Crocin attenuates cisplatin-induced liver injury in the mice

Abstract

Cisplatin (CDDP) is one of the most frequently used antitumor agents, but its application is significantly limited by its hepatotoxicity. In the present study, we investigated the effects of crocin against CDDP-induced oxidative stress and apoptosis in the liver of Kunming mice. Crocin was administered to the mice once daily for 7 consecutive days at the doses of 6.25 and 12.5 mg/kg body weight orally. On day 1, a single intraperitoneal injection of CDDP was given at the dose of 10 mg/kg body weight. Crocin treatment significantly improved CDDP-induced hepatic damage as indicated by serum aspartate aminotransferase and alanine aminotransferase levels. Crocin relieved CDDP-induced oxidative stress by reducing malondialdehyde level and recovering the levels of glutathione and antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. In addition, liver histopathology indicated that crocin alleviated CDDP-induced focal necrosis. Immunohistochemical staining and Western blot analysis showed that crocin significantly decreased the levels of phospho-p38 mitogen-activated protein kinase (MAPK), tumor protein 53 (p53), and cleaved caspase-3. Taken together, our data suggest that crocin provides protective effects against CDDP-induced hepatoxicity by attenuating oxidative stress and inhibiting the activation of p38 MAPK, p53, and caspase-3.

Introduction

Cisplatin (CDDP), a platinum (Pt) containing drug, is one of the most frequently used antitumor agents. However, the dose of CDDP is greatly limited for its toxicity. High doses of CDDP required for effective tumor suppression could lead to hepatotoxicity, which is also encountered during low dose repeated CDDP therapy. ^1,2 The precise mechanism of CDDP hepatotoxicity is not fully understood. It has been reported that oxidative stress through the generation of reactive oxygen species (ROS) decreased antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) in the liver and reduced non-enzymatic molecules such as glutathione (GSH) and malondialdehyde (MDA), which are the major alterations in CDDP hepatotoxicity. ³ Several lines of evidence suggest that CDDP is not specific in action against tumors but also exhibits cytotoxicity to rapidly dividing normal cells through the production of ROS to induce oxidative stress. ⁴ In addition, functional and structural mitochondrial damage and apoptosis play important roles in CDDP-induced hepatotoxicity. ⁵

The induction of oxidative stress and lipid peroxidation has been implicated in hepatotoxicity. ⁶ Therefore, there is a need to explore natural compound that can effectively diminish CDDP-induced toxicity to improve the chemotherapeutic efficacy of CDDP. Crocus sativus L., commonly known as saffron, is a plant cultivated in various parts of the world. The major biologically active ingredients of saffron are crocin, picrocrocin, and safranal. ⁷ Numerous studies have shown a variety of pharmacological effects of crocins, such as protection against cardiovascular diseases, inhibition of tumor cell proliferation, neuroprotection, and antioxidant effect. ⁸

Therefore, we hypothesized that crocin may have protective effects against CDDP-induced liver toxicity by interfering with oxidative stress and apoptotic pathway. In the present study, we investigated the effects of crocin against CDDP-induced oxidative stress and apoptosis in the liver of Kunming mice.

Methods

Animals and grouping

Kunming mice (18–22 g) were obtained from the Experimental Animal Center of Qingdao Institute for Drug Control, People’s Republic of China. All animals were fed with a standard laboratory diet and water ad libitum. They were maintained in a controlled environment at 20–25°C with 50 ± 5% relative humidity under a 12-h dark–light cycle. All the experiments were conducted under the guidelines of laboratory animal use and care of the European Community (EEC Directive of 1986; 86/609/EEC).

After 1-week acclimation period, 30 mice were randomly assigned to 5 different groups (6 animals in each). Group I received normal saline (0.2 ml, intraperitoneally (i.p.)) for 7 successive days. Group II received CDDP (10 mg/kg, single i.p.) on the first day. Group III and IV were treated with crocin (6.25 and 12.5 mg/kg per day, i.p., respectively) for 7 successive days after a single dose of CDDP (10 mg/kg, i.p.) on the first day. Group V (positive control) were treated with silymarin (100 mg/kg per day, orally (p.o.)) for 7 successive days after a single dose of CDDP (10 mg/kg, i.p.) on the first day.

