Sodium valproate, a histone deacetylase inhibitor, with praziquantel ameliorates Schistosoma mansoni-induced liver fibrosis in mice

    Sodium valproate, a histone deacetylase inhibitor, with praziquantel ameliorates Schistosoma mansoni-induced liver fibrosis in mice Mohamed G. Elsakkar, Maha M. Eissa, Wafaa A. Hewedy, Rasha M. Nassra, Soha F. Elatrebi PII: DOI: Reference:

S0024-3205(16)30472-6 doi: 10.1016/j.lfs.2016.08.010 LFS 14987

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

6 June 2016 7 August 2016 10 August 2016

Please cite this article as: Elsakkar Mohamed G., Eissa Maha M., Hewedy Wafaa A., Nassra Rasha M., Elatrebi Soha F., Sodium valproate, a histone deacetylase inhibitor, with praziquantel ameliorates Schistosoma mansoni-induced liver fibrosis in mice, Life Sciences (2016), doi: 10.1016/j.lfs.2016.08.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Sodium Valproate, a Histone Deacetylase Inhibitor, with Praziquantel Ameliorates Schistosoma Mansoni-Induced Liver Fibrosis in Mice.

PT

Authors Mohamed G. Elsakkar1, Maha M. Eissa 2, Wafaa A. Hewedy 1, Rasha M. Nassra 3, Soha F.

RI

Elatrebi1.

Department of Clinical Pharmacology, 2Department of Medical Parasitology, 3Department of

NU

1

SC

Affiliations

Medical Biochemistry, Faculty of Medicine, Alexandria University, Alexandria, Egypt.

MA

Corresponding author Dr. Wafaa A. Hewedy

TE

D

Tel: +201225071549

E-mail: [email protected]

AC CE P

Postal address: Almoassat medical Campus, Clinical Pharmacology department-Faculty of Medicine, Alexandria, Egypt

1

ACCEPTED MANUSCRIPT Abstract: Aims: This study explores the potential antifibrotic effect of sodium valproate (SV), an inhibitor

PT

of class I histone deacetylase (HDAC) enzymes, and/or praziquantel (PZQ) on Schistosoma

RI

mansoni (S. mansoni)-induced liver fibrosis in mice.

SC

Main methods: Male Swiss albino mice were divided into nine groups: group I- normal control (NC); group II- uninfected gum mucilage (GM) treated; group III- uninfected PZQ- treated;

NU

group IV- uninfected SV-treated; group V- control S. mansoni infected mice; group VI- infected GM-treated; group VII- infected PZQ-treated; group VIII- infected SV-treated; group IX-

MA

infected PZQ+SV treated. All SV administrations were 300 mg/kg/day orally and administered for five weeks beginning on the 5th week post infection (WPI). All PZQ administrations were

TE

D

500 mg/kg/day orally and administered for 2 consecutive days beginning on the 7th WPI. Serum transforming growth factor-beta 1 (TGF-β1), tumor necrosis factor-alpha (TNF-α), hepatic

AC CE P

hydroxyproline (Hyp) content, and liver function tests (AST and ALT) were determined. Specimens of the hepatic tissues were examined histologically. Key findings: Treatment of S. mansoni-infected mice with SV significantly decreased the serum levels of ALT, TGF-β1 and TNF-α, and the liver tissue hydroxyproline content compared with the S. mansoni infected untreated groups. Histologically, treatment with SV revealed regression of the granulomatous inflammatory reaction. Combined treatment with PZQ and SV produces more favorable biochemical results, and aborted granulomatous reaction compared with either drug alone. Significance: Sodium valproate is a promising anti-fibrotic agent. It demonstrated an antifibrotic effect in early stages of S. mansoni infection through downregulation of profibrogenic cytokines, and collagen deposition.

2

ACCEPTED MANUSCRIPT Keywords: histone deacetylase inhibitors, liver fibrosis, Schistosoma mansoni, sodium valproate, praziquantel.

PT

1. Introduction:

RI

Hepatic schistosomiasis is one of the most prevalent forms of chronic liver diseases in the world

SC

caused by the blood-dwelling trematodes of the genus Schistosoma. Schistosoma mansoni (S. mansoni), which is responsible for intestinal schistosomiasis, causes symptoms ranging from

NU

abdominal pain and bloody diarrhea to hepatosplenomegaly, periportal fibrosis and portal hypertension [1].

MA

Praziquantel (PZQ) is the mainstay treatment for all Schistosoma species in most endemic areas. However, PZQ is inactive against juvenile schistosomes and only has a limited effect on already

resistance to PZQ [2].

