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 Table of Contents  
Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 251-258

Pentoxifylline modulation hepatotoxicity and apoptosis induced by nitrosamine in rats

1 Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Department of Anatomical Sciences, Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran

Date of Submission06-Apr-2020
Date of Acceptance02-May-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Dr. Shiva Roshankhah
Department of Anatomical Sciences, Medical School, Kermanshah University of Medical Sciences, Kermanshah
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_54_20

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Background: Pentoxifylline (PEN) is a xanthine derivative with different functional characteristics including dilution of blood and increase in tissue oxygenation rate. Nitrosamines (NITs) are well known as strong carcinogenic agents. This study attempts to show the histopathological and biochemical effects of PEN against hepatotoxicity induced by NIT in rats. Methods: Sixty-four rats were assigned to eight groups including the groups of sham: NIT (40 mg/kg); PEN (25, 50, and 100 mg/kg); and NIT + PEN. Experimental treatments were applied either intraperitoneally (for NIT) or orally (for PEN) daily for 4 weeks. The relative expression level of p53 and Bax genes and hepatocyte apoptotic index were analyzed. Liver malondialdehyde (MDA), tissue ferric-reducing ability of plasma (FRAP), the diameters of hepatocytes (DH) and central hepatic vein (CHV), and biochemical liver function indicators (LFI) were investigated. Griess technique was hired for the determination of the level of serum nitrite oxide (NO). Results and Conclusions: NIT significantly increased the level of apoptotic gens and index, MDA, NO, diameter of CHV and DH, and LFI and decreased the FRAP level compared to the sham group (P < 0.01). All parameters in PEN and PEN + NIT groups significantly reduced (except FRAP level, which was decreased) in compared to the NIT group (P < 0.01) . By summarizing the results of this research, it is concluded that the PEN administration alleviates the hepatotoxicity due to oxidative stress produced by NIT in rats.

Keywords: Apoptosis, hepatotoxicity, nitrosamine, pentoxifylline

How to cite this article:
Salahshoor MR, Jalili C, Abdolmaleki A, Roshankhah S. Pentoxifylline modulation hepatotoxicity and apoptosis induced by nitrosamine in rats. Biomed Biotechnol Res J 2020;4:251-8

How to cite this URL:
Salahshoor MR, Jalili C, Abdolmaleki A, Roshankhah S. Pentoxifylline modulation hepatotoxicity and apoptosis induced by nitrosamine in rats. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Dec 3];4:251-8. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/251/294859

  Introduction Top

The term nitrosamine (NIT) refers to a specific carcinogenic compound with numerous therapeutic capabilities and functions.[1] Most of the NIT-based injuries are related to the presence of nitrites and secondary amines in their biochemical structures.[2] Nitrites can also be converted to NIT in the body and the acidic environment of the stomach.[3] Moreover, NIT exists in amines and nitrite-containing foods (naturally or as a preservative).[4] NIT induces the oxidative stress, genetic material damages, lipid peroxidation, and an excess protein deposit in the body tissues by the generation of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide.[5] The liver is a highly vulnerable organ to be affected by ROS damages, probably due to the abundance of polyunsaturated fatty acids available in the liver.[6]

Pentoxifylline (PEN) has antitumor necrosis factor-alpha (TNF-α) properties.[7] PEN is widely used in the treatment of cardiovascular disorders, diabetic nephropathy, alcoholic hepatitis, and venous leg ulcers.[8] PEN with the chemical name of 1-(5-oxohexyl)-3, 7-dimethylxanthine is a synthetic xanthine derivative, which inhibits the effects of xanthine oxidase enzymes.[9]

