MATERIALS AND METHODS

Chemicals and reagent kits: Carbon tetrachloride, curcumin, thiobarbituric acid, GSH, superoxide dismutase, epinephrine and 5,5’-dithio-bis-(2-nitrobenzoic acid; DTNB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Activities of GSH-Px, GSH-ST, glucose-6-phosphate dehydrogenase (G6PD) and catalase (CAT) were determined using GSH-Px, GSH-ST, G6PD and CAT commercial kits (Bio-Diagnostic Company, Egypt). All other chemicals were bought from nearby sources with the highest analytical degree.

Animals, experimental design and doses: A total of 30 healthy adult BALB/c male mice weighting 25-30 g/each were purchased from the Theodor Bilharz Institute (Imbaba, Cairo, Egypt) and assigned into groups. All experimental animals preserved in a specific pathogen-free environment. The experimental animal protocols were done according to guidelines accepted by the Institutional Animal Care and Use Committee, which were approved by Assiut University. All procedures concerning the experiment were performed according to the Declaration of Helsinki and the guidelines for the care and use of experimental animals proven by the National Institutes of Health (NIH). Prior to the experiment, all animals for one week were acclimatized in plastic cages (10 animals per cage) inside a well-ventilated room (animal room, Zoology Department, Faculty of Science, Assiut University, the experiment lasted from mid-January 2019 until mid-March of the same year). The animals were kept under typical laboratory conditions (temperature of 23°C, relative humidity of 60-70% and a 12 h light/dark cycle) and were fed a diet of standard commercial pellets (contain 20% crude protein and 11% crude fiber, rich in protein and energy) and water containing libitum. Later, 1 week after acclimation, mice were randomly separated into 3 groups (10 mice each, n = 10).

Animal treatments: Control mice were intraperitoneally injected (i.p.) with olive oil (vehicle; 0.1 mL kg^–1 b.wt.) 3 times/weekly for 8 weeks. The CCL₄ group received CCL₄ (0.1 mL kg^–1, i.p.) 3 times/weekly for 8 weeks. The CCL₄-curcumin group, intraperitoneally injected with CCL₄ (0.1 mL kg^–1) and curcumin (200 mg kg^–1) was intragastrically administered 3 times/weekly for 8 weeks. The third group (CCL₄-curcumin group) intragastrically received curcumin (200 mg kg^–1) prior 3 days of CCL₄ treatment.

In the second group, toxicity was stimulated in the liver and spleen. All animals received good human care and the study protocols were in agreement with Institutional Guidelines for the use of laboratory animals.

Collection and preparation of samples: All animals were sacrificed at day 57 post-CCL₄ injection and blood was collected from the heart to obtain plasma for biochemical studies. We stored the plasma at -80°C until use. Removed liver and spleen, washed in saline (4°C), cut into small pieces. Part was immersed in 10% neutral buffered formalin for histopathological study. Other samples were homogenized in 0.1 M cold phosphate buffered (PBS) (pH 7.4) using IKA Yellow line DI homogenizer (18 Disperser, Germany) at 6000 rpm for 1 h at 4°C. Supernatant was frozen at -20°C for biochemical assays. For RNA extraction and gene expression analysis another samples were suspended in trizol solution.

Analytical measurements: Alkaline phosphatase activity (ALP, IU L^–1) were determined as Belfield and Goldberg¹⁶, aspartate aminotransferase (AST, U L^–1) and alanine aminotransferase (ALT, U L^–1) activities were measured by standard spectrophotometric, according to the method described by Reitman and Frankel¹⁷. The total protein content in liver and spleen tissues were determined colormitically using the method of Lowry et al.¹⁸ Malondialdehyde (MDA) levels in liver and spleen tissues were estimated by the method of Ohkawa et al.¹⁹, using thiobarbituric acid (TBA). Glutathione (GSH) levels were measured deepening on the method of Beutler et al.²⁰. Superoxide dismutase (SOD) activity was estimated using the method of Misra and Fridovich²¹. The CAT, GSH-Px, GSH-ST and G6PD were determined according to the methods described by Aebi²², Paglia and Valentine²³, Habig et al.²⁴ and Kornberg et al.²⁵, respectively.