TE

D

developed liver and spleen lesions. Additionally, there are recent concerns about tolerance and/or

AC CE P

Complications of schistosomiasis are mostly due to egg-induced granulomatous lesions. The initiation, development, and regression of granuloma are mediated by cytokines and chemokines leading to recruitment of inflammatory cells against the antigenic stimuli. Granulomatous response evolves from an early type 1 helper (TH1) to a sustained and dominant type 2 helper (TH2) cells, where interleukin (IL)-4 and IL-13 being the main cytokines driving this reaction. Although many studies showed that recovery from advanced fibrosis is possible, drugs to prevent and treat fibrosis are only partially effective [3]. The key pathogenic event in hepatic fibrogenesis is activation of hepatic stellate cells (HSCs) [4]. HSCs undergo a process of trans-differentiation from the quiescent vitamin A-storing cells to myofibroblasts. The myofibroblasts are responsible for production of fibrogenic extracellular matrix (ECM) components within the space of Disse and becomes involved in inflammatory

3

ACCEPTED MANUSCRIPT signaling. These responses are tightly controlled by cytokine production both from the HSCs themselves as well as the neighboring cells [5]. Growth factors such as TGF- α or –β, reactive

PT

oxygen species and products of lipid peroxidation are triggers that activate HSCs [6].

RI

An important process in transcriptional regulation of HSCs is the modification of histones by histone modifying enzymes (HMEs) [7]. HMEs include histone deacetylases (HDACs), histone

SC

acetyltransferases, histone methyltransferases and histone demethylases. Acetylation by histone

NU

acetyltransferases (HAT) abolish the positive charge of the lysine and reduces chromatin compaction, favoring transcription. Deacetylation by the action of HDACs has the opposite

MA

effect [8]. However, histone acetylation also modulates binding of many proteins that regulate gene transcription [9]. Functionally, histone modifications have the potential to influence several

TE

D

biological processes including differentiation and transdifferentiation of HSCs in vitro [10]. HDAC inhibitors (HDACIs) are being investigated as drugs used in a range of diseases,

AC CE P

including cancers and infectious diseases such as HIV/AIDS, and several parasitic diseases. Recently, the applicability of HDACIs in treatment of fibrotic disorders has been also explored [11].

Sodium valproate (SV), which has been widely used as an antiepileptic and mood stabilizer drug, is found also to inhibit class I HDAC enzymes. Several types of HDACIs, including SV, are undergoing several clinical trials for HDAC-relevant diseases [12]. Based on available data; we aimed in this study to test the hypothesis that SV will be a novel adjuvant to PZQ, the classical anti-schistosomal drug, in treatment of S. mansoni-induced liver fibrosis in mice.

2. Material and methods 2.1. Drugs:

4

ACCEPTED MANUSCRIPT Sodium Valproate (Depakine) was purchased from Sanofi-Aventis France S.A. company (Paris, France). Praziquantel (Distocide) was obtained from the Egyptian International Pharmaceutical

PT

Industries Company (Eipico, 10th of Ramadan, Egypt).

RI

2.2. Animals:

The present study was conducted on seventy-two age-matched male Swiss albino mice of body

SC

weights ranging from 18-30 grams. At the start of the study, no significant differences were

NU

found between the mean weights of the animals in the different tested groups (p>0.05). The mice were purchased from the animal house of Misr University for Science and Technology (MUST),

MA

Egypt. The mice were housed in conditions in accordance with the Helsinki declaration for animals and care. The animals were kept under standard conditions of light and temperature,

TE

D

with free access to food and water ad libitum for one week before the start of the experiment as an acclimatization period. All experimental procedures were approved and performed in

AC CE P

compliance with the guidelines of the Local Ethical Committee of the Faculty of Medicine, Alexandria University.

2.3. Parasitic Infections:

The cercariae of S. mansoni were obtained from infected Biomphalaria alexandrina snails that have been purchased from the Schistosome Biologic Supply Center, Theodor Bilharz Research Institute, Giza, Egypt. The snails were allowed to shed their cercariae in dechlorinated water, by exposure to direct sun light for one hour. The cercariae were counted in 0.1 ml of dechlorinated water under dissecting microscope after adding one drop of Lugol’s iodine. The cercariae were used for infecting the animals by transferring each mouse to a clean sedimentation flask for caudal immersion in few milliliters of aged dechlorinated water, containing 80 freshly shed cercariae, for 90 minutes [13].

5

ACCEPTED MANUSCRIPT 2.4. Pilot Studies: A small dose-finding pilot study, with three animals for each dose, was conducted for SV. The

PT

drug dose that provided the best improvement in histological sections of the liver after S.

RI

mansoni infection was chosen.

A pilot study was conducted to test for the possible schistosomicidal activity of SV by studying

SC

its effect on adult worm count in S. mansoni infected mice. During this study, 12 mice were

NU

infected with S. mansoni cercariae and divided into two equal groups. The first group was left untreated as a control group while the second group was treated with SV 300mg/kg/day for 2

MA

weeks beginning on the 5th week post infection (WPI) [14]. The mice were then sacrificed and the adult S. mansoni worms were counted. The statistical analysis of the results revealed no

TE

D

significant antischistosomal effect for SV treatment (data not shown). 2.5. Experimental Procedure:

AC CE P

Thirty-two mice were randomly divided into 4 groups, with 8 mice in each group: group INormal control (NC) uninfected mice received distilled water orally; group II- uninfected mice received 2% gum mucilage (GM) orally; group III- uninfected mice were treated with PZQ; group IV- uninfected mice were treated with SV. The remaining forty mice were infected with S. mansoni cercariae. These animals were randomly divided into five groups, with 8 mice in each group: group V- control infected mice received distilled water orally; group VI- infected mice received 2% GM orally; group VII- infected mice were treated with PZQ; group VIII- infected mice were treated with SV; group IX- infected mice were treated with PZQ and SV in combination. All SV administrations were 300 mg/kg/day orally, and were dissolved in sterile distilled water at a final concentration of 15 mg/ml and administered for five weeks beginning on the 5th WPI.