The body has cellular defense systems against ROS, protecting the macromolecules and biological membranes against oxidation process by a disruption in ROS production. The antioxidant system consists of enzymatic and nonenzymatic low-weight compounds.[10] However, the body is not completely able in the prevention of damages caused by free radicals, especially in the acute incidence of the diseases. Thus, the application of antioxidants reduces cellular damage and prevents ROS-based diseases.[11] Antioxidants inhibit the oxidation process in cells and protect them against the harmful effects of environmental offensive factors.[12] PEN, as a noncompetitive inhibitor of phosphodiesterases, increases the amount of cyclic adenosine monophosphate (cAMP), activates the protein kinase A, and inhibits the TNF-α. According to the above-mentioned pharmacological properties of PEN, this substance reduces the production of leukotrienes, enhances the innate immunity, and thus finally leads to inflammation suppression.[13]

Because the PEN has diverse biochemical and antioxidant properties, it is capable to improve the capillary blood flow and tissue oxygenation rate.[14] The antioxidant effects of PEN on the neutrophils in response to superoxide are the result of NADPH oxidase.[15] Daily intake of food with high amounts of NIT-containing compounds along with the antioxidant effects of PEN made us to design a study in order to evaluate the probable effects of PEN on NIT-hepatotoxicity in male Wistar rats.

  Methods Top

Preparation of chemicals, drugs, and laboratory kits

Ether, formalin, ferric-reducing ability of plasma (FRAP), sodium acetate, ferric chloride, iron sulfate, hematoxylin and eosin stain (HE), and zinc sulfate powder were purchased from Sigma Corporation (St Louis, Missouri, USA). Commercial kits were obtained from Pars Azmoon Company (I. R. Iran). NIT, PEN, and all other buffer additives and solvents were obtained from Merck (Honenbrunn, Germany). PEN was dissolved in distilled water and diluted by normal saline (0.9%) to prepare different experimental doses. The stock solution was passed through a 0.45-mm pore size filter (Lida Manufacturing, Kenosha, Wis. USA). NIT, 200 μL from 1 g/mL stock solution in normal saline, was added to 19.8 mL of phosphate-buffered saline to prepare 40 mg/mL NIT solution for the injection.


Sixty-four male Wistar rats (220–250 g) (were purchased from the Pasteur Institute of Iran (IPI, Tehran, Iran). The animals were kept under standard conditions for 12 dark/12 light photocycle and at a temperature of 22°C ± 2°C in special plastic cages. Water and food were freely available to all animals. They were fed with standard pelletized food and treated with municipal water. All investigations were conformed to the ethical principles of animal research and were approved by the Ethics Committee of Kermanshah University of Medical Sciences (no. 1395.540).

Study design and treatment of animals

A total of 64 male Wistar rats were randomly divided into eight groups. The experimental groups included: Sham group, which received normal saline; NIT group, which received 40 mg/kg single dose of NIT; three different PEN groups, which received 25, 50, and 100 mg/kg of PEN for 4 weeks; and three different groups of NIT + PEN, which received a single dose of 40 mg/kg NIT followed by 25, 50, and 100 mg/kg of PEN for 28 days. The NIT and normal saline administration were applied intraperitoneally, and PEN was prescribed orally.[1]

Animal dissection and tissue sampling

The rats were anesthetized with ether inhalation. The heart was exposed by paramedian bilateral thoracotomy to access the right ventricle for 5 cc blood aspiration. The blood sample was incubated at 37°C for 15 min. Then, the clot was centrifuged at 3000 rpm for 15 min to serum separation. The serum was stored at −70°C for measurement of liver function indicators (LFI) parameters, blood nitrite oxide (NO) level, oxidative stress, and antioxidant capacity level. The animals' liver was also fixed in 10% formalin solution for histological and morphometric investigations.[6]