Crocin, silymarin, and CDDP were obtained from Sigma Chemical Co. (Missouri, USA). Seven days after CDDP insult, all the mice were anesthetized and killed. Blood samples were collected, and the liver was immediately removed and stored at −80°C for further use.

Determination of serum ALT and AST

Serum was separated by centrifugation at 2500 r/min at 4°C for 10 min. Activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by ALT and AST assay kits (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions and expressed in international unit per liter.

Biochemical analysis of the liver

Liver tissues were prepared in phosphate buffer (pH 7.4) to make 1:10 (w/v) homogenates. After centrifugation at 12,000g for 20 min at 4°C, the supernatants were collected for biochemical analysis. The levels of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx), and SOD were detected by assay kits purchased from Nanjing Jiancheng Bioengineering Institute (China). The values were normalized by the protein concentration of the sample, which measured using bicinchoninic acid (BCA) assay. MDA and GSH were expressed in nanomole per milligram protein and milligram per gram protein, respectively. SOD, CAT, and GPx were expressed as units per milligram of tissue protein.

Histopathological analysis of the liver

The liver was excised and then washed with saline. Liver samples were fixed in 10% neutral-buffered formalin solution for at least 24 h. The tissues were embedded in paraffin. The paraffin blocks were cut into 5-µm thick sections. The sections were stained with hematoxylin and eosin (H&E) for morphological evaluation under light microscope at 400 × magnification.

Immunohistochemical staining

The staining of phospho-p38 mitogen-activated protein kinase (MAPK), tumor protein 53 (p53), and cleaved caspase-3 in the liver tissue was performed using streptavidin–biotin–complex peroxidase kit (Boster, Wuhan, China). The slides were washed, dehydrated, and mounted for microscopic examination. Analysis of immunostaining in the liver was performed using a Media Cybernetics Image-Pro Plus analysis system linked to Olympus microscope. The cells stained positive for phospho-p38 MAPK, p53, and cleaved caspase-3 and were quantified by counting the yellow to brown cells and the total number of cells at five randomly selected fields at 400 × magnification.

Western blot analysis

Liver tissues were collected and lysed in radioimmunoprecipitation assay buffer supplemented with protease inhibitors to make 1:10 (w/v) homogenates and then were centrifuged at 12,000g for 20 min at 4°C. The supernatants were analyzed for protein concentrations using BCA kit (Beyotime Institute of Biotechnology, China). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked with phosphate-buffered saline with 0.05% Tween-20 (PBST) containing 5% nonfat dry milk for 1 h and then incubated at 4°C overnight with phospho-p38 MAPK, p53, caspase-3, or β-actin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, California, USA). Membranes were then washed with PBST, incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature, and then detected using an ECL kit Plus Western Blotting Detection System (Santa Cruz Biotechnology, Inc.). Protein levels were normalized to β-actin.

Stastical analysis

Data were presented as the mean ± SD and analyzed by one-way analysis of variance followed by Fisher’s least significant difference test using the Statistical Package for the Social Sciences (SPSS) 17.0 software (SPSS Inc., Chicago, Illinois, USA). Significant differences were defined as p < 0.05.

Results

Crocin attenuates liver injury induced by CDDP

Serum ALT and AST are biomarkers of liver injury. ⁹ Thus, we examined these biomarkers in experimental mice and found that serum AST and ALT levels were significantly higher in CDDP group than control group, indicating the severity of CDDP-induced liver injury. However, crocin significantly reduced the levels of serum AST and ALT compared to CDDP group (Figure 1).

Figure 1.

Crocin reduces serum ALT (a) and AST (b) levels in CDDP-treated mice. Data were expressed as mean ± SD (n = 6). (I) control group, (II) CDDP-treated group (10 mg/kg), (III) dose 1 of crocin (6.25 mg/kg body weight) + CDDP, (IV) dose 2 of crocin (12.5 mg/kg body weight) + CDDP, and (V) silymarin (100 mg/kg) + CDDP (10 mg/kg). *p < 0.05 versus control group; ** p < 0.05 versus CDDP-treated group. ALT: alanine aminotransferase; AST: aspartate aminotransferase; CDDP: cisplatin.

Crocin relieves oxidative stress induced by CDDP in the liver

Lipid peroxidation and levels of endogenous antioxidants are common index of oxidative stress. We found that CDDP increased hepatic MDA concentration significantly, which was inhibited by crocin and silymarin treatment (Figure 2(a)). Hepatic GSH content in CDDP group was significantly decreased compared to control group. Crocin with different doses and silymarin treatments significantly increased the level of hepatic GSH compared to CDDP group (Figure 2(b)).

