Sulphasalazine Prevents Fibrosis; Relevance of TGF β RI

Aims: The aim of this study is to investigate the protective effect of sulphasalazine on liver fibrosis. Besides; investigation of the expression pattern of transforming growth factor β receptor I (TGF β RI) in liver fibrosis and the possible modulatory effect of sulphasalazine on it. Study Design: Controlled experiment. Methodology: Five Sprague-Dawley rats groups were used for the experiment. Control group: Receiving corn oil; DMSO group: Injected with DMSO; 6 weeks group: Injected with 50% CCl 4 in corn oil; sulphasalazine group: Injected with sulphasalazine and CCl 4 and finally mesalazine group: were given mesalazine and CCl 4 . On the day after the last dose, rats were anesthetized with diethyl ether and blood samples were collected for measurement of blood chemistry. The animals then were euthanized, and livers were harvested and divided into 2 parts; one part was processed for standard histology and immunofluorescence techniques and the other was homogenized for oxidative status assessment. Results: TGF β RI has been shown to be upregulated in chronic liver injury; fibrosis stage; with expression occurring mainly in cell membrane of lesion area. This expression was decreased significantly with sulphasalazine treatment. Sulphasalazine has shown to have ability to diminish fibrosis in chronic liver injury. This decrease in fibrosis observed with sulphasalazine was in parallel with decrease in TGF β RI expression. Besides; this action of sulphasalazine has been suggested to be due to the whole molecule not to its moiety; mesalazine. Conclusion: TGF β RI may be used as a candidate marker for diagnosis and prognosis of chronic liver diseases. Besides; it may be used as a target therapy for chronic liver diseases. Moreover; sulphasalazine might be a promising adjuvant therapy for chronic liver diseases.


INTRODUCTION
Liver plays an unlimited role in the preservation and body homeostasis regulation. It is included in most biochemical pathways to growth, protection against disease, nutrient fund, energy facility and reproduction [1].Hepatotoxicity can be generated by certain common causes including therapeutic agents, natural chemicals, laboratory and manufacturing agents and herbal therapies [2].
Carbon tetra chloride (CCl 4 ) is one of the most communal models for inducing hepatotoxicity. It is transformed into a toxic CCl 3 radical by hepatic cytochrome P 450 2E1 (CYP2E1). Thus; it brings an acute Centro-lobular necrosis which starts a wound healing response (fibrosis) [5]. However, Contact to these chemicals in humans is rare and generally occurs in the manufacturing during fabrication and in places where these chemicals is usually used [6].
Hepatic stellate cells are the most important player in hepatic injury. Quiescent HSCs are characterized by significant desmin expression and vitamin A storage. Following liver injury, HSCs lose their vitamin A content, rise the expression of α-smooth muscle actin (α-SMA) and gain a myofibroblast-like phenotype [7].
These events are primarily triggered by mediators released by activated Kupffer cell and injured hepatocytes [8].
Sulfasalazine is a synthetic drug obtained from the grouping of sulfapyridine and 5-aminosalicylic acid (mesalazine), an antibiotic and an antiinflammatory agent, respectively. This drug is usually used in the inflammatory diseases of the large intestine and rheumatoid arthritis [9] Despite great knowledge about liver fibrosis, there are little drugs approved for management of it. So the aim of this work is to investigate the possible protective effect of sulphasalazine on liver fibrosis; an aspect that has gone side by side with investigation of expression pattern of TGFβRI in liver fibrosis and investigation of the possible modulatory effect of sulphasalazine on it.

Animal Model
Adult male Sprague-Dawley (SD) rats weighing 250-300 g and aging 70 days were used in the current study. The animals were obtained from the breeding colony maintained at the animal house of the Nile Company for pharmaceuticals, Cairo, Egypt. They were housed in the animal facility of Faculty of Pharmacy, Al-Azhar University in 20 X 18 X 25 cm plastic cages with stainless steel wire lids and mesh floor with 5 animals per cage. They were kept at 23±1°C, 55% relative humidity with 12:12-h light: Dark cycle. They were maintained on a standard rodent chow (El-Nasr Company, Abou-Zaabal, Cairo, Egypt) and allowed to food and water ad libitum. Animals were randomly divided into five groups; (1) control group: 9 rats received corn oil (2 mg/kg/twiceweekly/intraperitoneal(IP)/ repeated for 6 weeks); (2)DMSO group: 12 rats were injected with DMSO, the vehicle of sulphasalazine (1 ml/kg/day/IP/repeated for 6 weeks) and 50% CCl 4 in corn oil (4 ml/kg/IP/twice weekly/repeated for 6 weeks); (3) 6 weeks group:12 rats were injected with 50% CCl 4 in corn oil (4 ml/kg/IP/twice weekly/repeated for 6 weeks); (4) sulphasalazine group: 12 rats were injected with sulphasalazine (75 mg/kg/day/IP/repeated for 6 weeks) [10] and CCl 4 as previously indicated in 6 weeks group and (5) mesalazine group: 12 rats were given mesalazine (100 mg/kg/day/orally by oral gavage/repeated for 6 weeks) [11] and CCl 4 as previously indicated in 6 weeks group. At the end of experimental period, rats were anesthetized with diethyl ether and blood samples have been drawn from retro orbital plexus for measurement of blood chemistry. The animals then were euthanized, and tissue samples from the livers were harvested and divided into 2 parts; one part was processed by standard histology and immunofluorescence techniques and the other was homogenized in 0.15 M KCl for oxidative stress assay. All animal procedures were performed in accordance with the international guide for the care and use of laboratory animals [12].