Gene expression analysis: The effect of CUR administration on the expression for some selected genes implicated in the inflammation and toxicity of liver and spleen were studied using real-time quantitative PCR (RT-PCR). We designed the primers used for quantitative (RT-PCR) analysis using the Primer Express 1.5 software (Applied Biosystems). The mouse primers were designed as following: Nox2, Forward: 5’-TCCGTATTGTGGGAGACTGG-3’, Reverse: 5’-AAAGGGCGTG ACTCCAATC-3’; COX-2 Forward: 5’-CCTAGATAACAGAGCCGC TTTC-3’, Reverse: 5’-TTTCACCATAGAATCCAGTCCG-3’; 5-LO Forward: 5’-ATTGCCATCCAGCTCAACCAAACC-3’, Reverse: 5’-TGGCGATACCAAACACCTCAGACA-3’; TNF-α Forward: 5-ATGAGCACAGAAAGCATGA-3, Reverse: 3-AGTAG ACAGAAGAGCGTGGT-5; IL-6 Forward: 5-ACCGAGCTCTGTT GACAAG-3, Reverse: 3-TCCTCGCCACACTTCTCTTT-5; IL-1β Forward: 5-GCACTACAGGCTCCGAGATGAAC-3, Reverse: 3-TTG TCGTTGCTTGGTTCTCCTTGT-5 and normalized using GAPDH Forward: 5-GTTGTCTCCTGCGACTTCA-3, Reverse: 3-GGTGGTCC AGGGTTTCTTA-5. After performing the indicated treatments, RNA was extracted and then the corresponding cDNA was prepared and then real time PCR was applied for gene expression analysis as earlier designated²⁶. All cDNA samples were managed in a 96-well plate using the following cycling conditions: 10 min at 95°C and 40 cycles at 95°C for 15 sec ended by 1 min at 60°C. These data were analyzed according to Livak and Schmittgen²⁷.

Histopathological analysis: After blood withdrawal anesthetized animals were euthanized by cervical dislocation then dissected for extraction of liver and spleen. Pieces of each organs 2×2 mm were fixed in 10% buffered for 24 h and routinely processed for paraffin embedding.

Blocks has been prepared, then sections 5 were cut and stained with hematoxylin and eosin stain H and E²⁸. The stained slices were examined by the light microscope (Olympus CX31, Japan) and photographed using a digital camera (Olympus, Camedia C-5060, Japan).

Statistical analysis: By using one-way analysis of variance (ANOVA) followed by Newman-keuls Multiple Comparison Test as a post-test, we analyzed the results statistically. These analysis were executed using a computer prism program for Windows, version 7.0 (Graph Pad software, Inc, San Diego CA. USA). The level of significance between groups was accepted at p<0.05, 0.01 and the data were expressed as mean±Standard error (SE).

RESULTS

Analytical measurements
Serum function tests: The results indicate that mice administered with CCL₄ (0.1 mL kg^–1), showed marked hepatic damage (group 2) when compared to control (group 1) this was evidenced by a marked increase in the levels of serum liver enzymes, (AST, ALT and ALP) in (group 2) when compared to control (group 1), co-treatment of mice with CUR (group 3) resulted in a significant decrease in these enzymes (Table1).

Oxidative stress markers
A-Free radicals
A1-Lipid peroxidation (LPO): A significant increase of liver and spleen tissues of malondialdehyde (MDA) in concentrations was observed in group 2, (animals treated with CCL₄) as a product of lipid peroxidation, CUR treatment (200 mg kg^–1) reduced this CCL₄-induced increase in MDA (Table 2).

B-Antioxidants
B1-Enzymatic antioxidants: It was observed that treatment of animals with CCL₄ (group 2) resulted in a significant decrease in the activities of CAT, GSH-Px, GSH-ST and SOD in liver and spleen tissues, while activates of G6PD were significantly increased. Co-treatment with CUR (group 3) restored all these activities nearly to the control results (Table 2).

B2-Non-enzymatic antioxidants: The data shown in (Table 2) confirm the decrease in GSH level in CCL₄-treated mice, co-treatment with CUR significantly recovered the decreased total GSH level (Table 2).