Figure 2.

Crocin relieves oxidant stress in the liver of CDDP-treated mice. Changes in hepatic (a) MDA content, (b) GSH content, (c) SOD activity, (d) CAT activiy, and (e) GPx activity. Data were expressed as mean ± SD (n = 6). (I) control group, (II) CDDP-treated group (10 mg/kg), (III) dose 1 of crocin (6.25 mg/kg body weight) + CDDP, (IV) dose 2 of crocin (12.5 mg/kg body weight) + CDDP, and (V) silymarin (100 mg/kg) + CDDP (10 mg/kg). *p < 0.05 versus control group; ** p < 0.05 versus CDDP-treated group. CDDP: cisplatin; MDA: malondialdehyde; GSH: glutathione; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase.

In addition, we examined the activities of SOD, CAT, and GPx in the livers of all experimental mice. Crocin could increase SOD, CAT, and GPx enzyme activities in the livers of CDDP-treated mice (Figure 2(c) to (e)). Taken together, these data suggest that crocin relieves oxidative stress induced by CDDP in the liver.

Crocin improves liver morphology in CDDP-treated mice

Next, we performed histological assessment of hepatoprotective effects of crocin on CDDP-induced acute liver damage. In the liver sections of control animals, we observed normal hepatic cells with well-preserved cytoplasm, prominent nucleus and nucleolus, and visible central veins. Intraperitoneal injection of CDDP resulted in histological changes in liver morphology, including centrilobular necrosis, hepatic cord degeneration, and hepatocyte ballooning. However, hepatic lesions were markedly ameliorated by treatment with crocin (Figure 3). These data demonstrate that crocin improves liver morphology in CDDP-treated mice.

Figure 3.

Crocin improves hepatic morphology in CDDP-treated mice. Tissues sections were stained with hematoxylin and eosin by standard techniques. (I) control group, (II) CDDP-treated group (10 mg/kg), (III) dose 1 of crocin (6.25 mg/kg body weight) + CDDP, (IV) dose 2 of crocin (12.5 mg/kg body weight) + CDDP, and (V) silymarin (100 mg/kg) + CDDP (10 mg/kg). Magnification ×400. CDDP: cisplatin.

Crocin reduces the levels of phospho-p38, p53, and cleaved caspase-3 in the liver of CDDP-treated mice

Immunohistochemical analysis showed that the staining of phospho-p38, p53, and cleaved caspase-3 was much stronger in the livers of CDDP-treated group (group II) compared to control group (group I). However, crocin treatment reduced the staining of phospho-p38, p53, and cleaved caspase-3 in the livers of groups III, IV, and V compared to group II. In addition, there were no significant differences in the immunostaining of all proteins among groups III, IV, and V (Figure 4).

Figure 4.

Crocin reduces the staining of phospho-p38 MAPK, p53, and cleaved caspase-3 in the liver of CDDP-treated mice. Shown were representative immunohistochemical images of liver sections. (I) control group, (II) CDDP-treated group (10 mg/kg), (III) dose 1 of crocin (6.25 mg/kg body weight) + CDDP, (IV) dose 2 of crocin (12.5 mg/kg body weight) + CDDP, and (V) silymarin (100 mg/kg) + CDDP (10 mg/kg). Brown color indicated specific immunostaining and light blue color indicated nuclear hematoxylin staining. Magnification ×400. MAPK: mitogen-activated protein kinase; CDDP: cisplatin.

Furthermore, we examined the levels of phospho-p38, p53, and cleaved caspase-3 in the livers by Western blot analysis. Compared to control group, the levels of phospho-p38, p53, and cleaved caspase-3 were increased in the livers of CDDP-treated group (group II). However, their levels were reduced in the livers of mice treated with 6.25 and 12.5 mg/kg crocin or silymarin (Figure 5). Taken together, these data indicate that crocin could reduce the levels of phospho-p38, p53, and cleaved caspase-3 in the liver of CDDP-treated mice.

Figure 5.

