Therapeutic potential of a novel combination of Curcumin with Sulfamethoxazole against carbon tetrachloride-induced acute liver injury in Swiss albino mice

Background In the current study, we have investigated the effect of each of curcumin (CUR) and sulfamethoxazole (SMX) either separate or mixed together (CUR + SMX) on biochemical, hematological and histological alternations associated with carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Results CCl4, caused changes of several biomarkers, proving its hepatotoxic effects, such as an increase in aminotransferases liver enzymes alanine and aspartate transaminases (ALT, AST), malondialdehyde (MDA), and nitric oxide (NO) formation, with a decrease in superoxide dismutase (SOD), glutathione reductase (GSSG), total antioxidant capacity (TAO), glutathione (GSH), total protein, and albumin, compared to a negative control mice group. Compared to the CCl4 group of mice, the CUR and SMX separate and/or together (CUR + SMX) treatments showed significance in (p < 0.001), ameliorated liver injury (characterized by an elevation of (ALT, AST) and a decrease (p < 0.001) in serum albumin and total protein), antioxidant (characterized by a decrease in (p < 0.001) MDA, NO; an increase (p < 0.001) SOD, GSSG, TAO; and reducing GSH), hematological changes (characterized by a decrease (p < 0.001) in white blood cells count and an increase (p < 0.001) in platelets count, hematocrit levels, hemoglobin concentration, and (p < 0.05) red blood cells count), SDS-PAGE electrophoresis with a decrease in protein synthesis and changes in histological examinations. Conclusions CUR and SMX either separate or together (SUR + SMX) may be considered promising candidates in the prevention and treatment of liver fibrosis.


Impact statement
Liver diseases particularly liver fibrosis, cirrhosis, and hepatocellular carcinoma are major health problems worldwide and in Egypt. The current work reports on the impact of the therapeutic potential of curcumin (CUR) and/or sulfamethoxazole (SMX) against carbon tetrachloride-induced acute liver injury in Swiss albino mice. We demonstrated the CUR and SMX used separate or mixed together may be promising therapy in the prevention and treatment of liver fibrosis.

Background
The liver carries out an essential job in the control of various physiological processes and it controls some vital functions, for example secretion, storage, and metabolic activities. It is frequently exposed to different xenobiotic and therapeutic agents. It detoxifies exogenous (toxic compounds) and endogenous (waste metabolites) substances of organisms and integrates helpful operators [1]. As far as anyone is concerned, no reports are available regarding actual curative therapy agents for liver disorders or diseases and most of the existing remedies only aid in healing or regeneration of liver [2]. Fibrosis, a general clinical condition, can be seen in various organs, but mostly in the liver. It is regularly connected with the end phase's of chronic inflammatory diseases [3,4]. Liver fibrosis is the phenomena of the advancementof a hepatic scar because of chronic liver injury, coming about because of complex interconnected changes in cell population, extracellular matrix (ECM), and cytokines [5,6]. For this study, fibrosis utilized carbon tetrachloride (CCl 4 )-induced animal model [7,8], published reports [9], showed CCl 4 as xenobiotic to induce acute and chronic tissue injuries through bio-enactment of the stage I cytochrome P450 enzymes to deliver receptive metabolic trichloromethyl radicals (CCl 3 ) and peroxy trichloromethyl radicals (OOCCl 3 ). These receptive metabolites have potential to covalently bond with enormous particles, for instance, lipids, proteins, and nucleic acids, pushing them out of their typical crucial usefulness. CCl 4 injection lead to an expansion type of free radicals that are chemically highly reactive, and if they do not cause injury or necrosis, they bring about genuine unsafe impacts, such as hepatic fibrosis, copying the oxidative stress that has a fibrogenic effect on hepatic stellate cells (HSC) [3,5,6,10,11]. Curcumin, CUR ( Fig. 1): 1, 7-bis (4-hydroxy-3-methoxyphenyl) hepta-1, 6-diene-3, 5-dione. (C 21 H 20 O 6 , molecular weight = 368.38) is the most bioactive structure among the curcuminoid chemical compounds that are extracted from the powdered dry rhizome of Curcuma longa Linn (Zingiberaceae), which is a perennial herb widely cultivated in the tropical region of Asia, especially in India [12,13]. The medicinal value of curcumin has been well recognized. It has been proven that curcumin has anti-cancer [14], chemo preventive effect [15], antioxidant [16], antiproliferative [17], immunomodulatory [18], hepatoprotective [19], and anti-inflammatory properties [20]. Furthermore, CUR has shown to inhibit fibrosis; however, the specific mechanism of curcumin bioactivity still remains unclear [21].
In this study, we have investigated the effect of each of curcumin (CUR) and sulfamethoxazole (SMX) either separate or together (CUR + SMX) on induced liver fibrosis of carbon tetrachloride (CCl 4 ), and induced acute liver injury in Swiss albino mice. The study, also, included an investigation of the effects of these, previously mentioned, compounds on biochemical, hematological, and histological alternations associated with carbon tetrachloride (CCl 4 )-induced liver fibrosis in mice.

