Zoledronic acid prevents the hepatic changes associated with high fat diet in rats; the potential role of mevalonic acid pathway in nonalcoholic steatohepatitis

European Journal of Pharmacology 858 (2019) 172469

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Zoledronic acid prevents the hepatic changes associated with high fat diet in rats; the potential role of mevalonic acid pathway in nonalcoholic steatohepatitis

T

Reham Hussein Mohameda,∗, Maha Tareka, Ghada Galal Hamamb, Samar F. Ezzatb a b

Department of Pharmacology, Faculty of Medicine, Ain Shams University, Cairo, Egypt Department of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, Cairo, Egypt

A R T I C LE I N FO

A B S T R A C T

Keywords: NASH Zoledronic acid Free cholesterol TNF-α VEGF

The role of hepatic free cholesterol (FC) in nonalcoholic steatohepatitis (NASH) is raised up and the intervention with cholesterol synthesis will be a potential therapeutic target. This study investigated the hepatoprotective effect of mevalonic acid pathway inhibition by Zoledronic acid (ZA) on the hepatic changes associated with high fat diet (HFD) in rats.Thirty two male Wistar rats were used. They were divided into 2 groups: (I) control naïve (II) NASH: induced by HFD for 12 weeks, this group is subdivided into (A) NASH untreated (B)NASH + ZA (50ug/kg/week) i.p. for 12 weeks (C) NASH + ZA (100μg/kg/week) i.p. for 12 weeks. Portal pressure (PP), liver enzymes AST and ALT, serum glucose, lipid profile, hepatic levels of tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), FC and triglyceride (TG), histopathological changes and expression of both hepatic alpha smooth muscle actin (α-SMA) and Caspase-3 were measured. ZA significantly prevented portal hypertension, worsening in liver function, and dyslipidemia. The hepatic levels of TNF-α, VEGF, FC and TG were significantly decreased in comparison to NASH untreated group. ZA hindered the histopathological changes induced by HFD. ZA inhibited the expression of hepatic α-SMA and Caspase-3 with significant difference favor the high dose intervention. ZA in a dose related manner prevents the hepatic pathological effects of chronic HFD ingestion in rats. This may be largely mediated by its ability to reduce TNF-α and hepatic FC.

1. Introduction Nonalcoholic steatohepatitis (NASH) is a chronic progressive liver disease, which is mainly characterized by fat accumulation and infiltration of abundant inflammatory cells in the liver)Powell et al., (1990). NASH has high risk for progression to cirrhosis which is the most common risk factor for hepatocellular carcinoma)Bugianesi (2007). Several studies suggested that disturbed hepatic cholesterol homeostasis and liver free cholesterol (FC) accumulation are important for the pathogenesis of NASH and strongly associated with the progression and severity of liver damage (Puri et al., 2007; Caballero et al., 2009; Ioannou, 2016). FC accumulation leads to liver injury in NASH through the activation of intracellular signaling pathways that influence Kupffer cells (KCs), stellate cells (HSCs) and hepatocytes. FC accumulation in KCs and HSCs correlates with their activation. Activation of KCs causes the secretion of pro-inflammatory mediators (e.g., tumor necrosis factoralpha (TNF-α) that influence neighboring cells and induces

∗

inflammation. TNF-α plays a major role in the pathogenesis of NASH. TNF-α is reported to be significantly increased in NASH patients and its Level is well correlated with progression to NASH (Kakino et al., 2017; Pessayre et al., 2002). In addition, FC accumulation in HSCs and their activation are considered major causes of accelerated liver fibrosis (Friedman, 2008; Carpino et al., 2004; Teratani et al., 2012). Finally, FC accumulation in hepatocytes induces itself lipid peroxidation and lipotoxicity leading to direct apoptosis and necrosis (Arguello et al., 2015). In NASH, there are liver architectural disturbance, with distortion of vascular architecture by fibrosis, scarring, regenerative nodules, and functional hepatic microcirculation alterations which lead to increase portal pressure (PP) and development of portal hypertension (PHT) (Bosch et al., 2003; Bosch et al., 2003). Moreover, angiogenesis plays an important role in the pathophysiology of PHT, and it is observed that vascular endothelial growth factor (VEGF) promotes an extensive neovascularization in the PHT (Fernandez, 2015). Portal hypertension, a major complication of advanced liver fibrosis can cause

