Fibrosis of the liver is a state of complicated end stage alteration of structure and function due to different aetiologies. Fibrosis is a consequence of different prevalent mechanisms according to the diverse causes of parenchymal damage. Fibrosis caused by chronic viral infection is initially concentrated within and around the portal tract, while fibrosis secondary to toxic/metabolic damage is located mainly in the centrolobular areas 1.

Oxidative stress, characterized by the overproduction of reactive oxygen species (ROS), which overwhelm the levels of antioxidants, has been suggested as the pathogenic factor of a number of human diseases and was reported to cause tissue damage 2. ROS can react with cellular macromolecules such as nucleic acids, polyunsaturated fatty acids in cellular membranes and sulfhydryl bonds in proteins to cause mutagenesis, carcinogenesis and cell death.

Thioacetamide (TAA, CH₃-C[S] NH₂), a known fungicide used to control the decay of fruits 3 was shown to be S-oxidized at the thioamide group to TAA sulfoxide (CH₃-C[SO] NH₂) and subsequently di-Soxide (CH₃-C[SO₂] NH₂) in the liver. The reactive intermediates in this pathway covalently bind to hepatic macromolecules and eventually cause liver injury 45, whereby free radical-mediated lipid peroxidation contributes to the development of TAA induced liver fibrosis 67. Prolonged administration of TAA causes hyperplastic liver nodules, liver cell adenomas and hepatocarcinomas. The free radicals produced during TAA metabolism interfere with ribosomal activity, thereby hindering protein synthesis 8. The biochemical and morphological changes observed in TAA-induced rat liver injury resemble to a large extent human liver disease and could serve as a suitable model for studying the causes of human liver fibrosis and cirrhosis 9.

Tissue fibrosis is associated with the increase of the tTG activity and accumulation of ECM 10. In liver fibrosis induced in rats by carbon tetrachloride (CCl₄) and in human patients with an acute liver disease, Mirza et al. 11 found a dramatic rise in tissue transglutaminases (tTG) activity. The enzyme catalyzes the specific cross-linking of ε-amines and α-glutamyl residues among amino acids 12. This activity leads to the cross-linking of extracellular matrix (ECM) proteins thereby increasing the deposition 13 of such proteins and their resistance to proteolytic enzymes, which leads to tissue fibrosis 1415. Several studies specifically described the role of tTG in cross-linking of fibronectin, osteonectin, osteopontin, laminin and other extracellular matrix components 12.

The pathogenesis of liver fibrosis is not clear; however, it was suggested that an increase of ROS coupled with a decrease in body antioxidant system activity play an important role in the pathological changes, particularly in the cases of radiation exposure and liver toxicity 16. Radiation exposure may cause disruption of normal cell membranes as a result of direct interaction of radiation with cellular membranes or through the action of free radicals produced by radiation 17.

Several endogenous protective mechanisms may limit ROS and the damage caused by them 18. However, this protection may be insufficient. When the formation of ROS is excessive, additional protective mechanisms of dietary, antioxidants may help maintain liver functions. Several natural antioxidants were proposed to prevent and treat hepatopathies induced by oxidative stress 19. Rich in flavonoids, Fructus Piperis Longi (Bibo, long pepper) is used in Chinese medicine to treat various conditions such as jaundice and allergy 20. It has been demonstrated to be anti-tussive, anti-asthmatic, anti-allergic, anti-tubercular, antipyretic, hypotensive, hypoglycemic, antihelmentic and coronary vasodilatory 21. The aim of the present study is to investigate the hepatoprotective activity of Fructus Piperis Longi against liver fibrosis.

Administration of Fructus Piperis Longi ethanol extract (Plf ext) to rats, by force-feeding, for a period of five weeks, did not show significant changes in all the studied parameters, indicating that the extract did not affect the liver functions (Tables 1, 2, 3, 4, 5).

As shown in Table 1, TAA significantly increased (P = 0.0001) liver collagen content and tTG activity. Irradiated rats showed significantly increased liver collagen content (P = 0.019) and tTG activity (P = 0.0001). In the TAA + irradiation group, significant increases (P = 0.0001) in liver collagen content and tTG activity were observed. Treatment of Plf ext significantly ameliorated (P = 0.0001, no significance, 0.0001, 0.0001, 0.0001 and 0.0001 respectively) the increase of collagen content and tTG activities in the rats that received TAA or γ-irradiation or both (Table 1).