Analysis of curcumin (CUR)
The melting point (m.p.) of CUR was measured in an open capillary glass tube on a Gallenkamp melting point apparatus (Gallenkamp and CO, UK) and was uncorrected. Infrared (IR) spectrums of CUR was obtained on Perkin Elmer 1430 spectrophotometer with potassium bromide (KBr) disc, in the wavenumber range of 4000-400 cm −1 . Its 1 H nuclear magnetic resonance (NMR) (500 MHz, DMSO-d 6 ) and 13 C NMR (125 MHz, DMSO-d 6 ) spectra were determined on JEOL's NMR spectrometer (500 MHz, Japan), using DMSO-d 6 as solvent. Chemical shifts are expressed as δ values (ppm), using tetramethylsilane (TMS) as an internal standard. The following abbreviations were used to indicate the NMR-signals: s (singlet), d (doublet), and br (broad). Electron impact (EI) mass spectral (MS) analysis at 70 eV of curcumin was performed on Thermo Scientific Trace DSQ II GC-MS (50-400 m/z), (Waltham, MA) system.
Measuring the melting point and the IR analysis of curcumin were performed at the Chemistry Department, Faculty of Science, Egypt, while its 1 H and 13 C NMR and MS spectral analysis were performed at the Spectral Analysis Unit, Chemistry Department, Faculty of Science, Egypt.

Experimental animals
Adult male Swiss albino mice (total number, n = 180) were purchased from Zoology Department, Faculty of Science, Egypt, with average weight (25-30 gm each) and housed at the experimental animal house of the Faculty of Science. The animals were maintained in a properly controlled environment of temperature, humidity, and light. The mice were fed with autoclaved chow and filtered water and were adapted for a week prior to experimentation.

Experimental design
The Swiss male adult albino mice were divided into nine (I-IX) groups (20 mice/each) as shown in Table 1. At the end of the experiment, mice fasted for 12 h and then blood samples were taken from the heart puncture under light ether anesthesia. Blood samples were collected for biochemical and hematological analysis. Animals were sacrificed and the liver dissected out and washed with isotonic saline and divided into two parts, the first part was stored at -20°C until assay for estimation of antioxidant parameters. The second part of the liver tissue was fixed in (10%) formalin for histopathology assessment.

Biochemical analysis
Estimation of nitric oxide (NO) level was determined in liver tissues by using Biodiagnostic kit, Egypt (cat. no. NO2533), according to the method of Montgomery and Dymock [48]. Glutathione reductase (GSSG) was determined by using Biodiagnostic kit, Egypt (cat. no. GR2523), according to the method of Goldberg and Spooner [49]. The serum samples were collected for liver function tests. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) were estimated by using Biodiagnostic kits, Egypt (cat. no. AS1061 (45), cat. no. AL1031 (45), according to the method of Reitman and Frankel [50]. Serum total protein and albumin concentrations were estimated by using Biodiagnostic kits, Egypt (CAT.NO. TP2020, cat. no. AB1010), according to the method of Gornall et al. [51] and Doumas et al. [52]. Moreover, plasma samples were collected for antioxidant assays. Estimation of superoxide dismutase (SOD) activity was assayed according to the method of Nishikimi et al. [53]. Glutathione reduced (GSH) was estimated by using a commercial kit (Biodiagnostic Company, Dokki, Giza, Egypt), according to the method of Beutler et al. [54]. Total antioxidant activity (TAO) carried out according to the method of koracevic et al. [55]. Malondialdehyde (MDA) level was assayed using Biodiagnostic kit, Egypt (cat. no. MD2529), according to the method of Satoh [56] and Rubio et al. [57].

Hematological analysis Complete blood count (CBC)
A portion of retro-orbital blood samples was collected from each animal used for a complete blood count. Blood cell counts (white blood cells, red blood cells, and platelets) were performed with HORIBA Hematology analyzer (model: MICROS 60 OT) (France).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
Protein fractionation was done by using one-dimensional polyacrylamide gel electrophoresis according to the method of Leammli [58].