Corresponding author. Faculty of medicine, Ain Shams University Abbasia, Cairo, Egypt. E-mail address: [email protected] (R.H. Mohamed).

https://doi.org/10.1016/j.ejphar.2019.172469 Received 20 February 2019; Received in revised form 15 June 2019; Accepted 16 June 2019 Available online 22 June 2019 0014-2999/ © 2019 Published by Elsevier B.V.

European Journal of Pharmacology 858 (2019) 172469

R.H. Mohamed, et al.

gastroesophageal variceal hemorrhage, hepatorenal syndrome and hepatic encephalopathy (Bosch et al., 2003; Marks and Harbord, 2013). Cholesterol is the major product of the mevalonate pathway in the liver. Farnesyldiphosphate synthase (FDPS) is one of key enzymes involved in mevalonate pathway (Healey et al., 2015; Okin and Medzhitov, 2016). Zoledronic acid (ZA), one of bisphosphonates is primarily used in the management of several bone disorders (Russell et al., 1999). Morever, ZA has been reported to be inhibitor of farnesyldiphosphate synthase (FDPS) in mevalonate pathway in bone cells (Luckman et al., 1998). The present study was conducted to investigate the effect of ZA on PP, liver function, lipid profile, hepatic levels of TNF-α, VEGF, FC and TG and histopathological changes in Wistar rats fed on HFD.

AD instrument) pressure transducers connected to Computer which contained Lab Chart Software. This software was used to analyze portal venous pressure recordings.

2. Materials and methods

2.4.2.1. Determination of serum ALT and AST levels. The serum ALT and AST levels were measured by kits purchased from Diamond Diagnostic (Cairo, Egypt).

2.4.2. Biochemical and Molecular Studies After scarification of animals, blood was withdrawn from each rat from the retro orbital vein into tube and let about half an hour to clot then, centrifuged for 20 min at 1500 g to get serum samples. Livers were dissected. Right lobe of the liver was removed, cut into longitudinal sections 2-4 mm in thickness and kept in 10% formalin for histopathological and immunohistochemical examination. The other lobe kept freezed at −80 for Molecular Studies. The samples were assessed for the following parameters:

2.1. Chemicals & drugs Zoledronic acid supplied as a white powder dissolved in saline 0.9%, cholesterol and bile salts were purchased from sigma Chemical Company, Cairo, Egypt.

2.4.2.2. Measurement of lipid profile and serum glucose levels. Rats were fasted for 12 h before scarification for measurement of:

• Serum total cholesterol, triglycerides and HDL levels were measured

2.2. Experimental animals Thirty two male Wistar rats aged 9 weeks and weighed 150–200 g. Rats were purchased from National Research Institute (Cairo, Egypt) were housed in an animal room with temperature (22 °C) and lighting (12 h (light) – 12 h (dark) cycle) control. An adaptation period of 1 week was allowed before initiation of the experimental protocol. All animal procedures were approved by the Institutional Animal Ethics Committee for Ain Shams University, Faculty of Medicine.

• •

by automated spectrophotometric method on the Beckman DU-70, USA by commercial available kits (TECO diagnostics, USA and BioChain, USA, Randox, UK respectively). LDL Cholesterol level was calculated according to Friedewald formula in mg/dl (Friedewald et al., 1972) LDL = Total cholesterol(HDL + Triglyce ride/5). Serum glucose level was measured by glucose meter (AccuCheck; Roche Diagnostics, USA).

