INTRODUCTION

Liver plays important roles in metabolism, detoxification, secretion, and storage (^{Wallace et al., 2008}). Hepatotoxicity implies chemical or drug induced liver damage that is considered as a cause of acute and chronic liver diseases (^{Ostapowicz et al., 2002}). Histological patterns of hepatotoxicity vary from zonal necrosis, hepatitis, fibrosis, and neoplasm (^{Mumoli et al., 2006}).

Thioacetamide (TAA) is an organo-sulfur compound that is well known as hepatotoxin (^{Aydin et al., 2010}). It is commonly used as a fungicide to control the decay of citrus fruits especially orange (^{Lee et al., 2003}), as organic solvent in leather industry (^{Pohanish, 2012}) and in preparation of nano-composites (^{Housková et al., 2007}). So, potential occupational exposure to TAA may occur via dermal contact and/or inhalation (Al-Attar, 2011). Model of TAA induced liver injury is widely used to study the acute hepatotoxicity and chronic hepatitis and fibrosis (^{Bataller & Brenner, 2005}).

As the World Health Organization reported lack of safe modern drugs (^{Ayyanar et al., 2008}), there is a considerable interest in finding natural drugs for the treatment of liver diseases to be added in foods or medicinal materials instead of synthetic ones.

Probiotics are beneficial live microorganisms to health if administered in adequate amount (^{Sanders et al., 2014}). For animals, they are used as therapeutic, prophylactic and growth promoters (^{Anukam et al., 2006}). Recently, the intake of probiotics is recommended for those have disturbance of liver enzymes activities (^{Takei, 2013}).

Silymarin is one of the promising herbs showing activity in treating liver diseases (^{Stickel & Schuppan, 2007}). It has anti-oxidative, anti-inflammatory, anti-fibrotic, and liver regenerative effect (^{Thyagarajan et al., 2002}).

So, the present study aimed to evaluate the ameliorative potential of probiotics and/or silymarin in treating TAA induced hepatotoxicity in rats via histological and immunohistochemical assessment.

MATERIAL AND METHOD

RESULTS

Histological findings. Liver obtained from control negative animals revealed normal hepatic architecture where normal hepatocytes were arranged into radiating hepatic cords from the central veins. Besides, normal blood sinusoids were enclosed in between hepatic cords (Fig. 1A).

Liver sections of group II showed an abnormal histological feature. Numerous and thick interlobular connective tissue septa were identified (Fig. 1B). The stained sections with Masson's trichrome demonstrated pseudolobular parenchyma that was surrounded by obvious collagen fibers of the interlobular septa (Fig. 1C) in addition, it was infiltrated with inflammatory cells (Fig. 1D). Congestion of blood sinusoids and Macrophagocytus stellatus (Kupffer cells) proliferation were detected in between the hepatocytes that were degenerated and showed cytoplasmic vacuolation (Fig. 1D). The portal area manifested severe congestion of the portal vein, inflammatory cells infiltration (Fig. 1E), and fibrosis around the bile duct (Fig. 1F).

Fig. 1 Representative photomicrographs of livers obtained from control negative group (A) and thioacetamide treated group (B-F). CV, central vein; arrowhead, blood sinusoids; long arrow, interlobular septa; short arrow, inflammatory cells; PV, portal vein; BD, bile duct. All sections stained with H&E except Figs. C and F stained with Masson's trichrome stain. Scale bars: (A)= 100 µm; (B, C)= 200 µm; (D-F)= 50 µm. 

Liver sections of group III still showed detectable interlobular connective tissue septa (Figs. 2A, B) but, fewer and thinner than livers sections of positive control group. Slight reductions of fibrous tissue were detected within the portal area in comparison to positive control group (Figs. 2C, D). Moreover, congestion of the central veins (Fig. 2A) and portal vein and inflammatory cells infiltration (Fig. 2C) were still present. Also, the hepatocytes showed degeneration with cytoplasmic vacuolation (Fig. 2C).

Fig. 2 Representative photomicrographs of livers obtained from probiotics treated group (A-D). Long arrow, interlobular septa; CV, central vein; short arrow, inflammatory cells; PV, portal vein. All sections stained with H&E except Fig. D stained with Masson's trichrome stain. Scale bars: (A, B)= 200 µm; (C, D)= 50 µm. 

Liver section taken from group IV revealed no nodular parenchyma due to absence of detectable fibrous septa (Fig. 3A). The fibrous deposition was decreased and concentrated only in the portal area (Fig. 3B). Improvement of the hepatocytes was noticed especially around the central veins as well as few numbers of Macrophagocytus stellatus (Kupffer cells) were detected compared to positive control group but congestion of blood sinusoids was still present (Fig. 3C).

Fig. 3 Representative photomicrographs of livers obtained from silymarin treated group (A-C). PV, portal vein; CV, central vein; blood sinusoids, arrowhead. All sections stained with H&E except Fig. B stained with Masson's trichrome stain. Scale bars: (A, B)= 200 mm; (C)= 50 mm. 