6

ACCEPTED MANUSCRIPT All PZQ administrations were 500 mg/kg/day orally, and were suspended in 2% gum mucilage at a final concentration of 25 mg/ml and administered for 2 consecutive days beginning on the 7th

PT

WPI [15]. The prepared drug solutions and the vehicle were administered to the mice as 20

RI

ml/kg. 2.6. Estimation of the biochemical parameters:

SC

At the end of the 10th WPI, and on the last day of the experiment, the animals were fasted for 8 h.

NU

The mice were anesthetized, and blood samples were collected from the abdominal aortae of all animals, followed by separation of the sera. The sera were stored at −20 °C until used for

MA

biochemical studies. The mice were then killed by decapitation. The abdomen of each animal was opened, and the liver was rapidly excised. Each liver was dissected and divided into two

D

parts; one was fixed in formalin for histopathological examinations. The other part was frozen at

TE

−80 °C until be homogenized in distilled water using a polytron homogenizer for determination

AC CE P

of hydroxyproline (Hyp) content.

2.6.1. Estimation of serum biomarkers of liver injury: The serum aminotransferases were assayed using colorimetric kits (Spectrum Diagnostics, Cairo, Egypt). The serum alanine aminotransferase (ALT) and the serum aspartate aminotransferase (AST) were determined according to the methods described by Reitman and Frankel [16]. The results are expressed as units per liter (U/L). 2.6.2. Estimation of serum markers of liver fibrosis: The serum transforming growth factor beta1 (TGF-β1), and tumor necrosis factor α (TNF-α) were assayed using mouse ELISA kits (eBioscience, Vienna, Austria) [17, 18]. The kits employ the quantitative sandwich enzyme immunoassay technique and the absorbance of the products of

7

ACCEPTED MANUSCRIPT the reactions was measured at 450 nm. The results are expressed as picograms per milliliter (pg/mL).

PT

2.6.3. Estimation of hepatic hydroxyproline content [19]:

RI

In brief, aliquots of standard hydroxyproline samples were hydrolyzed in alkali (sodium hydroxide 2N final concentration). The hydrolyzed samples by autoclaving at 120 °C for 20 min

SC

were then mixed with a buffered chloramine-T reagent, and the oxidation was allowed to

NU

proceed for 25 min at room temperature. The chromophore was then developed with the addition of Ehrlich's reagent by incubation at 65 °C for 20 min., and the absorbance of the reddish purple

MA

complex was measured at 550 nm using a spectrophotometer. Absorbance values were plotted against the concentrations of standard hydroxyproline and the concentrations of hydroxyproline

D

in tissue samples per gram liver was determined from the standard curve. The results are

TE

expressed as micrograms per milliliter (μg/mL).

AC CE P

2.7. Histopathological examination

For histological examination, specimens of the hepatic tissues were fixed in buffered 10% formalin. They were processed for routine paraffin block preparation. From every specimen, 5-7 randomly selected sections were taken from different depths to give a representative appreciation of the whole liver. Sections of 5μm were cut and stained with haematoxylin and eosin stain (H&E), for ordinary histopathological study, and Masson’s trichrome stain to demonstrate fibrous tissue and collagen fibers [20, 21]. The histological slides were coded and the pathologist was unaware of the origin of the material during examination. 2.8. Statistical analysis Statistical analyses were performed using Graph Pad InStat software (Version 3.02). Data were presented as the mean±SD. ANOVA one-way test was used for the statistical analysis of the

8

ACCEPTED MANUSCRIPT data. The Post Hoc test, Bonferroni’s multiple comparison test, was used for comparing means of two independent groups after a significant ANOVA. A level of p<0.05 was defined as being

PT

statistically significant.

RI

3. Results

SC

3.1. Biochemical results

No significant difference was found in any of the biochemical parameters or the histological

NU

changes between vehicles (distilled water or gum mucilage) administered and drugs (PZQ or SV) treated uninfected mice. Similarly, administration of the vehicles (GM) to S. mansoni infected

MA

mice did not reveal significant difference in the biochemical or the histological sections tested compared with the infected mice (group I). Infection of mice with S. mansoni resulted in a

TE

D

significant elevations in serum levels of ALT and AST compared with the control groups (groups I and II). Treatment of S. mansoni-infected mice with PZQ alone significantly decrease the liver

AC CE P

transaminases tested. Although SV alone failed to significantly decrease serum AST levels, it significantly reduced serum ALT levels in infected mice. AST was only reduced when PZQ was added to SV treatment. Adding PZQ to SV treatment significantly reduced ALT levels compared with PZQ treatment alone (Table 1). The levels of TGF-β1 were found to significantly increase in the sera taken from S. mansoni infected mice compared with their levels in the control mice. Treatment of infected mice with PZQ, SV, or alternatively the combination of both drugs resulted in a significant decrease in the serum levels of TGF-β1 compared with the infected untreated groups. Treatment with SV significantly decreased TGF-β1 compared with the PZQ treated group. Adding SV to PZQ treatment significantly augmented its suppressive effect on TGF-β1 levels (Table 2).