Oxidative stress

The level of oxidative stress in the liver was assessed by colorimetric analysis. Thus, the thiobarbituric acid-reactive species were measured using malondialdehyde (MDA) as the final product of lipid peroxidation. Briefly, three separate solutions each with a volume of 1400 μl including acetic acid, TBA, and sodium dodecyl sulfate, were added to 100 μl of liver homogenate, and the mixture was stirred for 50 min (previous materials were supplied by Sigma, USA). 4 ml of 1-butanol (Sigma, USA) was added to the previous mixture and centrifuged at 5000 rpm for 15 min. The absorbance of the higher layer was measured at 532 nm (Spectro; Germany), and sequential concentrations of tetra ethoxy propane (Sigma, USA) were used as the external standard. The liver antioxidant capacity was measured using the FRAP assay. The FRAP substance consisted of 30 ml of acetate buffer (Sigma, USA) and 1.5 ml of chloride ferric (Sigma, USA). Briefly, 60 μl of liver homogenate was added to 1.5 ml of newly prepared solution. The FRAP substance (Sigma, USA) available in a test tube was incubated at 37°C for 10 min. The absorbance of the blue-colored complex was read against a blank at 593 nm. Sequential concentrations of FeSO4.7H2O (Sigma, USA) were used as an external standard.[1]

Biochemical liver function indicators assessment

The liver was split and turned into a homogenous solution. To separate the biological enzymes, the obtained solution was centrifuged twice at 10,000 rpm for 15 min. The supernatant was separated for enzyme measurement. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were examined by the Reitman and Frankel method. Alkaline phosphatase (ALP) activities were determined according to the procedure set out in the practical laboratory manual.[11]

Nitrite oxide test

NO assessment applied by the means of microplate technique is measured by Griess assay. For this purpose, the sample was defrosted and the supernatant (400 μl) was deproteinized by 6 mg zinc sulfate under the condition of centrifugation. The solution was mixed with 100 μl of vanadium chloride, 50 μl of N-(1-naphthyl) ethylenediamine dihydrochloride, and 50 μl of sulfonamide. 0.1 M sodium nitrite was used for the standard curve, and increasing concentrations of sodium nitrite (5, 10, 25, 50, 75, and 100 μM) were prepared. The Greiss solution was added to all microplates containing sodium nitrite and supernatant. The reading was done through an ELISA reader (Stat Fax 100, Miami, FL, USA) at a wavelength of 540 nm.[1]

Morphological and histopathological examinations

For histological examinations, a vertical incision was made just below the right lobe of the liver. The liver sample was washed and placed in 10% formalin for a week. For tissue preparation, the sample was dehydrated in ascending concentrations of ethanol, cleared by xylene solution, and then embedded in paraffin. Thin 4-μm sections were sliced using a microtome (Leica RM 2125, Germany) and stained with H and E staining. Images were captured by the 40x objective lens. In the images, the full cellular area, hepatocyte outline, and maximum and minimum axes of each hepatocyte (to obtain the mean axis) were measured. At least 50 hepatocytes from each zone were assessed in each liver. A separate measurement diameter of central hepatic vein (CHV) was performed using the same assay too. The histological data were collected using an Olympus BX-51T-32E01 research microscope (Leica RM 2125, Nussloch, Germany) connected to a DP12 Camera (Olympus Optical, Tokyo, Japan) with a 3.34-million pixel resolution and Olysia Bio-software (Olympus Optical Co. LTD, Tokyo, Japan).[11]

Apoptosis assay

The TUNEL test was performed according to the manufacturer's procedure (Roche, Germany). Paraffin-embedded blocks were prepared using an automatic tissue processor. Five-μm histological slices were cut through a microtome (Leica, Germany), and five slices per rat were selected. The tissues were subjected to deparaffinization. After routine deparaffinization and blocking, the slices were exposed to terminal deoxynucleotidyl transferase (TdT) with digoxigenin (DIG). DIG labeling and counterstaining were then carried out. To quantitate the rate of apoptosis, the number of positive cells within the liver was calculated.[6]