Liver sections obtained from group V showed nearly normal feature of the liver, where there was no identified interlobular septa (Fig. 4A) but, only few amount of fibrous deposition within the portal area was identified (Fig. 4B). The hepatocytes showed improvement where the cells were arranged into normal pattern of hepatic cords (Fig. 4A) as well as, the hepatocytic cytoplasm showed no vacuolation especially those around the central veins (Fig. 4C). Congestion of blood sinusoids (Figs. 4A, C) and portal vein (Fig. 4B) were still identified.

Fig. 4 Representative photomicrographs of livers obtained from probiotics and silymarin treated group (A-C). PV, portal vein; CV, central vein; blood sinusoids, arrowhead. All sections stained with H&E except Fig. B stained with Masson's trichrome stain. Scale bars: (A, B)= 200 µm; (C)= 50 µm. 

Immunohistochemical findings. Ki-67 immunostaining was localized in the nuclei of the positive cells. The liver of control negative group showed few Ki-67 immunopositive cells (Fig. 5A), while the liver from probiotic and/or silymarin treated groups (Figs. 5C-E) revealed more Ki-67 immunopositive cells with mitotic figures compared to those of the control negative (Fig. 5A) and TAA groups (Fig. 5B).

Fig. 5 Ki-67 immunohistochemical expression in livers from control negative group (A), thioacetmide treated group (B), probiotics-treated group (C), silymarin treated group (D) and probiotics and silymarin treated group (E). Positive ki-67 immunostaining (arrows). Scale bars = 50µmm. 

Caspase 3 immunostaining was localized in the cytoplasm of the positive cells. In the TAA intoxicated liver, immunopositive cells were higher in number and showed more intense staining (Fig. 6B) than those in the control negative (Fig. 6A) and the other treated groups (Figs. 6C-E). The most affected hepatocytes were those around the central veins as they showed intense caspase 3 immunostaining (Fig. 6B).

Fig. 6 Caspase 3 immunohistochemical expression in livers from control group (A), thioacetmide treated group (B), probiotics-treated group (C), silymarin treated group (D) and probiotics and silymarin treated group (E). Positive caspase 3 immunostaining (arrows). Scale bars = 50 µm.silymarin treated group (E). Positive caspase 3 immunostaining (arrows). Scale bars = 50 µm. 

DISCUSSION

The current study showed abnormal histological features of the liver of rats treated with TAA in comparison to normal liver. ^{Sadasivan et al. (2006}), described the hepatic damage, after TAA administration, ranging from parenchymal cell necrosis and liver cell proliferation, to the production of pseudolobules and nodular cirrhosis. In our study, liver of rats treated with TAA showed degeneration of hepatocytes with vacuolar cytoplasm, and periportal inflammatory cell infiltration with intense centrilobular necrosis, and congestion of blood sinusoids and central veins that was similar to finding of ^{Chen et al. (2008}). Also, pseudolobular hepatic parenchyma and numerous thick interlobular septa were detected in our study that was in consistence with Al-Attar, (2011). In our study, fibrosis of the portal area, inflammatory cells infiltration, and Macrophagocytus stellatus (Kupffer cells) proliferation were noticeable and identical to findings of ^{Nada et al. (2015}) and ^{Wang et al. (2016}a). Both inflammatory and Kupffer cells play a critical role in the regulation of liver inflammation (^{Wynn & Barron, 2010}). Additionally, Macrophagocytus stellatus (Kupffer cells) release fibrogenic transforming growth factor (TGF-β1) leading to collagen deposition (Bataller & Brenner). These marked hepatotoxic effect of TAA is not for the compound itself but due to its metabolites (TAA-S-dioxide) which initiates necrosis (^{Yogalakshmi et al., 2010}) and bind to other intracellular molecules causing oxidative stress (Wang et al., 2016b) generating reactive oxygen species (ROS) responsible for oxidative stress (^{Pallottini et al., 2006}).

In probiotic treated group, the hepatic damage caused by TAA was slightly improved as well as, a slight reduction of fibrous tissue deposition was noticed compared to TAA group. However, ^{D'Argenio et al. (2013}) reported that FB (L. paracasei B21060, L- glutamine, Arabinogalactan and Xilooligosaccharides) has a significant marked reduction effect on liver fibrosis in rats injected with ccl4. Also, Velayudham et al. and ^{Gupta et al. (2013}) found that probiotics reduce the progression of fibrosis in a model of steatohepatitis in rats. This variety in the effectiveness of probiotics in the different studies may depend on formulation of probiotics (^{Paolella et al., 2014}). Furthermore, Sanders et al. stated that different doses, strain combinations, and other components like, prebiotics may be affect the health outcomes of probiotics. Both ^{Tilg & Hotamisligil (2006}) and Takei reported the hepatoprotective effect of probiotics intake on liver activity in probiotics treated group compared to TAA-treated group. This improvement may occur as probiotics prevent the production and/uptake of lipopolysaccharides in the gut therefore, reducing levels of low grade inflammation (^{Gratz et al., 2010}).