TAA induced significant decreases (P = 0.0001) in liver GSH content, SOD and Cat activities (Table 2), which were parallel to significant increases in LP (P = 0.0001), LPH (P = 0.003) and CD (P = 0.0001) content (Table 3). Irradiated rats showed significant decreases (P = 0.0001) in liver GSH content and Cat activity in association with significant increases in LP (P = 0.0001), LPH (P = 0.008) and CD (P = 0.002) content (Tables 2 and 3). In the TAA + irradiation group, significant decreases (P = 0.0001) in liver GSH content, SOD and Cat activities in association with significant increases in LP (P = 0.0001), LPH (P = 0.001) and CD (P = 0.0001) content were observed. Treatment of Plf ext significantly reduced (P = 0.0001) oxidative stress in the rats that received TAA or γ-irradiation or both.

Significant increases (P = 0.0001) of plasma ALT, AST, ALP and GGT activities were observed in rats that received TAA or γ-irradiation or both. Treatment of Plf ext ameliorated (P = 0.0001) these increases (Table 4).

Significant increases (P = 0.0001) in the content of total, direct and indirect bilirubin were observed in the rats that received TAA or γ-irradiation or both. Treatment of Plf ext ameliorated (P = 0.0001) these increases (Table 5).

Figure 1 shows a significant increase in the liver weight of the rats that received TAA or γ-irradiation or both. Administration of Plf ext did not significantly ameliorate liver weight.

In the present study, parameters of liver fibrosis induced by TAA with or without radiation exposure were shown as an increase of liver weight, significant increases in liver tTG activity and collagen content associated with significant decreases in GSH content, SOD and Cat activities and increases in LP, LHP and CD content. Hepatic damage was indicated by significant increases of serum AST, ALT, ALP activities and bilirubin content.

The increase in tTG activity may be attributed to the increased binding of the nuclear factor-kappaB (NF-κB) to the NF-κB motif of the tTG promoter, where tTG gene expression increases during hepatic injury and fibrosis 38. The concomitant increase of both hepatic collagen and tTG activity may be explained by the dual effect exerted by the NF-κB, which is induced by oxidative stress 39. Nevertheless, the association between tTG activity and fibrosis may involve other factors such as the factor-beta (TGF-β), major fibrogenic growth factors, where tTG activates the latent TGF-μ1, which in turn leads to de novo synthesis of tTG 40. The increase of tTG activity may also be a consequence of GSH depletion and mitochondrial dysfunction 41.

The depletion in GSH content may be due to the oxidation of sulfhydryl group and diminished activity of glutathione reductase 42. The significant decrease in the activity of antioxidant enzymes may be caused by cell membrane damage and alterations in dynamic permeability of membranes due to peroxidation, followed by the release of intracellular enzymes to the blood stream 43. In addition, an excess of ^•OH causes oxidative damage to enzymes, resulting in the modification of their activities 4344. The marked increase in MDA levels is likely to be a result of the inactivation of scavenger enzymes induced by ROS 444546. According to Ueda et al. 47, the generation of lipid peroxide and its appearance in the animal's liver may be a result of a chain of reactions or may be initiated by an indirect mechanism that enables the escape from anti-oxidation.

Fructus Piperis Longi has recently been proposed as a chemopreventive agent for its antioxidant activities 4849. The present study showed that treatment of Plf ext significantly reduced liver fibrosis as evidenced by significant decreases of tTG activity and collagen content, with concomitant enhancement of the antioxidant status and improvement of liver functions. The results are consistent with other reports on the role of polyphenols against oxidative stress 39 because Plf ext is rich in polyphenols 50 which may up-regulate the antioxidant 515253, thereby decreasing the free radical-induced lipid peroxidation 51.

The increased resistance of liver tissue against liver fibrosis and oxidative stress after treatment was shown by the significant decreases in serum ALT, ALP and AST activities and bilirubin content. The results are consistent with a previous study which demonstrated that the ethanolic extract of Fructus Piperis Longi possessed hepatoprotective activity lowering serum enzymes ALT and AST 54. Further studies NF-κB, tTG gene expression and TGF-B would help elucidate the mechanism of action

Treatment of the ethanolic extract of Fructus Piperis Longi ameliorated the increase of the activity of tTG enzyme and enhanced the antioxidant activities in fibrotic liver.

tTG: transglutaminase; Plf ext: Fructus Piperis Longi ethanol extract; TAA: thioacetamide; LP: lipid peroxides; LHP: lipid hydroperoxides; CD: conjugated dienes; GSH: reduced glutathione; SOD: superoxide dismutase; Cat: catalase; AST: aspartateaminotransaminase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; GGT: gamma-glutamyltransferase; ROS: reactive oxygen species; SD: standard deviation; NF-κB: nuclear factor-kappaB; TGF-β: tumour growth factor-beta; Abs₂₃₄: Absorbance at 234 nm.

The authors declare that they have no competing interests.

SZM designed the study, supervised the experiments, prepared Plf ext and wrote the manuscript. HEK performed the animal experiments. Both authors supervised the research assistants to carry out clinical chemistry assays. Both authors read and approved the final manuscript.