9

ACCEPTED MANUSCRIPT Infection of mice with S. mansoni resulted in a significant increase in the serum levels of TNF-α compared with the control groups. Treatment of S. mansoni infected mice with PZQ resulted in a

PT

significant decrease in the serum levels of TNF-α compared with the infected group. Treatment with SV alone or in combination with PZQ significantly reduced the serum level of TNF-α

RI

compared with the infected untreated groups. A significant decrease in TNF-α was found with

SC

treatment of the infected mice with SV in combination with PZQ compared with PZQ treatment

NU

alone (Table 2).

Liver hydroxyproline levels were found to increase significantly in the mice infected with S.

MA

mansoni compared with the levels in the control groups. Treatment of the infected mice with PZQ, SV or alternatively the combination of the 2 drugs resulted in significant decreases in the

TE

D

liver hydroxyproline content compared with the S. mansoni infected untreated groups. Combining PZQ with SV treatments significantly decreased the liver hydroxyproline content

AC CE P

compared with the use of PZQ alone (Figure 1). Table 1. Effect of treatment with PZQ and/or SV on serum ALT and AST levels in S. mansoni infected mice (n=8).

Uninfected mice treated with

S. mansoni infected mice treated with

NC

GM

PZQ

SV

Infected

GM

PZQ

SV

PZQ+SV

(group

(group

(group

(group

control

(group

(group

(group

(group

I)

II)

III)

IV)

(group V)

VI)

VII)

VIII)

IX)

ALT

11.41

12.0

11.97

12.27

21.38

20.44

15.64

14.96

9.75

(U/L)

±1.21

±3.55

±1.55

±2.95

±4.14a

±5.1

±2.05b

±4.53b

±2.55bce

AST

4.8

4.66

4.2

5.29

33.56

31.99

6.82

29.63

7.02

(U/L)

±1.25

±1.71

±1.33

±1.59

±16.7a

±19.6

±1.72be

±7.98

±1.7be

GM: gum mucilage; SV: Sodium Valproate; PZQ: Praziquantel; NC: Normal control. ap<0.05 versus group I; bp<0.05 versus group V; cp<0.05 versus group VII; ep<0.05 versus group VI. 10

ACCEPTED MANUSCRIPT

Table 2. Effect of treatment with PZQ and/or SV on serum TGF-β1 and TNF-α levels in S.

PT

mansoni infected mice (n=8). S. mansoni infected mice treated with

NC

GM

PZQ

SV

Infected

GM

(group

(group

(group

(group

control

I)

II)

III)

IV)

(group V)

TGF-β

83.38

85.2

84.55

87.13

1452.6

(pg/mL)

±12.02

±8.76

±13.7

±5.67

TNF-α

21.62

23.11

21.21

28.62

(pg/mL)

±4.17

±6.99

±7.5

±5.68

RI

Uninfected mice treated with

SV

PZQ+SV

(group

(group

(group

(group

VI)

VII)

VIII)

IX)

1339.91

683

300.63

113

±240.8a

±305.10

±123.1be

±59.35 bc

±20.08bce

94.37

99.33

57.50

27.50

28.50

±39.16a

±38.70

±13.08be

±5.88bc

±7.23bce

MA

NU

SC

PZQ

D

GM: gum mucilage; SV: Sodium Valproate; PZQ: Praziquantel; NC: Normal control. ap<0.05

AC CE P

TE

versus group I; bp<0.05 versus group V; cp<0.05 versus group VII; ep<0.05 versus group VI.

Figure 1. Effect of treatment with PZQ and/or SV on liver hydroxyproline levels in S. mansoni infected mice (n=8). Data are expressed as means± SD. GM: gum mucilage; SV: 11

ACCEPTED MANUSCRIPT Sodium Valproate; PZQ: Praziquantel; NC: Normal control. ap<0.05 versus group I; bp<0.05

PT

versus group V; cp<0.05 versus group VII; ep<0.05 versus group VI.

RI

3.2. Histological results

Microscopic examination of the livers from the control and the vehicle administered mice;

SC

groups I and II; revealed the classical pattern of branching and anastomosing cords of

NU

hepatocytes radiating from central veins and limiting the sinusoidal spaces lined by endothelial cells and Von Kupffer cells. The hepatocytes appeared polyhedral with eosinophilic granular

MA

cytoplasm and rounded centrally located vesicular nuclei with prominent nucleoli. Portal areas were seen at the angles of the lobules containing branches of hepatic artery, portal vein and bile

TE

D

ducts (Figures 2-A, and 3-A).