Real-time quantitative polymerase chain reaction

The real-time polymerase chain reaction evaluated the liver gene expression of p53 and Bax. The tissue was frozen in nitrogen and then stored in at −70°C freezer. RNA was removed using RNeasy mini kit (Qiagen, Tehran, Iran) based on the manufacturer's instructions. DNA was treated via DNase set kit (Qiagen, Tehran, Iran). The RNA was hired for cDNA production through cDNA Synthesis Kit. The expression level of the related genes was measured through glyceraldehyde-3-phosphate dehydrogenase primer as endogenous control by SYBR Green (Fermentas, Tehran, Iran) through comparative Ct method. The primer nucleotide sequences are listed in [Table 1].[16]
Table 1: Primers used in real-time polymerase chain reaction

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Statistical analysis

Kolmogorov–Smirnov test was first conducted to confirm the data compliance of the normal distribution. One-way analysis of variance (one-way ANOVA) was used for statistical analysis, and comparison among the different groups was made using Tukey post hoc test. SPSS Software (SPSS, New York: IBM, SPSS version 16.0) was used for data analysis, and the results were expressed as the mean ± standard error. P < 0.05 was considered statistically significant.

  Results Top

Oxidative stress

The results of the oxidative stress assessments indicated that the level of liver MDA statistically significantly increased in the NIT group compared to the sham control group (P < 0.01). The liver MDA level decreased statistically significantly in all NIT + PEN experimental groups compared to the NIT group (P < 0.01). Similarly, the NIT statistically significantly decreased the liver FRAP level of the NTI group in comparison with that of the sham group (P < 0.01). Administration of PEN statistically significantly increased the FRAP level in all NIT + PEN groups compared to the NIT group (P < 0.01). Treatment with PEN in all groups had no statistically significant differences in the liver FRAP and MDA levels compared to the sham group (P > 0.05) [Figure 1].
Figure 1: Comparison of NIT, sham, and PEN groups of (a) hepatic malondialdehyde level and (b) tissue ferric-reducing ability of plasma level. *Significant difference compared to the sham group (P < 0.01).*Significant difference compared to the NIT group (P < 0.01).Significant difference compared to the NIT group (P < 0.01). NIT: Nitrosamine, PEN: Pentoxifylline

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Biochemical liver function indicators evaluation

PEN led to a statistically significantly increased level of ALT, AST, and ALP enzymes in comparison with the sham group (P < 0.01). The mean concentration of ALT, AST, and ALP enzymes showed no statistically significant differences in all PEN groups compared to the sham group (P > 0.05). In addition, in all PEN and NIT + PEN groups, significant decrease was recorded in the mean of ALT, AST, and ALP enzymes in comparison with the NIT group (P < 0.01) [Table 2].
Table 2: Different liver enzymes among the treatment groups

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Nitrite oxide

The results of blood serum NO measurement showed a statistically significant increase in the NIT group compared to the sham group (P < 0.01). The mean NO level showed no statistically significant differences in all PEN groups compared to the sham group (P > 0.05). In addition, the mean level of NO was statistically significantly reduced in all doses of PEN and NIT + PEN groups compared to the NIT group (P < 0.01) [Figure 2].
Figure 2: Effects of PEN, NIT, and PEN + NIT on the average nitrite oxide levels. *Significant difference compared to the sham group (P < 0.01).*Significant difference compared to the NIT group (P < 0.01).Significant difference compared to the NIT group (P < 0.01). NIT: Nitrosamine, PEN: Pentoxifylline

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Morphometric assessment

The mean diameter of CHV and diameter of hepatocytes (DH) in the experimental groups showed a significant increase among the sham and NIT groups (P < 0.01). The mean diameter of CHV and DH showed no significant differences in all PEN groups compared to the sham group (P > 0.05). Further, PEN and NIT + PEN significantly reduced the mean diameter of CHV and DH in all the treated groups in comparison with the NIT group (P < 0.01) [Figure 3].
Figure 3: Effects of NIT, PEN, and PEN + NIT administration on the diameter of CHV (a) and DH (b). *Significant difference compared to the sham group (P < 0.01).*Significant difference compared to the NIT groups (P < 0.01).Significant difference compared to the NIT group (P < 0.01). NIT: Nitrosamine, PEN: Pentoxifylline, CHV: Central hepatic vein, DH: Diameter of the hepatocyte