Gene expression analysis (anti-inflammatory and pro-inflammatory markers): The mRNA expression of TNF-α, IL-6, IL-1, NOX2, COX-2 and 5-LO levels of liver and spleen were significantly increased in mice administrated with CCL₄ compared to control group. Significant reduction in all raised parameters was observed when CUR was given along with CCL₄ compared with CCL₄ treated group (Fig. 1-4).

Fig. 1:

Effect of CCL4 and curcumin on IL-1, IL-6 and TNF-α mRNA expression, measured using real time PCR in BALB/c mice liver Data were reported as Mean±SE, n = 8-10, *p<05, **p<0.01, ***p<0.001, aSignificance difference between control group and CCL4 group, bSignificance difference between CCL4 group and curcumin co-treated group

Fig. 2:

Effect of CCL4 and curcumin on NOX2, COX-2 and 5LO mRNA expression, measured using real time PCR in BALB/c mice liver Data were reported as Mean±SE, n = 8-10, *p<05, **p<0.01, ***p<0.001, aSignificance difference between control group and CCL4 group, bSignificance difference between CCL4 group and curcumin co-treated group

Fig. 3:

Effect of CCL4 and curcumin on IL-1, IL-6 and TNF-α mRNA expression, measured using real time PCR in BALB/c mice spleen Data were reported as Mean±SE, n = 8-10, **p<0.01, ***p<0.001, aSignificance difference between control group and CCL4 group, bSignificance difference between CCL4 group and curcumin co-treated group

Fig. 4:

Effect of CCL4 and curcumin on NOX2, COX-2 and 5-LO mRNA expression, measured using real time PCR in BALB/c mice spleen Data were reported as Mean±SE, n = 8-10, **p<0.01, ***p<0.001, aSignificance difference between control group and CCL4 group, bSignificance difference between CCL4 group and curcumin co-treated group

Histopathological analysis: Histopathological findings of the liver were evaluated by light microscopy. Examination of the liver tissues of control rats had normal histoarchitecture of hepatocyte (Fig. 5a, b). Nevertheless, histopathological examination of HE-stained sections revealed that the treatment of male rats with CCL₄ showed vascular and cellular changes. The vascular changes were in the form of congestion and dilatation of the central veins (Fig. 5c). The cellular changes were in the form of an infiltration by mononuclear inflammatory cells and diffuse hyperplasia of kupffer cells (Fig. 5d). Moreover, hepatocytes were showed by rarified areas of cytoplasm (Fig. 5e), while examination of liver sections of curcumin treated group showing normal histological appearance (Fig. 5f).

Examination of the spleen tissues of control and olive oil treated rats had normal histoarchitecture (Fig. 6a, b). Histopathological examination of male rats treated with _CCL4 showed scattered necrotic areas in the parenchyma and increased number of macrophages were seen (Fig. 6c, d). In addition, there was an apoptotic change in megakaryocyte with fragmented nuclei (Fig. 6e), while examination of spleen sections of curcumin treated groups showed histological appearance nearly similar to the control (Fig. 6f).

DISCUSSION

In this study, the significant rise in serum AST, ALT and ALP activities in CCL₄ group as compared with the control group is matched with the findings of Prakash et al.²⁹, Kone et al.³⁰. This outcome is in harmony with the extensive hepatocellular damage noted in the histopathological section of the current study because these hepatic enzymes are cytoplasmic in position and are released into circulation after loss of cell membrane integrity³¹. Results of the current study demonstrated that CUR caused a significant reduction in serum ALT, ALP and AST activities which return to normal along with healing of hepatic parenchyma and renewal of hepatocytes. Prevention of hepatic enzymes from leakage into the circulation can be attributed to the antioxidant, anti-inflammatory and anti-apoptotic properties of CUR^32,33 which has an important role in maintaining the membrane integrity against the cytotoxicity of CCL₄.

The CCL₄ is metabolized in the endoplasmic reticulum of kupffer cells and hepatocytes involving two phases. In the first phase, it is metabolized by cytochrome P450 gives rise reactive trichloromethyl radicals which in presence of oxygen produces trichloromethyl peroxyl. These free radicals are unstable and immediately react with the membrane components initiating a chain reaction that leads to lipid peroxidation³¹ which is considered to be one of the main mechanisms of hepatic injury by CCL₄. In this study, we found a clear increase in TBARS, an index of LPO, in the liver and spleen of CCL₄ challenged mice as previously observed in liver by Ritesh et al.³⁴ and in liver and spleen by Al-Zeiny and Abbas³⁵. Given that GPX reduces lipid peroxides and other organic hydroperoxides, the reduction in this enzymatic antioxidant following exposure to CCL₄ based on these findings might encourage overproduction of these cytotoxic mediators³².