The Livers from mice infected with S. mansoni showed large fibrocellular granulomas in the

AC CE P

hepatic parenchyma with a central zone of schistosoma ova surrounded by inflammatory cells, and an outer zone of fibrous tissue. Evidence of collagen deposition was detected by Masson Trichrome stain (Figures 2-B, and 3-B). Treatment with PZQ decreased granuloma sizes with degenerated calcified ova surrounded by giant cells, epithelioid cells, lymphocytes, plasma cells, and fibrous tissues (Figures 2-C, and 3-C). Livers of infected mice treated with SV revealed regression of the granulomatous inflammatory reaction and markedly improved architecture of hepatic lobule. The granulomas were few, small, aborted and ill formed around schistosoma eggs (Figures 2-D, and 3-D). Livers of infected mice that received the combination of PZQ and SV showed few lymphocytic cell infiltrations associated with apparent improvement of architecture of the hepatic lobules. Few small granulomatous reactions were also observed (Figures 2-E, and 3-E).

12

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

13

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 2: Representative photomicrographs of H&E stained liver tissue sections (x400) (A) Photomicrograph of a liver section from the normal control group (group I) showing normal

D

architecture.

TE

(B) Photomicrograph of a liver sections from the infected untreated group (group V) showing a well-developed shistosoma granuloma.

AC CE P

(C) Photomicrograph of a liver section from the infected group treated with PZQ (group VII) showing calcified ova.

(D) Photomicrograph of a liver section from the infected group treated with SV (group VIII) showing an aborted granulomatous reaction (→) composed of lymphocytes and histiocytes with absence of schistosoma eggs. (E) Photomicrograph of a liver section from an infected group treated with SV+PZQ (group IX) showing one aborted granuloma with absence of schistosoma eggs (→).

14

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

15

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 3: Representative photomicrographs of Masson’s Trichrome stained liver tissue

D

sections (x400).

TE

(A) Photomicrograph of a liver section from the normal control group showing no fibrosis. (B) Photomicrograph of a liver section from group V showing collagen fibers deposited around

AC CE P

schistosomal granuloma. A Schistosoma mansoni egg is surrounded by fibrous deposition. (C) Photomicrograph of a liver section from group VII showing reduction in size of granuloma and collagen deposition.

(D) Photomicrograph of a liver section from group VIII showing an aborted granuloma (→). (E) Photomicrograph of a liver section from group IX showing an absence of collagen fibrous deposition in an aborted granuloma (→).

4. Discussion It is well known that the main cause of mortality and morbidity in human schistosomiasis is hepatic fibrosis [22]. In the continuing absence of an effective vaccine against this devastating disease, the only viable strategy is the mass treatment of populations in endemic areas using the currently available drug, PZQ. Effective PZQ treatment results in elimination of the parasite but a slight reduction in the liver fibrosis; mostly due to schistosomal eradication [23]. For that reason, it

16

ACCEPTED MANUSCRIPT may be beneficial to combine PZQ with an anti-fibrotic drug in the treatment of schistosomiasis to eliminate worm and reduce liver fibrosis at the same time.

PT

In the present study, S. mansoni infected mice is used to model human schistosomiasis. Mice are

RI

among the most susceptible host species for schistosoma growth and egg lying [24]. Murine infections differ from the human infections in the number of adult worms per unit of body

SC

weight, and the distribution of ova between the liver and mesenteric circulation. The density of

NU

eggs per gram of tissue is much higher in mice than it is in the most heavily infected human subjects [25]. This may affect the outcomes of experimental murine schistosomiasis when it is

MA

used to model human schistosomiasis. However, these species develop similar hepatic

TE

down-regulated spontaneously [26].

D

granulomatous inflammations, as regards dynamics and cellular compositions, and are similarly

Despite of these dissimilarities between murine hosts and humans in some aspects of the disease,

AC CE P

the use of mice hosts, makes it easy to understand many phenomena of the disease. They continue to be a suitable model for preclinical development of novel chemotherapeutic agents and vaccines [27].

The present study revealed that S. mansoni infection was associated with histological changes consistent with liver inflammatory reaction and fibrosis induced by schistosoma ova deposition. This granulomatous reaction was shown to induce liver injury as evidenced by the increase in serum levels of AST and ALT. These findings are in contrast with that associated with human schistosomiasis that causes periportal fibrosis usually with preservation of the hepatic functions in the early-compensated schistosomiasis [28]. However, a previous study reported similar increase in serum transaminases in S. mansoni infected animals, which may be explained by the

17

ACCEPTED MANUSCRIPT hepatocellular injury induced by the heavy egg deposition in this mice model of schistosomiasis [25, 29].

PT

The remarkable increase in the serum level of TGF-β1 and TNFα, detected in the present study,

RI

reflects the immunological response of the liver tissues to ova deposition. The initial reaction to Schistosoma is Th1 dominated, due to worm derived antigens, with oesinophils and neutrophils

SC

secreting TNF-α, IL1, IL6, and IFN-γ [30]. Then the immune response switches to Th2

NU

inflammation, primarily directed against egg-derived antigens with secretion of IL4, IL5, IL10 and IL13 that drive the formation of the granulomas [31]. IL4 and IL13 are necessary for TGF-

MA

β1 activation; and a positive feedback loop of IL4/IL13 and TGF-β1 propagates the disease [32]. Error! Bookmark not defined.