Silymarin treated group showed almost normal hepatic parenchyma without appearance of pseudolobulation. This finding indicates the ability of silymarin to inhibit the stage of pseudolobule formation and decrease the amount of fibrous tissue that was supported by ^{Popescu et al. (2010}) and ^{Fatima et al. (2013}). This effect occurs due to silymarin inhibits transformation of stellate cells into myofibroblasts reducing the collagen fibers deposition (^{Abdo et al., 2016}). From previous studies, we can conclude that silymarin has a good recovery effect on fibrosis regardless the causative agents where Fatima et al. stated the effect of silmaryin of fibrosis induced by TAA while Popescu et al., stated that in ccl4 and alcohol induced fibrosis. Also, improvement of the hepatocytes was noticed where the vacuolar degeneration and inflammatory cells infiltration were decreased that was in consistence with ^{Amin et al. (2012}). This improvement was due to the effects of silymarin as cell membrane stabilizer, permeability regulator that prevents hepatotoxic agents from entering hepatocytes, promoter of ribosomal RNA synthesis, and enhancer of liver regeneration (^{El Mesallamy et al., 2011}; Abdo et al.) as well as, it inhibits the cytochrome p450 detoxification system and prevents the metabolism of toxic TAA (^{Papetti et al., 2002}). Beside its antioxidant properties (^{Gazák et al., 2007}), silymarin has anti-fibrotic and anti-inflammatory effects (^{Karimi et al., 2011}).

The combination of probiotics and silymarin in treatment of TAA induced hepatotoxicity showed more improvement of the histological structure of the liver than treatment with probiotics or silymarin alone. No interlobular septa, normal pattern of hepatic cords, and normal hepatocytes especially around the central veins were detected. However, few fibrous tissues within the portal area, vacuolar degeneration in hepatocytes close to portal area, and congestion of portal vein were still identified. It was interesting that, we can conclude that combination of probiotics and silymarin potentiate each other as anti-inflammatory, antioxidant and anti-fibrotic agents.

To assess the amelioration potential of probiotics and/ or silymarin on treatment and recovery of TAA intoxicated liver, immunohistochemical staining of Ki-67 and caspase 3 was performed to evaluate the proliferative and apoptotic status of the hepatocytes.

In the current work, the limited number of Ki-67 immunopositive cells in the liver of control group was in agreement with ^{Youssef et al. (2012}), which reflects a normal homeostasis turnover of the liver. In TAA intoxicated liver, hepatocytes shows low proliferative capacity where their replication activity is reached after many cycles of injury and repair (^{Trak-Smayra et al., 2004}). Also, ^{Clouston et al. (2005}), indicated that proliferation of hepatocytes and liver regeneration are suppressed in fibrotic injured livers compared to non-fibrotic ones. The significantly higher numbers of Ki-67 immunopositive cells in probiotics and/or silymarin treated groups compared to control and TAA groups was supported by ^{Tani et al. (2001}), who revealed suppressed cellular proliferation in liver of rats intoxicated with TAA. Such finding suggests that probiotics and/or silymarin enhance the regeneration of TAA-intoxicated livers via increasing the expression of ki-67 (proliferation marker) that is critical tool in evaluation of liver regeneration (^{Gerlach et al., 1997}; ^{Pizem et al., 2001}). On contrary, ^{Arffa et al. (2016}), stated that livers of TAA intoxicated rats have increased ki-67 expression than treated groups. The findings of ki-67 immunolabeling were interesting as it revealed a higher regeneration activity of using silymarin and combination of silymarin with probiotics in treatment of TAA intoxicated liver than using probiotics alone. This result matches the histological findings suggesting that silymarin exerts more potent effect on liver regeneration predominantly through enhancing the proliferation of hepatocytes.

It is common that TAA administration in rats induces hepatic cell necrosis and apoptosis promoting fibrosis (^{Wang et al., 2016}b) therefore, we used caspase 3 immunostaining as a tool for apoptosis detection. Our results demonstrated more intense caspase 3 immunostaining in hepatocytes around the central veins indicating a centrilobular necrosis of the liver intoxicated by TAA as mentioned by ^{Omneia et al. (2014}). Inactivation of caspases protects hepatocytes against apoptosis, reduces cytokines involved in inflammatory signaling, and ameliorates fibrogenesis (^{Dixon et al., 2012}; ^{Thapaliya et al., 2014}). It was interesting that probiotics and/ or silymarin have apoptosis inhibitor effect where the livers from probiotics and/or silymarin treated groups revealed low apoptotic rats compared to TAA intoxicated group. In contrast to our results, ^{Kadir et al. (2013}) detected low apoptotic rate immunohistochemically in liver of TAA group.

Stimulation of hepatocytes proliferation and inhibition of apoptosis are tightly correlated with the restoration of liver structure. Our results showed an increase of Ki-67 expression (marked as liver regeneration) but a decrease of caspase 3 expression (marked as liver apoptosis) was detected in livers of rats treated with probiotics and/or silymarin treatment than TAA intoxicated rats.

CONCLUSION

The current work reveals the ameliorative potential effect of probiotics and/or silymarin on the structure of TAA intoxicated livers in rats through enhancing proliferation and reducing apoptosis of hepatocytes in rats. Also, it was remarkable that treatment with silymarin and combination of probiotics and silymarin are more potent as antifibrotic agents than using probiotics alone that was proved histologically and immunohistochemically.