CUR significantly reduced the CCL₄-induced increase in hepatic and splenic LPO as appeared in the current study, suggesting that CUR could reduce the free radicals production or remove these pro-oxidant molecules³⁶. In fact, the increase in hepatic SOD, CAT and GPX activities in this experimental model denotes improvement in the ability of antioxidant defense system to scavenge superoxide and hydrogen peroxide radicals³².

Fig. 5(a-f):

(a, b) Liver of control rats showing normal histological architecture, (c-e) Liver of CCL4 treated group, (c) Showing congestion and dilatation of the central vein (arrow), (d) Magnified part of (c) showing mononuclear cell infiltration (black arrow) and hyperplasia of kupffer cells (red arrow), (e) Showing hepatic cells with positively stained cytoplasmic clumps separated by rarified areas of cytoplasm and (f) Liver of curcumin treated group showing normal appearance

Fig. 6(a-f):

(a, b) Spleen of control rats showing normal parenchyma (normal architecture) consists of white pulp (WP), red pulp (RP) and megakaryocyte with characteristic lobulated nucleus and acidophilic cytoplasm (arrow), (c-e) Spleen of CCL4 treated group showing scattered necrotic areas in the parenchyma (arrows), (d) Large number of macrophages are present (arrows), (e) Apoptotic changes in megakaryocyte with fragmented nuclei (arrow) and (f) Spleen of curcumin treated group showing cellular architecture nearly similar to the control

Liver GSH was significantly reduced in CCL₄ treated mice in the present study and this could be due to enhancing substrate utilization by glutathione peroxidase, as there is a direct correlation between GSH depletion and elevated lipid peroxidation³⁷. A significant reduction in the activities of GST, GSH-Px, SOD and CAT had been recorded in the liver and spleen of CCL₄ treated mice. it is well known that GST implicated in preserving the cells against ROS mediated injury by detoxification of lipid hydro peroxides formed due to oxidative damage³⁸, it can be hypothesized that the reduction in its activity add another contributory factor in stimulating the lipid peroxidation in the current hepato- and splenotoxic mice model. Such alterations were also recorded by many studies^39-41 and these results suggested that CCL₄ administration make the liver and spleen more vulnerable to the attack of free radicals.

Consumption of CUR in the present study led to recovery of the redox balance state by reactivating the impaired activity of important antioxidant enzymes and restoring the levels of non-enzymatic antioxidants as observed in other hepatocellular toxicity models induced by cadmium⁴² or paracetamol⁴³. This result may attribute to up-regulation of antioxidant enzyme gene expression as that found in aflatoxin B1-intoxicated rats treated with CUR³⁵.

The second phase of CCL₄ hepatotoxicity involved the inflammatory responses which play an important role. Some hepatic cells as kupffer cells, hepatic stellate cells (HSCs) and sinusoidal endothelial cells can be activated to release cytokines which moderate liver fibrogenesis, activate of HSCs, regulate expression and deposition of matrix proteins and modify the renewal of hepatocytes ^{44, 45}.

The present study approved that the pro-inflammatory cytokines IL-1β, TNF-α and IL-6 were highly expressed in the liver and spleen of CCL₄-treated mice as previously estimatesd⁴⁶. These cytokines act both as a part of immune reactivity to hepatocellular damage and as a cytopathological mediator incriminated in inducing further hepatic histoarchitecture deteriorations⁴⁷. In this study, the elevation in the expression levels of these cytokines is a clear biochemical footprint parallel to the perturbations in hepatic and splenic histopathological features. The potential increase in oxidants secondary to the suppression in antioxidant defense mechanism in the current experiment is suggested to induce the gene expression of pro-inflammatory cytokines, e.g., hydrogen peroxide induces the transcript levels of IL-1β and TNF-α⁴⁸.