TE

D

Hellerbrand et al. [33] indicated that TGF-β1, the most abundant isoform in both the normal and fibrotic liver [34], accelerates the activation of hepatic stellate cells. The activated HSCs are

AC CE P

predominantly responsible for the increased synthesis of the extracellular matrix (ECM) protein during hepatic fibrogenesis [35]. Hepatic injury induces the HSCs to undergo activation, transformation to a myofibroblast-like cell with loss of cellular retinol stores and the appearance of smooth muscle actins (α-sma), increased synthesis of ECM proteins, and contractility [36]. In agreement with previous studies [37, 38], we found a remarkable increase in hepatic Hyp content which reflects the level of collagen deposition and fibrosis in the S. mansoni infected mice. The current study revealed that treatment of infected mice with PZQ significantly improved liver functions and decreased granuloma size with calcification of schistosoma eggs. This may be a consequence of the reduced serum levels of TGF-β1, TNF-α, and hepatic Hyp. These effects of PZQ could be attributed to its lethal effects on viable worms that abort further egg deposition.

18

ACCEPTED MANUSCRIPT The antigens derived from these eggs are considered the triggering stimulus for the multiple inflammatory and fibrogenic signalling pathways in this model [39]. PZQ may also influence the

PT

immune response of the host, and hence exerts dual effects on the infection. However, only high

RI

doses of PZQ was found to have a clear antifibrotic effect in a study conducted in mice infected with S. japonicum [40].

SC

Since its introduction into clinical use in 1968, SV has become one of the most commonly

NU

prescribed antiepileptic drugs. Recently SV has been reported to be a class I HDACI [41]. A potential antifibrotic effect of SV was investigated in different experimental animal models of

MA

fibrosis [42].

In the present study, the anti-fibrotic effect of SV was observed histologically as a major

TE

D

reduction in granuloma formation. The improvement of liver injury with SV treatment was shown by the normalization of the serum ALT levels. However serum AST did not improve

AC CE P

significantly with the improvement of the liver histology. This may be explained by the specificity of ALT as an indicator of liver inflammation and damage in comparison to the less specific AST, which is also found in other tissue sites like cardiac and skeletal muscle and red blood cells [43, 44]. The observed abortion of granuloma formation, in SV treated mice, may be explained by the reduced expression of TNF-α, TGF-β1, and hepatic Hyp. TNFα plays a central role in the inflammatory process and as an accelerator factor of cell proliferation. It may participate in the granuloma formation through its proinflammatory and profibrogenic effects. The decrease in serum level of TNF-α, noticed in the present study, points to the anti-inflammatory effects of SV. The suppressive effect of SV on TNF-α production was suggested by Ichiyama et al. [45] to be the result of inhibition of nuclear factor κB (NF-κB).

19

ACCEPTED MANUSCRIPT TGF-β1 is considered as one of the key mediators of fibrogenesis. Watanabe et al. [46] found that SV exerted an anti-fibrogenic activity in a human HSC line by blocking the TGF-β1 autocrine loop

PT

and inhibiting TGF-β1-induced collagen type 1 α1 expression.

RI

A recent study suggested that treatment of mice with valproic acid decreased collagen deposition and in-vivo activation of stellate cells. This effect may be, at least in part, due to inhibition of

SC

HDAC. However, the involvement of non-histone deacetylase targets in the HSCs activation

NU

process can’t be ruled out [47]. The results of the present study showed that, SV significantly decreased hepatic hydroxyproline content indicating its efficiency in this specific animal model.

MA

The paucity of schistosomal eggs in the liver sections obtained from the infected mice treated with SV deserves further study concerning the influence of SV on fecundity of female S.

TE

D

mansoni. Moreover, Dubois et al. [48], reported that treatment with histone deacetylase inhibitors, including SV, induces mortality and apoptosis in schistosomula in-vitro.

AC CE P

The present study showed also that the most significant reduction in liver fibrosis was in the group of mice treated with SV combined with the traditional treatment of hepatic schistosomiasis, PZQ. This was reflected in further reduction in serum ALT levels. The combination of SV with PZQ clearly showed better anti-fibrotic effects through normalization of the serum TGF-β1 and TNF-α level, and the liver hydroxyproline content. The mechanism of this effect is probably through inhibition of new liver injury induced by parasite egg deposition and interruption of hepatic inflammation and collagen type I synthesis with attenuation of preexistent collagen.

Conclusions:

20

ACCEPTED MANUSCRIPT From the results of the present study, it could be concluded that SV/PZQ combination has a hepatoprotective effect against schistosomal hepatic fibrosis. Further studies are needed to

PT

investigate the actual pathways responsible for all different activities of SV.

RI

Although, the results obtained in murine hosts may not translate accurately to the case of human, however it is a first step in screening drug leads in the chemotherapy of human schistosomiasis.

SC

For that reason, we recommend investigating the possible beneficial effect of adding SV to the

NU

standard antihelminthic PZQ treatment to ensure prevention of the liver fibrotic complications of hepatic schistosomiasis in human.

MA

Acknowledgements:

We are grateful to Professor Dr. Nagwa Eweis, Professor of Pathology, Alexandria Faculty of

References:

AC CE P

work in this research.

TE

D

Medicine, Alexandria University, for her participation in accomplishment of the histological

[1] Parkin DM. The global burden of urinary bladder cancer. Scand J Urol Nephrol Suppl 2008; (218):12-20.