2.4.2.3. Measurement of hepatic TNF-α and VEGF. TNF-α was measured using the rat TNF-α ELISA kit (BioChain, USA) for the quantitative measurement of TNF-α in liver tissues according to the manufacturer's instructions. VEGF protein concentration was determined using the Quantikine rat VEGF Immunoassay (R&D Systems) according to the manufacturer's recommended protocol.

2.3. Experimental procedures 2.3.1. Induction of NASH model The rats received HFD with 71% of energy from fat, 11% from carbohydrates and 18% from protein. The major components of HFD (g/100 g diet) were 87.8% standard chow diet, 2% cholesterol and 20% lard (Wang et al., 2008) and 0.2% Bile salts were added according to Deng et al. (2007). The whole cocktail was mixed together using water then pellets were made and left to dry. The diet was prepared every 5 days and stored in the refrigerator.

2.4.2.4. Quantification of hepatic FC and TG level per tissue weight mg. Hepatic FC was measured using the rat CH ELISA Kit (BioChain, USA) and TG was measured using Wako Chemicals (Richmond, VA, USA) according to the manufacturer's instructions.

2.3.2. Drug administration Thirty two male Wistar rats were randomly distributed into 2 main groups, Group I (Control Naïve): Rats were ad libtum and fed ordinary chow diet for 12 weeks and Group II (HFD): Rats were fed HFD for 12 weeks.HFD group was further subdivided into 3 equal subgroups:(A) NASH untreated: received weekly i.p. injection of Saline 0.9%. (B) NASH + ZA in dose 50ug/kg/week, i.p. for 12 weeks (C) NASH + ZA in dose 100ug/kg/week, i.p. for 12 weeks. ZA was administrated starting from the beginning of the experiment (Olejnik et al., 2016).

2.4.3. Histopathological and immunohistochemical studies (Suvarna et al., 2013; Scudamore, 2014): Liver sections were cut at 5 μm and stained with H&E and Mallory's trichrome stain. Other paraffin sections were cut on positively charged slides and were subjected to modified Avidin-Biotin immunoperoxidase technique for α-SMA. Positive immunoreactivity for α-SMA appeared as brown cytoplasmic staining of varying degrees. Positive control was done by staining a section of the colon. Negative control was done by omitting the step of primary antibody. Antibodies against α-SMA were purchased from Calbiochem Biotechnology, San Diego, CA, United States. Caspase-3 immune reaction; Paraffin sections of the liver and of a positive control (tonsils) were processed. Negative control was processed according to the same protocol, except for the use of the primary antibody. Positive expression of Caspase-3 was detected as brown cytoplasmic reaction with some nuclear staining. The kit was supplied by Lab Vision Corporation. Kato Road, Fremont, USA.

2.4. Assessment of the effects of treatment 2.4.1. In vivo measurement of portal pressure (PP) (Tsuia et al. 2003) At the end of 12th week, rats were anesthetized by intraperitoneal injection of xylazine ketamine mixture (ketamine 100 mg/kg + xylazine hydrochloride 5–10 mg/kg). Shaving and disinfection of skin were done. The abdomen was opened via a midline abdominal incision; part of the intestinal loops was gently shifted towards the left side and covered with moistened gauze. The measurement of PP was performed through a small cannula (20G) irrigated with heparinized saline every 3 min and inserted into the distal part of the portal vein. This cannula was connected to Power Lab data acquisition systems via the Ultra Pressure-Volume Unit. PowerLab acquisition data systems (Powerlab

2.5. Morphometric study An image analyzer Leica Q win V.3 program installed on a computer in the Department of Histology and Cell Biology, Faculty of Medicine, Ain Shams University, was used. The computer was connected to a Leica DM2500 microscope (Wetzlar, Germany). Animals from all 2

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serum glucose levels were significant decrease with both doses of ZA (50, 100 μg/kg/wk) by 26.3% & 40% respectively compared to NASH untreated group. ZA in dose (100/kg/wk) showed significant decreased of serum glucose level compared to ZA in dose 50ug/kg/week.

groups were subjected for morphometric study. Measurements were taken from three different slides obtained from each animal. Five haphazardly selected non-overlapping fields were examined for each slide at objective lens X 40 to measure. Mean area percentage of collagen fibers in Mallory's trichrome stained sections, and mean area percentage of positive immune reaction for α-SMA and caspase-3 were measured.