In our study, the significant inhibition in the expression of CCL₄ –induced pro-inflammatory cytokines under investigation following CUR consumption is consistent with that found in C57BL/6 mice⁴⁹. CUR down-regulates TNF- α by suppressing its various inducers⁵⁰, modulating methylation of its promoter⁵¹, inhibiting p300/CREB-specific acetyl transferase⁵² and decreasing NF-kB⁵³.

The expression of 5-LO in the liver is restricted to macrophages, that is required for the development of acute liver failure⁵⁴. In our study marked elevation in mRNA 5-LO expression in liver and spleen have been found in CCL₄-treated mice.

Inhibition of 5-LO gene expression by CUR supplementation in our study could play a fundamental role in arresting the growth and inducing the apoptosis in kupffer cell, thereby inhibits hyperplasia in these cells and subsequently prevents hepatotoxic necro-inflammatory injury induced by CCL₄^{55, 56}.

The increase in the production of pro-inflammatory cytokines (e.g., IL-1β and TNF-α) may cause up-regulation of COX-2 expression⁵⁷, following CCL₄ exposure in our experimental model. The suppression in COX-2 gene expression by CUR administration might indicate its ability to excrete hepatoprotective effect on CCL₄-induced liver damage via inhibition of prostaglandin synthesis and amelioration of inflammatory responses. Inhibition of nuclear factor-Kappa B is supposed to be one of the etiological factors implicated in down-regulation⁵⁸ of COX-2.

In this study, over expression of NOX2 could enhance nuclear factor kappa B activation which explains the concomitant over expression of COX-2 in the liver and spleen of CCL₄ challenged mice and resulted in ROS-mediated liver fibrosis⁵⁹.

The obvious reduction in NOX2 gene expression in the liver and spleen of CCL₄ intoxicated mice following administration of CUR might suppress free radicals generation and lipid peroxidation trying to protect against hepatic fibrosis⁶⁰.

The histopathological changes in the hepatic features following CCL₄ intoxication is in parallel with that observed by Ma et al.⁶¹ and Khedr and Khedr ⁶². Mononuclear cell infiltration in the liver and spleen of intoxicated mice reflects the non-specific immune response of leucocytes and macrophages to the foreign bodies. As soon as, tissue damaged, leukocytes rapidly move to the injury sites initiating an inflammatory response. Therefore, leukocyte infiltration was considered as a marker of inflammatory response⁶³. The CUR efficaciously suppressed leucocyte infiltration, so it was suggested that CUR not only dampen the inflammatory response on the level of pro-inflammatory mediators but also on the level of cellular microenvironment⁶⁴. Kupffer cell hyperplasia observed in the CCL₄ challenged liver represents one of defensive tools of detoxification and is contributed to hepatic oxidative stress⁶⁵. Activation of JNK, c-Jun and caspase-3, increased expression of pro-apoptotic proteins and reduced expression of anti-apoptotic protein (Bcl-2)⁶⁶, are the potential underlying mechanistic ways behind appearance of apoptotic changes in megakaryocyte with presence of fragmented nuclei in the spleen of CCL₄ exposed mice.

It was very clear in this study that he antioxidant, anti-inflammatory and anti-apoptotic properties of CUR played a pivotal role in returning the cellular architecture of liver and spleen of CCL₄-induced toxic model nearly similar to the control as previously reported⁶². The CUR can be beneficial for preventing spleen toxicity by suppressing the cellular reactive oxidants generation, up-regulating the levels of the cellular antioxidant enzymes and protecting the cells against oxidative damage of proteins and lipids, protects cells from stress induced MAPK dependent and p53 mediated intrinsic apoptosis⁶⁷. Inhibition of kupffer cells activation and hepatic oxidative stress and arresting the intrinsic apoptotic pathway, are believed to contribute to the ability of CUR to limit hepatocellular damage mediated by CCL₄^68,69.

CONCLUSION

These findings provide compelling evidences that CCL₄ disrupted the redox potential, caused up-regulation in pro-inflammatory cytokines and cytopathogenic inducers and histoarchitecture deteriorations in the liver and spleen of mice. CUR effectively limited the above mentioned disorders and therefore can be used as a promising therapeutic strategy offering new opportunities for treatment of hepatotoxic and spleenotoxic-associated CCL₄ affections. However, further studies are highly recommended to investigate in depth the other molecular pathways underlying the antioxidant and anti-inflammatory effects of CUR in this experimental model.