[2] Doenhoff M, Cioli D, Utzinger J. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Curr Opin Infect Dis 2008; 21: 659-667. [3] Fallowfield JA, Kendall TJ, Iredale JP. Reversal of fibrosis: No longer a pipe dream?. Clin Liver Dis 2006; 10: 481–97. [4] Marra F. Hepatic stellate cells and the regulation of liver inflammation. J Hepatol 1999; 31: 1120–30. [5] Gutierrez-Ruiz MC, Gómez-Quiroz LE. Liver fibrosis: searching for cell model answers. Liver International 2007; 27: 434–439. 21

ACCEPTED MANUSCRIPT [6] Galli A, Svegliati-Baroni G, Ceni E, Milani s, Ridolfi f, Salzano R, et al. Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated

PT

mechanism. Hepatology 2005; 41: 1074–1084.

RI

[7] Pierce RJ, Dubois-Abdesselem F, Lancelot J, Andrade L, Oliveira G. Targeting Schistosome Histone Modifying Enzymes for Drug Development. Curr Pharm Des 2012; 18(24):3567-78.

NU

deacetylation. Annu Rev Biochem 2007; 76:75–100.

SC

[8] Shahbazian MD, Grunstein M. Functions of site-specific histone acetylation and

[9] Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000; 403:41–

MA

5.

[10] Pierce RJ, Dubois-Abdesselem F, Caby S, Trolet J, Lancelot J, Oger F, et al. Chromatin

Cruz 2011; 106(7):794-801.

TE

D

regulation in schistosomes and histone modifying enzymes as drug targets. Mem Inst Oswaldo

AC CE P

[11] Andrews KT, Haque A, Jones MK. HDAC inhibitors in parasitic diseases. Immunology and Cell Biology 2012 90; 66–77.

[12] Tan J, Cang S, Ma Y, Petrillo RL, Liu D. Novel histone deacetylase inhibitors in clinical trials as anticancer agents. J Hematol Oncol 2010 4; 3: 5. [13] Olivier LJ. Infectivity of Schistosoma mansoni cercariae. Am J Trop Med Hyg 1966; 15: 882-5. [14] Cansu A, Erdogan D, Serdaroglu A, Take G, Coskun ZK, Gurgen SG. Histologic and morphologic effects of valproic acid and oxcarbazepine on rat uterine and ovarian cells. Epilepsia 2010; 51: 98-107. [15] Gonnert R, Andrews P. Praziquantel, a new broad spectrum antischistosomal agent. Zeitschrift fur Parasitenkund 1977; 52: 129–50.

22

ACCEPTED MANUSCRIPT [16] Reitman S, Frankel S. Colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am J Clin Path 1957; 28: 56-63.

PT

[17] Ferreira Rde C, Montenegro SM, Domingues AL, Bandeira AP, Silveira CA, Leite LA, et

RI

al. TGF beta and IL13 in schistosomiasis mansoni associated pulmonary arterial hypertension; a descriptive study with comparative groups. BMC Infect Dis 2014; 14: 282.

SC

[18] De Souza Rda P, Cardoso LS, Lopes GT, Almeida MC, Oliveira RR, Alcântara LM, et al.

NU

Cytokine and Chemokine Profile in Individuals with Different Degrees of Periportal Fibrosis due to Schistosoma mansoni Infection. J Parasitol Res 2012;2012:394981.

MA

[19] Reddy GK, Enwemeka CS. A Simplified Method for the Analysis of Hydroxyproline in Biological Tissues. Clin Biochem 1996; Jun 29(3): 225-9.

TE

D

[20] Drury RB , Willington EA. Histological techniques, 5th ed. Oxford, New York, Toronto: Oxford University Press 1980; 140-249.

AC CE P

[21] Culling CFA. Tissues requiring special treatment or techniques. In: Handbook of Histopathological and Histochemical Techniques, 3rd ed. London: Butterworth &Co 1974; 40566.

[22] Andrade ZA. Schistosomal hepatopathy. Mem Inst Oswaldo Cruz 2004; 99: 51-7. [23] Homeida MA, el Tom I, Nash T, Bennett JL. Association of the therapeutic activity of praziquantel with the reversal of Symmers’ fibrosis induced by Schistosoma mansoni. Am J Trop Med Hyg 1991; 45:360-5. [24] Warren KS, Peters PA. Comparison of penetration and maturation of Schistosoma mansoni in the hamster, mouse, guinea pig, rabbit, and rat. Am J Trop Med Hyg 1967; 16:718–722. [25] Cheever AW. Quantitative comparison of the intensity of Schistosoma mansoni infections in man and experimental animals. Trans R Soc Trop Med Hyg 1969; 63:781–795.

23

ACCEPTED MANUSCRIPT [26] Stavitsky AB. Regulation of granulomatous inflammation in experimental models of schistosomiasis. Infect Immun 2004; 72:1–12.