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Histopathological changes

Histological evaluation showed physiologic liver structure in the sham and PEN treatment groups. After treatment with LPD in the NIT group, the liver showed obvious histological damage changes. These anomalies included the increment in white blood cells (indicating inflammation), increased pathological irregularities, sinusoidal dilatation, and the vacuolization in hepatocytes (showed necrosis process). Treatment with NIT + PEN in all the experimental groups reduced the liver damage caused by the NIT [Figure 4].
Figure 4: Microscopic images of the liver in different groups (5 μm, H and E, ×100). Micrograph of the liver section in the sham groups (a), normal liver structure. Micrograph of the liver section in NIT group (b), increased white blood and macrophage cells (inflammation) (black arrows), central hepatic vein dilatation (blue arrows) and hyperemia (yellow arrows), due to the oxidative stress caused by NIT. Micrograph of the liver section in PEN (100 mg/kg) group (c), normal liver structure. Micrograph of the liver section in PEN + NIT (100 mg/kg) group (d), normal liver structure. NIT: Nitrosamine, PEN: Pentoxifylline

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Apoptotic index

The apoptotic index (AI) was significantly higher in NIT group compared to the sham group (P < 0.01). No statistically significant differences were similarly found in the AI in PEN groups as compared to the sham group (P > 0.05). Furthermore, whole several doses of PEN in NIT + PEN groups represented a statistically significant decline in the AI as compared to the NIT group (P < 0.01) [Figure 5].
Figure 5: Apoptotic induction (a-d) and index (e). Apoptosis induction (a-d) (400 × magnifications, TUNEL staining): sham group (a), PEN group (100 mg/kg) (b), NIT group (40 mg/kg) (c) and PEN (100 mg/kg) + NIT (40 mg/kg) group (d). Red nuclei refer to apoptotic cells. Apoptotic index (e): *Significant difference to the sham group (P < 0.01).*Significant difference to the NIT group (P < 0.01).Significant difference to the NIT group (P < 0.01). NIT: Nitrosamine, PEN: Pentoxifylline

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Apoptotic gene expression levels

Upregulated changes of p53 and Bax genes in the NIT group compared to the sham group were statistically significantly detected (P < 0.01). In addition, a significant downregulation of these genes similarly was distinguished in all doses of PEN and NIT + PEN groups as compared to the NIT group [Figure 6].
Figure 6: Relative gene expression following PEN and NIT administration. *Statistically significant (P < 0.01) between NIT and sham groups.*Statistically significant (P < 0.01) between PEN and NIT groups.Significant modifications in PEN + NIT groups as compared to the NIT group (P < 0.01). NIT: Nitrosamine, PEN: Pentoxifylline

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  Discussion Top

PEN improves blood flow and oxygenation in vessels due to its various biochemical and antioxidant properties.[8] NIT can cause a wide range of histological damages such as disruption in CNS and retinal cell development, hepatic and cardiovascular diseases, autoimmune disturbances, and allergic reactions.[1]

In the present study, the effect of PEN on the NIT-induced oxidative stress was investigated. The findings of the current survey suggest that NIT administration has adverse and destructive effects on the histological structure, apoptotic gene index, function of the liver, oxidant–antioxidant equilibrium, and increase in the NO level. On the other hand, the PEN has an antioxidant relief effect on some liver parameters in NIT-administered animals. We also concluded that the PEN can reduce the level of lipid peroxidation and increase antioxidant capacity in liver tissue.