Sample preparation
One gram of each sample (liver tissue) was ground in 1 ml homogenate buffer (0.02 M Tris-HCl pH~7.5) using a mortar. The content was transferred to a new Eppendorf tube, centrifuged at 10000 rpm for 10 min at 4°C, and then the supernatant was kept frozen at − 20°C until required. Protein samples were denatured by boiling in a water bath for 5 min with SDS sample buffer. Then, they were loaded into the wells of gel which was composed of 5% stacking gel and 12% polyacrylamide separating gel next to the molecular weight marker protein. The separated protein on the polyacrylamide gel was stained with Coomassie blue R-250 Andrews [59]. After that, the gel  was removed from the distaining solution, placed between two transparent sheets, and was left to air dry. Then, the gel was scanned and analyzed by the Gel-Pro Analyzer program http://meyerinst.com/imaging-software/image-pro/gel/index.htm (Media cybernetics, Georgia, USA).

Histopathological examinations
Mouse liver tissues were fixed in 10% neutral formalin, then dehydrated, and further embedded with paraffin. Paraffin-embedded liver samples were sectioned to 3μm-thin slices, which were stained with hematoxylineosin (HE) staining and Masson staining according to standard protocols of Oner-lyidogan et al. [60].

Statistical analysis
Data were evaluated by one-way analysis of variance (ANOVA) by "SPSS" 14.0 for Microsoft Windows, SPSS Inc. and considered statistically significant at a two-sided p < 0.05. Numerical data were expressed as mean ± SD.

Nuclear magnetic resonance spectroscopy of curcumin
The 1 H and 13 C NMR spectra of curcumin are shown in Figs. 4 and 5, respectively. The NMR data of curcumin, whose interpretation confirmed its structure and its effectiveness, concurring with the literature [42,64,67,68].

Mass spectrometry (MS) of curcumin
The MS analysis has confirmed the structure of curcumin, showing its molecular ion peak at m/z = 368 with a relative intensity of 0.92%.

Curcumin and sulfamethoxazole relieved CCl 4 -induced on liver enzymes in Swiss mice
Measurement of liver enzyme activities demonstrated a significant increase in ALT and AST activities in CCl 4positive control group (III,  Fig. 7c, d.