3.2.3. Effect on hepatic TNF-α and VEGF levels According to fig. 2A-B ZA in both doses exhibited a significant decrease of hepatic TNF-α and VEGF compared to NASH untreated group. Also, ZA (100/kg/wk) showed significant decreased of the hepatic TNFα and VEGF by 48.6% and 83.3% respectively compared to ZA in lower dose.

2.6. Statistical analysis All values in the results will be expressed as means ± standard deviation (S.D.). Statistical difference among groups was determined using one way analysis of variance; ANOVA followed by Tukey's multiple comparison test. P values < 0.05 will be considered statistically significant. Statistical analysis will carried out using Graphpad prism, software program, version 5.0. (2007). Inc., CA, USA.

3.2.4. Effect on hepatic FC and TG levels As shown in fig. 2C-D, ZA in both doses exhibited a significant decrease in hepatic FC and TG levels compared to NASH untreated group. Moreover, ZA (100/kg/wk) showed significant decreased of hepatic FC and TG levels by 33.3% and 26.8% respectively compared to ZA in lower dose.

3. Results

3.3. Histopathological and immunohistochemical studies

3.1. Effect on portal pressure (PP)

Examination of H&E stained sections of control liver showed the general appearance of classic hepatic lobules formed of central vein and peripheral portal tracts, Fig. 3A. Cords of hepatocytes were seen radiating from the central vein in branching and anastomosing pattern. Hepatocytes appeared polygonal in shape with acidophilic cytoplasm and central rounded vesicular nuclei. Some hepatocytes were seen binucleated. Blood sinusoids were slit like and were lined with flat endothelial cells, Fig. 3B. Portal tracts contained bile duct, branches form hepatic artery, and portal vein, Fig. 3C. In untreated NASH group, dilated congested portal venin branches were seen in most portal tracts, Fig. 3D. Most hepatocytes were seen with vacuolated cytoplasm and shrunken deeply stained nuclei. Some of these nuclei were pushed peripherally. Disturbed radial arrangement of hepatic cords and obliterated blood sinusoids were also frequently seen, Fig. 3E. Cellular infiltration and dilated proliferated bile ducts were seen in most portal tracts, Fig. 3F. In NASH + ZA in dose 50ug/kg/week group, expanded portal tracts (Fig. 4A), congested central veins and intracellular vacuoles were noticed in most hepatocytes (Fig. 4B)near the portal tracts(Fig. 4C).In NASH + ZA in dose 100ug/kg/week group, normal hepatic architecture was seen in most lobules (Fig. 4D). Hepatocytes were seen with acidophilic cytoplasm and vesicular nuclei (Fig. 4E).Some portal vein branches were seen dilated (Fig. 4F). Examination of Mallory's trichrome stain, the control groupshowed minimal amounts of collagen fibers around central veins, portal tracts and in-between hepatocytes (Fig. 5A). In untreated NASHgroup, an apparent increase amount of collagen fibers was seen in most portal tracts (Fig. 5B). In NASH + ZA in dose 50ug/kg/week group, an apparent increase in the amount of collagen fibers was seen in some portal tracts (Fig. 5C). While, in NASH + ZA in dose 100ug/kg/week group, apparently few collagen fibers were seen around most portal tracts (Fig. 5D). Immunohistochemical examination of control liver showed positive immune reaction for α-SMAin smooth muscles of blood vessels in the portal tract (Fig. 6A). In un treated NASH group, an apparent increase in the immune reaction in the portal tracts and in-between hepatocytes (Fig. 6B).In NASH + ZA in dose 50ug/kg/week group, mild positive reaction was seen in-between hepatocytes and in the portal tracts (Fig. 6C). While NASH + ZA in dose 100ug/kg/week group, showed negative reaction between hepatocytes (Fig. 6D). Immunohistochemical examination of Caspase-3 in the control liver, showed caspase-3negative reaction in all hepatocytes (Fig. 7A). In untreated NASH group, few hepatocytes were seen with caspase-3 positive immune reaction in most lobules(Fig. 7B).In NASH + ZA in dose 50ug/ kg/week group, some caspase-3 positive hepatocytes were seen