PT

[27] Abdul-Ghani RA, Hassan AA. Murine schistosomiasis as a model for human

RI

schistosomiasis mansoni: similarities and discrepancies. Parasitol Res. 2010; 107(1):1-8. [28] Lambertucci JR. Revisiting the concept of hepatosplenic schistosomiasis and its challenges

SC

using traditional and new tools. Rev Soc Bras Med Trop 2014; 47(2): 130-6.

NU

[29] Dkhil MA. Role of berberine in ameliorating Schistosoma mansoni-induced hepatic injury in mice. Biol Res 2014; 47(1): 8.

MA

[30] Boros DL, Whitfield JR. Enhanced Th1 and dampened Th2 responses synergize to inhibit acute granulomatous and fibrotic responses in murine schistosomiasis mansoni. Infect Immun

TE

D

1999; 67: 1187–93.

[31] Burke ML, Jones MK, Gobert GN, Li YS, Ellis MK, McManus DP. Immunopathogenesis of

AC CE P

human schistosomiasis. Parasite Immunol. 2009; 31: 163–76. [32] El-Banhawey MA, Ashry MA, El-Ansary AK, Aly SA. Effect of Curcuma longa or parziquantel on Schistosoma mansoni infected mice liver: Histological and histochemical study. Indian J Exp Biol 2007; 45: 877-89. [33] Hellerbrand C, Stefanovic B, Giordano F, Burchardt ER, Brenner DA. The role of TGF beta1 in initiating hepatic stellate cell activation in vivo. J Hepatol 1999 Jan; 30(1):77-87. [34] de Bleser PJ, Niki T, Rogiers V, Geerts A. Transforming growth factor beta gene expression in normal and fibrotic rat liver. J Hepatol 1997; 26: 886-93. [35] Friedman SL. Cellular sources of collagen and regulation of collagen production in liver. Semin Liver Dis 1990; 10: 20-9.

24

ACCEPTED MANUSCRIPT [36] Schmitt-Graff A, Chakroun G, Gabbiani G. Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis. Virchows Archiv B Cell Path 1993; 422:99 107.

PT

[37] El-Lakkany N, el-Din SS, Ebeid F. The use of pentoxifylline as adjuvant therapy with

RI

praziquantel downregulates profibrogenic cytokines, collagen deposition and oxidative stress in experimental schistosomiasis mansoni. Exp Parasitol 2011 129; 152-7.

SC

[38] Attia YM, Elalkamy EF, Hammam OA, , Mahmoud SS, El-Khatib AS. Telmisartan, an AT1

NU

receptor blocker and a PPAR gamma activator, alleviates liver fibrosis induced experimentally by Schistosoma mansoni infection. Parasit Vectors 2013; 6; 199.

MA

[39] Graham BB, Chabon J, Gebreab L, Poole J, Debella E, Davis L et al. Transforming growth factor-β signaling promotes pulmonary hypertension caused by Schistosoma mansoni.

TE

D

Circulation 2013; 128: 1354-64.

[40] Liang Y-J, Luo J, Yuan Q, Zheng D, Liu YP, Shi L, et al. New Insight into the Antifibrotic

e20247.

AC CE P

Effects of Praziquantel on Mice in Infection with Schistosoma japonicum. PLoS ONE 6(5):

[41] Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc Natl Acad Sci USA 2004; 101: 18030–5. [42] Van Beneden K, Mannaerts I, Pauwels M, Van den Branden C, van Grunsven LA. HDAC inhibitors in experimental liver and kidney fibrosis. Fibrogenesis Tissue Repair 2013; 6(1):1. [43] Kim WR, Flamm SL, Di Bisceglie AM, Bodenheimer HC; Public Policy Committee of the American

Association

for

the

Study

of

Liver Disease.

Serum activity of alanine

aminotransferase (ALT) as an indicator of health and disease. Hepatology 2008; 47(4):1363-70. [44] Limdi JK, Hyde GM. Evaluation of abnormal liver function tests. Postgrad Med J. 2003; 79(932):307-12

25

ACCEPTED MANUSCRIPT [45] Ichiyama T, Okada K, Lipton JM, Matsubara T, Hayashi T, Furukawa S. Sodium valproate inhibits production of TNF-alpha and IL-6 and activation of NF-kappaB. Brain Res 2000; 857:

PT

246-51. [46] Watanabe T, Tajima H, Hironori H, Nakagawara H, Ohnishi I, Takamura H, et al. Sodium

RI

valproate blocks the transforming growth factor (TGF)-β1 autocrine loop and attenuates the

SC

TGF-β1-induced collagen synthesis in a human hepatic stellate cell line. Int J Mol Med 2011 28;

NU

919–25.

[47] Mannaertes I, Nuytten NR, Rogiers V, Vanderkerken K, van Grunsven LA, Geerts A.

MA

Chronic Administration of Sodium Valproate Inhibits Activation of Mouse Hepatic Stellate Cells In Vitro and In Vivo. Hepatology 2010; 51: 603-14.

TE

D

[48] Dubois F, Caby S, Oger F, Cosseau C, Capron M, Grunau C, Dissous C, Pierce RJ. Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and up-regulation of gene

AC CE P

transcription in Schistosoma mansoni. Mol Biochem Parasitol. 2009; 168(1):7-15.

Conflict of interest:

The authors declare no conflict of interest.

26