Thus, it appears that PEN with its antioxidant properties could reduce MDA and increase FRAPS levels. These important biochemical changes occur by the inhibitory effect of PEN on ROS production. Attention to the changes in NO levels revealed a significant increase in the level of NO in the NIT group compared to the sham group. In this study, administration of PEN significantly changed the NO level to a reduced state. It appears that NIT triggers the NO generation by stimulating the release of noradrenaline in the paraventicular and amygdala nucleus.[17] On the other hand, the antioxidant molecules can attenuate the activity of NO production system including enzymatic proteins, substrates, and cofactors.[18] The results of this study are in agreement with the findings of Jalili et al.,[1] which showed that the PEN can cause a reduction in NO level in the state of NIT-induced renal damages. Garcia et al.[19] reported that treatment with different doses of PEN in diabetic rats reduced the expression level of NO, which is also being in agreement with the results of the present study. It seems that oxidative stress available in cells will launch the NO production enzymes, which consequently leads to a decrease in cell survival.[17]

Due to the high consumption of oxygen, the mitochondrial dysfunction, especially in the liver, may increase the production of free radicals such as NO too.[11] PEN also recovers the cell damage offered by reduced MDA level, rate of oxidation, and hepatic histological alterations. The current study also shows that the PEN can reduce the lipid peroxidation (decreased state of MDA) and increase the antioxidant capacity of the liver, causing a reduction in oxidative stress. Consistent with these findings, a large body of studies has shown the antioxidant properties of PEN.[1],[7],[8] PEN seems to inhibit the liver lipid peroxidation induced by Tert-Butyl hydroperoxide.[20] Further, the PEN as a lipophilic molecule can inhibit the lipid peroxidation by the Fenton reaction.[21] Surveys such as that conducted by Donate-Correa et al. showed that PEN attenuated the shock of oxidative stress and lipid peroxidation in diabetic renal disease, which is in line with the results of the present study.[22] Thus, it appears that PEN with its antioxidant properties could reduce the MDA in the treatment groups by inhibiting the ROS production.[23] The present study also indicates the recovery effects of PEN on some liver parameters as well as decreasing the oxidative stress due to the decline in the MDA level. The toxicity of NIT administration can lead to changes in biochemical and antioxidant content in the blood and liver tissue. Therefore, the mechanism of NIT toxicity is implemented by oxidative stress.[24]

Our findings showed an increasing trend of DH and CHV values in NIT-treated animals. The treatment of animals with PEN in all doses decreased the DH and CHV variables compared to the NIT group. Another important finding is some explicit hepatic changes in the NIT group including the enhanced state of the sinusoidal spaces, macrophage accumulation around the CHV and the lymphoid cells penetrated in the portal space, and also CHV diameter enlargement. It seems that the invasion of free radicals to hepatocytes causes necrosis in the cells of liver parenchyma.[11] These cells can induce hepatic inflammatory responses, which lead to tissue damage by mononuclear inflammatory cells. The necrotic cells also release pro-inflammatory mediators which can exacerbate the poison-induced liver injuries.[25] Apparently, the macrophages are switched to active status in response to the tissue injury. They release stimulant mediators, such as the TNF-α, interleukin-1, and NO.[26] In the present study, the macrophages are actually the same as copper cells located in the hepatic sinuses. It may seem that the copper cell aggregation and the secretion of toxic mediators, with no symptoms of cell death, are involved in the state of hepatotoxicity and necrosis of hepatocytes.[16] Moreover, free radical production and subsequent presence of oxidative stress can be of the most critical and essential causes of the hepatocyte death.[11]