As shown in fig. (1), i.p injection of ZA in doses (50, 100 μg/kg/wk) exhibited a significant decrease in PP respectively by 52.6% and 82.9% compared to NASH untreated group. 3.2. Biochemical and Molecular Studies 3.2.1. Effect on serum ALT and AST levels As shown in Table (1), ZA in both doses (50, 100 μg/kg/wk) exhibited a significant decrease in ALT and AST respectively by 73.2% & 55.18% respectively compared to NASH untreated group. ZA in dose (100/kg/wk) showed significant decreased of serum liver enzymes compared to ZA in dose 50ug/kg/week. 3.2.2. Effect on lipid profile and serum glucose levels As regard table (1), ZA in both doses produced a significant decrease in total cholesterol, TG, LDL and a significant increase in HDL compared to NASH untreated rats. Moreover, NASH treated by (100/kg/wk) showed significant decreased of the total cholesterol, TG, LDL and a significant increase in HDL by 55.8L%, 87%, 37% & 9.1% respectively compared to NASH treated ZA group in dose 50ug/kg/week. Also,

Fig. 1. Portal pressure change in naïve control, untreated NASH, NASH + ZA (50μg &100μg) groups. Data are mean ± S.D. (n = 6). ∗P < 0.05 significant compared to naïve control group, #p < 0.05, significant compared to untreated NASH group by one-way ANOVA with Tukey's test. NASH: nonalcoholic steatohepatitis, ZA: Zoledronic acid. 3

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Table 1 Serum liver transamiases, serum lipid profile and serum glucose in naïve control, untreated NASH, NASH + ZA (50μg &100μg) groups. Data are mean ± S.D. (n = 6). aP < 0.05 significant compared to naïve control group, bp < 0.05, significant compared to untreated NASH group, cp < 0.05, significant compared to NASH + ZA 50ug by one-way ANOVA with Tukey's test. NASH: nonalcoholic steatohepatitis, ZA: Zoledronic acid, ALT: alanine aminotransferase, AST: aspartate aminotransferase, HDL: high-density lipoprotein, LDL: low-density lipoprotein. NASH + ZA 100ug 41.03 49.83 43.17 46.83 33.50 21.50 104.9

± ± ± ± ± ± bc

bc

2.01 2.64 1.84 2.23 0.55 1.37

bc bc bc bc bc

NASH + ZA 50ug b

69.33 ± 1.03 77.33 ± 2.73 b 67.83 ± 1.84 b 78.67 ± 2.61 b 30.33 ± 0.52 b 29.50 ± 0.55 b 129 ± 2.72 b

Untreated NASH 112.2 121.5 118.2 109.2 23.50 71.50 174.8

± ± ± ± ± ± ±

a

4.12 2.74 4.02 5.12 0.55 2.74 8,11

a a a a a a

Naïve control 29.83 33.83 40.33 34.50 37 ± 14.00 97.92

± 0.04 ± 2.23 ± 0.82 ± 4.68 0.02 ± 0.63 ± 7.13

Serum ALT (U/L) Serum AST (U/L) Total Cholesterol(mg/dl) Triglyceride(mg/dl) HDL (mg/dl) LDL (mg/dl) Serum glucose level (mg/dl)

Fig. 2. A. hepatic TNFα level, B. hepatic VEGF level,C. hepatic FC level, D. hepatic TG level in naïve control, untreated NASH, NASH + ZA (50μg & 100μg) groups. Data are mean ± S.D. (n = 6). ∗ P < 0.05 significant compared to naïve control group, #p < 0.05, significant compared to untreated NASH group, $p < 0.05, significant compared to NASH + ZA 50ug by one-way ANOVA with Tukey's test. NASH: nonalcoholic steatohepatitis, ZA: Zoledronic acid, TNFα: tumor necrosis factor alpha, VEDF: vascular endothelial growth factor, Fc: Free Cholesterol, TG: Triglycerides.