Biochemical Analysis
Serum enzymatic activities of transaminases (ALT and AST) were estimated by kinetic method according to the method of international federation of clinical chemistry (IFCC) [13,14]. Alkaline phosphatase (ALP) activity was assayed according to the method of IFCC [15-17] and serum albumin concentration according to the method described by Gendler [18]. The liver homogenate was used for the determination of the oxidative stress parameters. The level of thiobarbituric acid-reactive substances was assayed as malondialdehyde (MDA) as described by Mihara and Uchiyama [19]. The activity of SOD was determined using the method described by Marklund [20].

Immunofluorescence Analysis
Liver tissues sections were handled according to method described by Abdel-Bakky et al. [21]. The primary TGFβR1 antibody was diluted in blocking solution in the suitable dilution (1:400) and left overnight in 4°C. Secondary antibody (cyanine red conjugated) diluted in the blocking solution was incubated for 30 min and the nuclei were counterstained using DAPI. Finally, all slides were mounted with the fluoromount solution, covered by covering slips, and allowed to stand for detection by immunofluorescence microscope (Leica DM 5500B).

Histopathological Examination
Liver samples were taken from rats belonging to the different groups and fixed in 10% neutral buffered formalin for 24 hours. Washing was done in tap water, and then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven for 24 h. Paraffin wax tissue blocks were prepared for sectioning at 4 microns thickness by sledge microtome.
The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin & eosin stain and then examination was done through a light electric microscope [22]. Histopathological grading was achieved by subjective scoring approved by pathology department, faculty of pharmacy, Al-Azhar university, Cairo, Egypt with assistance of attached clues.

Statistical Analysis
Data were presented as the mean ±SE. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer as a post test, according to the number. of groups. The 0.05 level of probability was used as the criterion of significance using GraphPad Prism software version 5 (GraphPad Software Inc, CA, USA).

Liver Functions and Oxidative Status Assessment
To assess liver functions; we have performed liver function tests including serum transaminases, ALP, and albumin. The oxidative status of liver tissues has been determined through MDA and SOD assay as indicated in Table 1 and Table 2.
As indicated in Table 1 and Table 2; Serum ALT and AST activity of control group rats were 46.6±2.85 and 54.1±2.65 respectively. Administration of CCl 4 I.P. alone or combined with DMSO for 6 weeks significantly increased ALT and AST activity compared to control group. Interestingly concomitant administration of CCl 4 with sulphasalazine reduced serum activity of AST compared to DMSO group.
As shown in Table 1 and Table 2; ALP activity of control group rats was 312±21.9. Administration of CCl 4 I.P. for 6 weeks increased ALP significantly by 83% if given only and by 86% in combination with DMSO compared to control group. Concomitant administration of sulphasalazine with CCl 4 decreased ALP activity by 10% compared to DMSO group.
Serum albumin of control group was 3.81±0.08 g/dl. A significant decrease was observed only in mesalazine group as shown in Table 1 and  Table 2.
As shown in Table 1 and Table 2; the hepatic MDA content of control group was 4.73±0.318 nmol/g tissue. This content was significantly increased on CCl 4 treated groups to be 31.9±1.44 (nmol/g tissue) for 6 weeks group, 31.7±1.82 (nmol/g tissue) for DMSO group and 25.7±1.5 (nmol/g tissue) for sulphasalazine group. On the other hand; combination of CCl 4 with mesalazine showed MDA content of 9.86±0.339 respectively; results that showed significant decrease compared to 6 weeks CCl 4 treatment although they appeared nonsignificantly changed from control group. Finally sulphasalazine showed a significant decrease in MDA content compared to DMSO group. Table 1 and Table 2 show the M ± SEM for SOD of studied groups. The control group showed tissue SOD activity of 526±25.2 U/mg tissue. This activity was significantly decreased in other groups to become 242±15.4, 223±14, 281±16 and 225±9.65 for DMSO, 6 weeks, sulphasalazine, and mesalazine groups. Fig. 1 and Graph 1 show that TGFβR1 protein showed minimal expression in normal liver tissues. The tissue expression was showed to be maximal in 6 weeks CCl 4 groups. It is noted that the expression increased by about 146% in 6 weeks CCl 4 treatment group. It is also observed that the expression was located in the cell membrane of epithelial hepatic tissue in areas that shows great lesions.