A recent study reported by Khan and Alghamdi[27] also support our data, in which it was said that the hepatic injuries and apoptosis induction in hepatocytes can be caused by NIT consumption. NIT induced-production of free radicals may invade hepatocytes and cause necrosis in parenchymal cells. These cells can trigger the inflammatory responses in the liver and cause infiltration of mononuclear inflammatory cells.[28] The necrotic cells by the pro-inflammatory mediator's release can exacerbate the toxin-induced hepatic injuries.[16] Together, the studies provide the important insight into this fact that the oxidative stress induced by NIT administration can produce active ROS with notable examples of hydroperoxides, singlet oxygen, hydrogen peroxide, and superoxide. These chemical substances lead to the destruction of the cell, DNA, proteins, and intracellular lipids, and ultimately the liver tissue leads to injury.[29] PEN appears to own a protective effect against hepatic fibrogenesis. This effect is due to the following features of polyphenolic capacity, inhibitory effects on stellate cell activity, disorganization of the signal transduction pathways, and expression of the cell cycle proteins.[30] Stellate cells play a crucial role in the establishment of hepatic fibrosis and oxidative stress conditions.[11]

Recent evidence suggests that PEN can inhibit the MAPK phosphorylation in activated microglia and exert its anti-inflammatory effects on nuclear factor-kappa β (NF-κβ) pathway.[31] PEN can inhibit the NF-κβ by reducing H2O2 production, inhibiting the IKβ kinase, and phosphorylation and depleting of P65.[32] Previous research findings by Hammerman et al.[9] illustrated that PEN inhibits the induction of xanthine oxidase-induced ischemia–reperfusion injury, which is in consistent with the results of the current study.

The results of this study indicate that there are significant diversities among liver antioxidant capacity and liver enzymes levels in the NIT and the sham control groups. Similarly, there is a negative correlation between hepatic antioxidant capacity in the NIT group and AST, ALT, and ALP levels in the groups which received PEN and NIT + PEN. The increment in the activity index of hepatic enzymes that exist in blood serum indicates the liver injury in the current study. Moreover, the findings of Peng et al.[33] confirmed the results of this study in that the PEN could decrease the serum levels of ALT, AST, and ALP. These enzymes can be released into the blood flow due to the incidence of necrosis or cell membrane damages.[11] Data from several sources have identified that the NIT can induce damage to the integrity of the cell membrane by the inhibition of 1–4 respiratory chain reaction complexes.[34] The results are in agreement with Rajamanickam et al.'s[29] findings, which revealed that the NIT administration in male rats for 1 week induces the increased activity of liver enzymes and conversely reduces the total antioxidant capacity. PEN by prevention of lipid peroxidation stabilizes the cell membranes and prevents the enzyme leakage.[35] PEN effects on antioxidant enzymes activity, lipid metabolism and reduction in lipid peroxidation.[1]

In this study, the PEN also showed decreased effects of Bax and p53 (apoptotic genes) gene expression and apoptotic hepatocyte index. P53 makes the mitochondrial membrane permeable to the influx of cytochrome-c into the intracellular matrix. Accordingly, p53 adjusts the function of apoptotic elements such as Caspase and Bax.[17] As cell death is seen in hepatocytes, it is stated that the NIT has upregulatory effects on apoptotic factors.[36] In addition, PEN directly translocates within the intranuclear space to induce downregulation of related genes.[37] Elgendy also found that the apoptotic genes are expressed significantly after NIT administration in hepatocellular carcinoma.[38] In the current study, the PEN reduced apoptotic cell index and apoptotic gene expression to prevent cell death. Based on the obtained results, it can be indicated that the PEN own positive antioxidant effects on molecular function and histological construction of liver cells.

  Conclusions Top

The results of this study indicated that PEN may recover some liver dysfunctions in rats treated with NIT. It was found that PEN reduces ROS, cell apoptosis, expression of apoptotic gens, activating antioxidant agents, and detoxifying enzymes. Therefore, PEN might be considered to recover the functional and histological of the hepatocyte exposed to NIT. This could help the liver of individuals who have been exposed to NIT to act more effectively. The antioxidant properties of PEN may be the main reason for its positive effect on liver parameters; however, additional studies are required to define its exact molecular mechanism of action.


We are thankful to the director of the Department of Anatomical Sciences, Medical School of Kermanshah University of Medical Sciences, for providing technical inputs.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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