Fig. 3. [A-C] Naïve control group [A] the general appearance of classic hepatic lobules. Central vein (↑), and portal tract (▲). [B] Central vein (CV), binucleated hepatocytes (↑) and blood sinusoids (S). [C] The portal area contains bile duct, branches from the hepatic artery (A) and portal vein (V). [DF] untreated NASH group [D] dilated congested portal vein branch (V) in the expanded portal tract (▲). [E] hepatocytes with vacuolated cytoplasm and shrunken deeply stained nuclei. Hepatocytes with peripheral dense nuclei (↑). Disturbed radial arrangement of hepatic cords and obliterated blood sinusoids (▲). [F] dilated congested portal vein branch (V). Hepatocytes are seen with extensive vacuolated cytoplasm (↑) and pyknotic deeply stained nuclei. Inset: cellular infiltration in the portal tract (*) and dilated proliferated bile ducts (↑). H&E: [A& D]X 100, [B, C, E &F] X 400. 4

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Fig. 4. [A-C] NASH + ZA 50ug group: central vein (↑) and expanded portal tract (▲). [B] hepatocytes with intracellular vacuoles (↑) and congested central vein (CV). [C] few hepatocytes near the portal tract with intracellular cytoplasmic vacuoles (↑). [D-F] NASH + ZA 100ug group: [D] central vein (↑) and portal tract (▲). [E] hepatocytes with acidophilic cytoplasm and vesicular nuclei. [F] dilated portal vein branch (V). H&E: [A&D]X 100, [B, C, E &F] X 400.

Furthermore, administration of ZA for 12 weeks produced a significant reduction in a hepatic FC and TG levels dose dependently compared to NASH untreated subgroup. This can be explained partially by ability of ZA to decrease in serum levels of total cholesterol, TG and LDL and increase in HDL. Arguello et al. (2015) and Wouters et al. (2008), documented that hepatic FC accumulation is related to hyperlipidemia and plays a pivotal role in the pathogenesis of NASH. Also, it was found that dyslipidemia consequently leads to fat accumulation in the liver led to hepatic steatosis and fibrosis of the liver (Kampschulte et al., 2014). Moreover, ZA may inhibit hepatic FC by inhibiting the activity of FDPS enzyme in the mevalonic acid pathway. Cholesterol is the major product of the mevalonate pathway in the liver (Healey et al., 2015; Okin and Medzhitov, 2016). Cholesterol accumulation in the liver results in inflammation which facilitates the progression to steatohepatitis through inflammatory cell infiltration and fibrosis. There is another potential mechanism of hepatic FC in the pathogenesis of NASH include apoptotic cell death which is a central feature of lipotoxic liver injury (Malhi and Gores, 2008). Hepatocyte apoptosis correlates with disease severity in NASH (Feldstein et al., 2003; Arguello et al., 2015). In current work, administration of ZA in doses 50ug and 100ug for 12 weeks produced a significant decrease in hepatic TNF-α compared to NASH untreated subgroup. ZA in dose 100ug produced a significant decrease in hepatic TNF-α compared to ZA in lower dose. It was reported that ZA inhibits the production of TNF-α by macrophages (Wolf