Fig. 1. Immunofluorescence staining of liver sections of studied groups showing minimal TGFβR1 expression in control group. Six weeks CCl 4 treated group shows maximal expression located mainly in cell membrane (yellow rectangle). It is noted also that there is non-significant expression change between 6 weeks CCl 4 treatment alone or combined with DMSO. Combination of CCl 4 with sulphasalazine or mesalazine show decrease in the expression compared to 6 weeks CCl 4 treatment with maximal decrease occurring with sulphasalazine
It is also noted that concomitant administration of DMSO and CCl 4 for 6 weeks produced nonsignificant change from CCl 4 only. On the other hand; Concomitant administration of sulphasalazine and CCl 4 decreased the expression by about 46% compared to DMSO group while concomitant administration of mesalazine with CCl 4 reduced the expression by about 36% compared to 6 weeks CCl 4 group.

Histopathological Findings
Effects of CCl 4 treatment on histopathological findings of liver tissue are represented in Table 3 and Fig. 2; the control group shows normal hepatic architecture. On contrast, six weeks CCl 4 treatment shows expanded portal tract with fibrous septa extending from a portal tract to another (yellow arrows), marked micro-and macro-vesicular steatosis. The hepatocytes exhibit some aspects of single cell necrosis with dilated central veins and mild interface activity.
It is noted that sulphasalazine had minimal effect on steatosis. Interestingly; despite sulphasalazine group animals show marked steatosis, they also show completely diminished underlying fibrosis in their liver tissues.
Finally; combination of mesalazine with CCl 4 results in some alleviation in injury signs. However; it seems not to have positive effects regarding steatosis or fibrosis.

DISCUSSION
Administration of I.P. CCl 4 for 6 weeks has significantly elevated serum transaminases enzymes activities suggesting hepatocellular damage. These results agreed with previous reports that CCl 4 significantly increases serum transaminases [23][24][25]. This CCl 4 induced hepatic damage has been reported to be due to oxidative stress [26,27]. Concomitant administration of sulphasalazine with CCl 4 has reduced AST not ALT activity compared to DMSO group suggesting the positive effect toward liver injury. These effects might be due to the anti-inflammatory action of sulphasalazine.
This disagrees with what has been reported by Jennings and his coworkers a result that was interpreted by the relationship of hepatic injury, steatosis, fibrosis and cirrhosis to ulcerative colitis not sulphasalazine medication [28].
ALP activity caused by IP CCl 4 administration for 6 weeks and DMSO group has showed significant increase compared to control which was less than 3 times as control; a result that might suggest hepatotoxicity and mild biliary toxicity. This matches with that previously reported by Posen and Doherty [29]. Concomitant administration of mesalazine and CCl 4 for 6 weeks has significantly elevated ALP activity compared to 6 weeks CCl 4 treatment.
This result might suggest the toxic effect of mesalazine with the mentioned regimen on hepatic tissue or synergistic effect of mesalazine on CCl 4 toxicity; a suggestion that might be clear upon ALP and albumin result.
Meanwhile; it was clarified that free radicals production and oxidative stress are main players in liver injury especially CCl 4 induced liver injury [26,27]. In our study; administration of CCl significantly elevated tissue content of MDA and reduced serum SOD activity. These results  It has been found that concomitant administration of sulphasalazine and CCl 4 had markedly cleared fibrosis; major findings seen in CCl 4 . However, there is no positive effect on steatosis. This may be an expected result according to in-vitro study reports of Oakley et al. [33] concerning the inhibitory effect of sulphasalazine on the machinery and machinery of fibrous tissue synthesis.
As an interesting matter; the decrease in histopathological scores observed in mesalazine group didn't agree with sulphasalazine group although it was documented that sulphasalazine is a prodrug for mesalazine in some pharmacological actions [34]. This might suggest that the action of sulphasalazine previously reported by Oakley et al. [33] may be related to the sulphasalazine molecule at all not related to its moiety, mesalazine.
The TGF-β is the principal regulator in chronic liver injury sharing in all stages of disease progression [35]. Its action begins by binding to its receptor TGFβRI and TGFβRII [36]. It has been observed that TGFβR1expression occurred in the cell membrane. This agrees with Massague and Chen reports [37]. It has been also shown that TGFβR1 expression was upregulated only in chronic liver toxicity; specifically phases that showed some extent of tissue remodeling prescribed by Devaux et al. [38] suggesting involvement of TGFβR1in cellular processes involved in chronic not acute liver injury. This was similar to the previous reports in myocardial infarction (MI) by Devaux et al. [38]. Besides; they also showed that the maximal expression occurs in lesion areas suggesting the direct relationship between TGFβR1expression and lesions grade; a result that also is quite similar to Devaux et al. [38]. It was also observed that TGFβR1expression was nearly diminished in sulphasalazine group suggesting the tight linkage between TGFβR1 expression and fibrosis but not steatosis. This result can be explained by pro apoptotic effect of sulphasalazine toward activated HSC, the major cellular promoter of fibrogenesis [39].

CONCLUSION
Our findings indicate, for the first time, that TGFβR1 content is increased in chronic liver injury. TGFβR1 is upregulated in injury combined with underlying fibrosis; a result that may suggest usage of TGFβR1 as a candidate marker for diagnosis and prognosis of chronic liver diseases and a target for liver disease therapy. Moreover, sulphasalazine exhibits a fibroprotective effect in experimental liver fibrosis. This suggests a possible use of sulphasalazine as adjuvant in therapy of chronic liver diseases.