Discussion
Curcumin (CUR) is a widely examined natural compound that has demonstrated imposing in vitro therapeutic potential. Disregarding the way that the experimental efficacy of natural curcumin is frail due to its low bioavailability and high metabolism in the gastrointestinal tract because of its poor solubility in water and quick in liver and intestinal digestion which add to its fast excretion, researchers have focused on ameliorative bioavailability in the most recent decade [69,70]. Sulfonamides are the central and historically most prominent class of carbonic anhydrase inhibitors, being discovered in 1940, with many representatives in clinical use for decades [71]. Thus, we used sulfamethoxazole with curcumin in combination group (VIII, Table 1) to circumvent the problem of rapid metabolism and to improve its pharmacokinetics profile. The purpose of this study is to investigate the effect of each of curcumin (CUR) and sulfamethoxazole (SMX) either separate or together (CUR + SMX) on biochemical, hematological, and histological alternations associated with carbon tetrachloride (CCl4)-induced liver fibrosis in mice, and to explain the underlying mechanism. The CCl 4 is a causative agent of numerous disarranges through its capabilityof causing oxidative stress and inflammation. CCl 4 uses, through enzymes of the CYP 450 system, producing hepatotoxic metabolites for example, trichloromethyl radicals and trichloroperoxyl radicals. These metabolites cause hepatotoxicity through induction of lipid peroxidation that all together gives malondialdehyde (MDA) products that lead to hepatic injury [72]. According to our present data, CCl 4 caused changes of several biomarkers which proved its hepatotoxic effects such as an increase in aminotransferases liver enzymes (ALT, AST), MDA, and NO, with a decrease in SOD, GSSG, TAO, GSH, total protein, and albumin compared to the negative control group (I, Table 1). Oxidative stress is the most widely recognized instrument in organ injury. Oxygen is the primary particle for all cells for the creation of ATP, but it might, likewise can change into harmful species, for example reactive oxygen species. During aerobic respiration, the creation of free radicals could could bring about aging and cell death. Oxygen molecules are reduced by mitochondria to create superoxide ions or hydrogen peroxide (H 2 O 2 ). The superoxide and peroxide further respond with metal ions and produce hydroxyl radicals, which react with cell components including DNA and proteins. Polyphenolic curcumin is essentially appended to its antioxidant properties. CUR is seen as ten times more antioxidants than vitamin E [73]. CUR causes an increase in GSH concentration and activity of glutathione peroxidase and SOD. The glutathione and glutathione peroxidase act synergistically to scavenge free radicals. SOD scavenges superoxide radicals by changing over superoxide free radicals into H 2 O 2 while staying away from the development of hydroxyl radicals. Thus, the H 2 O 2 formed is expelled by the job of catalase or glutathione peroxidase. In this manner, SOD protects against free radical damage [74]. CUR has antioxidant properties that protect against CCl 4 -induced hepatic damage. It can repair all biomarkers against hepatic damage, i.e., decreasing level of ALT and AST, suppressing lipid peroxidation, decreasing MDA level, recovery of redox balance, and increasing level of reduced GSH [75].
Our results revealed that curcumin-treated group (IV, Table 1) had an increase in SOD, GSSG, TAO, GSH, total protein, and albumin, while it had a decrease in ALT, AST, MDA, and NO in comparison to CCl 4 group (III, Table 1) which agreed with Zhao et al. [76], who reported that there was an increase in serum biomarkers of ALT, AST, and a decrease in the levels of albumin and the total protein , after treatment with CUR [76]. Indicators in liver disease, ALT and AST are hepatocyte cytosolic enzymes. An increase in levels of ALT and AST usually confirm liver injury. Albumin and total protein levels tend to be reduced in chronic liver injury because of the impaired ability of liver cells to synthesize proteins [76,77]. Peng et al. [78] showed that curcumin pre-treatment at the doses of 50, 100, and 200 mg/kg markedly alleviated the expansion of MDA level brought about by CCl 4 and it can up-manage the activities of SOD and levels of GSH. Momeni and Eskandari [79] showed that the use of curcumin alone significantly increased the total antioxidant capacity of serum in comparison with the control. Its direct antioxidant activity, CUR may enhance the synthesis of glutathione and improve the antioxidant defense system, a significant increase in the serum concentration of MDA. Curcumin affected the levels of peripheral blood parameters through increasing HGB, RBC s , PLT, and decreasing WBCs, which agreed with the results of Yin et al. [80]; however, the combination between CUR and SMX group (VIII, Table 1) had a significant increase in HGB, RBCs, and PLT and a significant decrease in WBCs, so the combination was more effective than curcumin alone. Sulfamethoxazole-treated group (VI, Table 1) caused an increase in SOD, GSSG, GSH, total protein, and albumin but caused a decreased in ALT, AST, MDA, and NO compared to the CCL 4 group (III, Table  1) which was in agreement with Sahyon et al. [47]. Gupta et al. [81] reported that (sulfamethoxazole + selenium) treatment resulted in an increase in the activities of SOD, but caused a decrease (p < 0.001) in the levels of GSH when compared to diethylnitrosamine-treated animals and showed an increase in the levels of MDA. Also, Bottari et al. [40] reported that the treatments with SMX + trimethoprim, resveratrol, and inclusion complex in free forms or co-administered were able to reduce the total oxidation status and oxidation protein products levels in hepatic tissue of infected animals. In comparison, our results showed that curcumin and sulfamethoxazole combination have significantly, decreased MDA The present data demonstrated an increase in protein synthesis in the CCl 4 group with a corresponding decrease in the curcumin and/or sulfamethoxazole treated groups. This agreed with Thaloor et al. [82], who reported that the synthesis of proteins (including 60, 36, and 30.7 kDa proteins) was inhibited in the presence of 25 μM of curcumin. In a comparable study, Qin et al. [83] showed that the protein levels were significantly decreased in the CUR-treated groups. This, again, is in concurrence with our results. The histological examination of liver tissues showed that the liver of the control mice exhibited normal lobular architecture around the portal area. Treatment with CCl 4 alone caused an increased number of fibroblasts and fibrosis. This agreed with Ahmad et al. [84] who reported that CCl 4 treated group showed heavy cell infiltration, across the board vacuolated cytoplasm, darkly stained pyknotic and peripheral placed nuclei. Our results, also, agreed with Zhaoa et al. [21] which showed that the liver in CCl 4 treated mice have a large number of hepatocytes necrosis, leukocytes infiltration, and damaged lobule structure and the liver in CUR treated mice have reduce leukocytes infiltration and reduced hepatocytes necrosis compared to the mice of the CCl 4 -positive control group, which agreed with our CUR treated group and showed cell swelling of hepatocytes within the centrilobular area (arrow), in addition to moderate degree of vacuolation of hepatocytes within the periportal area. The liver of the mice, in the curcumin and sulfamethoxazole combination group (CUR + SMX), showed cell swelling and mild vacuolation of hepatocytes. While the mice group that was treated with sulfamethoxazole (SMX) alone showed cloudy swelling, vacuolar degeneration of hepatocytes, and mononuclear cells infiltration. The above-mentioned results, clearly demonstrated the highly significant effect of curcumin and/or sulfamethoxazole against the harmful effect of CCl 4 in dealing with Swiss mice group. However, further studies with different dosages and/or concentrations of curcumin and sulfamethoxazole will be done.

Conclusions
Treatment with curcumin and sulfamethoxazole either separately or together can reduce CCl 4 hepatotoxicity in mice. Each of curcumin and sulfamethoxazole could be considered a promising candidate in the prevention and treatment of liver fibrosis.