(Fig. 7C). While in NASH + ZA in dose 100ug/kg/week group, negative caspase-3 reaction was seen (Fig. 7D). There was a significant increase in the mean area percentage of collagen fibers, and the mean area percentage of α-SMA and Caspase-3 positive immune reaction, in untreated NASH group compared to control group, treated NASH groups with ZA in dose 50 & 100 μg/Kg/ Week showed a significant decrease in all measured parameters compared to untreated NASH group as shown in Table (2). 4. Discussion In the present work, IP administration of ZA for 12 weeks in doses (50μg/kg/wk) and (100μg/kg/wk) produced a significant improvement of lipid profile compared to NASH untreated group with preferable effect of ZA (100μg/kg/wk). This effect can be as a result of ability of ZA to block the activity of FDPS a key regulatory enzyme in the mevalonic acid pathway which critical to the production of cholesterol (Drake et al., 2008). Several clinical studies reported the great efficacy of ZA to reduce serum lipids level which is time and dose dependent (Adami et al., 2010; Montagnani et al., 2003; Gozzetti et al., 2008). Hyperlipidemia is considered one of important pathological factors that help in progression from simple steatosis to steatohepatitis as result of inflammatory changes and cytotoxicity in the liver (Subramanian et al., 2011).

Fig. 5. Mallory`s trichrome stain X100. [A] Naïve control group: few collagen fibers in the portal tract (▲) and around the central vein (↑). [B] Untreated NASH group: an apparent increase amount of collagen fibers in the portal tracts (▲). [C] NASH + ZA 50ug group: apparent increase collagen fibers in the portal tract (▲). Few collagen fibers are seen around the central vein (↑). [D] NASH + ZA 100ug group: few collagen fibers around the central vein (↑) and portal tract (▲).

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Fig. 6. Anti- α-SMA antibody X 400. [A] Naïve control group: showing positive immune reaction for α-SMA in smooth muscles of blood vessels at portal tract (▲). [B] Untreated NASH group: showing positive immune reaction in smooth muscles of branches of portal vein and hepatic artery, in the portal tract (▲). Inset: positive reaction between hepatocytes (↑). [C] NASH + ZA 50ug group: positive immune reaction in smooth muscles of portal vein branch, in portal tract (▲) and between hepatocytes (↑) [D] NASH + ZA 100ug group: positive immune reaction in smooth muscles of branches of portal vein and hepatic artery (▲).

In the current work, i.p administration of ZA for 12 weeks in both doses (50μg/kg/wk) and (100μg/kg/wk) produced a significant improvement in liver function revealed by decrease in serum levels of liver enzymes AST and ALT, and glucose compared to NASH untreated group. This can be explained by decrease hepatic level of level TNF-α and FC which reduce inflammation and deterioration of liver function. Zolidronic acid exhibited significant decrease in degenerated hepatocytes, inflammatory cellular infiltration, and amount of collagen fibers in dose dependent manner compared to NASH untreated subgroup. Also, there was significant decrease in mean area percentage of α-SMA and Caspase-3 positive immune reaction. α-SMA is clearly a proven marker for assessing myofibroblast differentiation which is a key marker involved in hepatic fibrogenesis by deposition large amounts of ECM (Rockey et al., 2013). Moreover, Caspase 3 has been identified to be the main player of apoptotic cell death in NASH and selective inactivation of caspase 3 prevent progression of steatohepatitis and hepatic fibrosis (Thapaliya et al., 2014). The significant effect of ZA on histopathological and immunohistochemical studies can be due to

et al., 2006). However, Dicuonzo et al. (2003), demonstrated that ZA increases level of TNF-α but HATTORI et al. (2015), detected that ZA increases level of TNF-α in experimental animals bearing tumor and not in normal experimental animals. TNF-α is proinflammatory cytokine involved in the earliest events of liver injury. TNF-α induces the production of other cytokines from hepatocytes and KCs that, together, recruit inflammatory cells and trigger hepatic inflammation, a key aspect of NASH (Subramanian et al., 2011). TNF-α has role in progression of hepatic inflammation to steatohepatitis and liver fibrosis through the activation of HSCs and TNF-α induced apoptosis (Wobser et al., 2009). It was reported that NASH patients with significant fibrosis exhibited increased expression of TNF-α mRNA in comparison with those with minimal or non-existent fibrosis through regulation Kupffer cell activation (Crespo, 2001; Tomita, 2006). The effect of ZA on hepatic TNF-α in current study may be due to reduction of hepatic cholesterol. Kleemann et al. (2007), reported that cholesterol accumulation increase TNF-α signaling pathways that play central roles in the evolution of cholesterol-induced inflammation in the liver.

