson Trichome GA

Effect of GA on liver fibrosis grade Histopathological examination of liver sections from (a) normal untreated rats showing normal hepatic architecture, (b) TAA-intoxicated (200 mg/kg) rats showing disturbances in architecture of the hepatic tissue S4 stage (hepatic cirrhosis) with formation of variable sized regenerating nodules (arrows) (c) silymarin-treated (50 mg/kg) rats showing almost the normal hepatic architectural, mild thin fibrous bands (%)(arrows) (d) GA-treated (50 mg/kg) rats showing normal hepatic architecture, mild thin fibrous bands (arrows). (Sirius red x50).

Effect of GA on PCNA, -SMA and Caspase-3 in liver tissue B4 B3 C4 C3 B1 C1 C2 B2 α-SMA TAA Normal Silymarin GA Immunohistochemical examinations of PCNA (A1-4), α-SMA (B1-4) and Caspase-3 (C1-4) expressions (magnification: 20×) in rat liver sections (n=6).

Summary & Conclusion

In vitro results showed that GA not only inhibits the activation or proliferation of rat HSCs cell lines in a dose-dependant manner without impairing parenchymal cell viability, but also stimulates the apoptosis of these cells. Furthermore, GA reduced TGF-β1 production in culture media in a concentration dependent manner as well as reduction in number of activated HSCs and expression of α-SMA. Such results were in accordance with the in vivo findings where GA treatment alleviated the up-regulation of hepatic TGF-β1 and PDGF-BB production as well as α-SMA expression that resulted from long-term of TAA administration, ultimately, alleviating the pathological damage induced by oxidative stress. In the same context, GA inhibited proliferation of damaged hepatocytes as indicated by the significant reduction in PCNA staining, an index of cellular proliferation.

GA reduced both TIMP-1 and HP, consequently reducing collagen deposition and fibrosis score from S4 to S2, which may help in prevention of liver fibrosis. GA or silymarin displayed equivalent results in terms of reductions in serum AST, hepatic MDA, PDGF-BB and TIMP-1, collagen content, fibrosis score, α-SMA and caspase-3 expression. On the contrary, GA exerted advantageous benefits than silymarin in terms of improvement in serum ALT, hepatic GSH stores, TGF-β1 and PCNA expression reflecting hepatocyte regeneration and progression towards fibrosis reversal. In conclusion, GA is an ideal antifibrotic therapy inducing HSC apoptosis with no harmful effects on hepatocytes and liver functioning, which would greatly enhance its clinical application.

Aknoledgements Walaa H. El-Maadawy Sayed H. Seif el-Din Pharmacology Dept, TBRI Sayed H. Seif el-Din Olfat A. Hammam Pathology Dept, TBRI Salwa H. Mohamed Immunology Dept, TBRI Shahira M. Ezzat Pharmacognosy Dept, Cairo University Seham S. El-Hawary Marwa M. Safar Pharmcol &Toxicol Dept, Cairo University Samira Saleh Sanaa S. Botros & Zeinab Demerdash Consultants, TBRI

Theodor Bilharz Research Institute