Fig. 7. Anti-Caspase-3 antibody X 400. [A] Naïve control group: negative immune reaction for caspase3 reaction. [B] Untreated NASH group: positive immune reaction in cytoplasm of hepatocytes (↑). [C] NASH + ZA 50ug group: positive caspase-3 immune reaction (↑) in few hepatocytes [D] NASH + ZA 100ug group: negative caspase-3 immune reaction.

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Table 2 Showing the comparison between different groups as regard mean area percentage of collagen fibers, α-SMA and Caspase-3 immune reaction.

a

Mean area percentage of Caspase-3

Mean area percentage of α-SMA

Mean area percentage of collagen fibers

0 5.08 ± 1.2a 2.87 ± 0.562b 0b

4.6 ± 0.7 24.84 ± 4.2a 5.75 ± 1.3b 3.2 ± 0.6b

5.92 16.3 7.02 6±

± 0.8 ± 0.8a ± 0.7b 0.9b

Naïve control group Untreated NASH group NASH + ZA 50ug group NASH + ZA 100ug group

P < 0.05 significant change compared to naïve control group. p < 0.05, significant change compared to untreated NASH group. NASH: nonalcoholic steatohepatitis, ZA: Zoledronic acid, α-SMA: alpha smooth muscle actin.

b

References

significant reduction of hepatic TNF-α and FC and lipid profile levels. This results in prevention of hepatic inflammation and fibrosis in dose dependent manner. In the current work, administration of ZA in both doses for 12 weeks produced a significant decrease in hepatic VEGF level compared to NASH untreated subgroup. In consistent with our result, Wood (2002), reported that ZA dose dependently inhibited the angiogenic response induced by VEGF. VEGF has important role in the pathogenesis of NASH and blockage the action of VEGF attenuates hepatic steatosis, inflammation, angiogenesis and fibrosis (Coulon et al., 2013). There is close relationship between liver angiogenesis, fibrogenesis and portal hypertension in NASH (Bocca et al., 2015). In present study, i.p administration of ZA in doses 50ug and 100ug for 12 weeks produced a significant decrease in portal pressure NASH untreated subgroup. This can be partially explained by significant decrease in hepatic VEGF and partially due to significant decrease in the mean area percentage of collagen fibers, and the mean area percentage of α-SMA. Disturbance of liver architectural, with distortion of vascular architecture by fibrosis lead to development of PHT (Bosch et al., 2003). Furthermore, there is Correlation between the hepatic venous pressure gradient and α-SMA expression in NASH (Sanyal et al., 2016). In conclusion, ZA in a dose related manner prevents the hepatic inflammation, deterioration in liver function, fibrosis and PHT induced by HFD in rats. This may be largely mediated by mevalonic acid pathway inhibition which consequently leads to significant reduction in lipid profile, hepatic TNF-α, VEGF, FC and TG levels induced by HFD.

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Declaration of interest 'Declarations of interest: none'. Conflicts of interest The authors declare that they have no competing interests. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CRediT authorship contribution statement Reham Hussein Mohamed: Conceptualization, Methodology, Validation, Writing - review & editing, Supervision, Resources, Investigation, Project administration. Maha Tarek: Conceptualization, Data curation, Writing - original draft, Formal analysis, Project administration, Investigation, Resources. Ghada Galal Hamam: Writing original draft, Methodology, Resources, Supervision. Samar F. Ezzat: Writing - original draft, Methodology. Acknowledgement None. 7

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