Crocin reduces protein levels of phospho-p38 MAPK, p53, and cleaved caspase-3 in the liver of CDDP-treated mice. Left panel: Western blot analysis of liver sections. Right panel: quantitation of the relative protein levels of phospho-p38 MAPK, p53, and cleaved caspase-3 in the liver. β-actin was loading control. (I) control group, (II) CDDP-treated group (10 mg/kg), (III) dose 1 of crocin (6.25 mg/kg body weight) + CDDP, (IV) dose 2 of crocin (12.5 mg/kg body weight) + CDDP, and (V) silymarin (100 mg/kg) + CDDP (10 mg/kg). *p < 0.05 versus CDDP-treated group. MAPK: mitogen-activated protein kinase; CDDP: cisplatin.

Discussion

In this study, we demonstrated the protective effects of crocin against CDDP-induced hepatotoxicity in the mice and found that the protective effects of crocin were associated with the amelioration of oxidative stress and apoptotic damage in the liver of CDDP-treated mice.

CDDP is one of the most potent anticancer drugs used in chemotherapy. Unfortunately, the clinical use of CDDP is often limited by side effects such as nephrotoxicity and hepatotoxicity. During the aggressive treatment protocols, higher doses of CDDP that may be required for effective tumor suppression could lead to hepatotoxicity, which is also encountered during low dose repeated CDDP therapy. ¹⁰

The increase in ALT and AST levels has been attributed to the damaged structural integrity of the liver. Kadikoylu et al. reported that CDDP administration induced significant increase in ALT and AST levels. ¹¹ In this study, we found that in CDDP-induced acute liver damage models, the levels of serum ALT and AST were significantly reduced by crocin, and the inhibitory effects of crocin (6.25 and 12.5 mg/kg) on serum ALT and AST levels were similarly to those of silymarin (100 mg/kg). Histopathological lesions supported these biochemical results. Hepatocyte and hepatic cord degeneration and focal necrosis infiltration in mice livers were induced by CDDP, which could be effectively ameliorated by crocin treatment.

The exact mechanism underlying CDDP toxicity is not fully understood, but the plausible mechanism may be through the generation of ROS, which may lead to oxidative stress. CDDP-induced ROS may lead to DNA damage and apoptotic cell death. ¹² Heme oxygenase and CAT are important protective responses against CDDP toxicity in the livers of tumor-bearing mice. ¹³ In the present study, CDDP treatment caused increased lipid peroxide levels and decreased activities of antioxidant enzymes that protect against lipid peroxidation in the liver. Lipid peroxidation is a marker of oxidative stress. ¹⁴ MDA is one of the main lipid peroxidation products, and elevated MDA level could reflect the degrees of lipid peroxidation injury in hepatocytes. ¹⁵ Remarkable elevation in MDA level has been observed after CDDP treatment. ¹⁴ Similarly, our results showed that there was remarkable increase in liver MDA level in mice treated with CDDP and that treatment with crocin significantly reduced MDA level.

Antioxidants protect against oxidative stress. Endogenous antioxidants such as GSH, GPx, SOD, and CAT act as free radical scavengers. ¹⁶ Our results showed that CDDP-induced GSH depletion and crocin treatment significantly attenuated GSH depletion. Additionally, we observed that the activities of antioxidant enzymes including SOD, CAT, and GPx were diminished in mice treated with CDDP. Treatment with crocin significantly recovered the activities of these antioxidant enzymes.

It is evident that apoptosis is involved in CDDP-induced liver injury. ¹⁶ It has already been reported that CDDP-induced toxicity was closely associated with the generation of ROS. ¹⁷ CDDP damages cellular DNA by forming Pt-DNA adducts, and CDDP-generated ROS augments DNA damage within the cells, leading to the activation of p38 MAPK. ¹⁸ Oxidants are potent activators of MAPKs, including p38 MAPK. ¹⁹ In our study, phospho-p38 MAPK level, indicative of p38 MAPK activation, was increased in CDDP-treated group compared to control group, while treatment with crocin significantly attenuated phospho-p38 MAPK level. These results further support the involvement of oxidative stress in CDDP-induced toxicity.

Caspase-3 is one of the caspases that play a central role in the execution of apoptosis. ²⁰ Our results showed that after CDDP treatment, considerable amount of cleaved caspase-3 was detected, suggesting that apoptotic effect of CDDP to hepatic cells could be related to the induction of caspase activation. Crocin treatment significantly decreased the level of cleaved caspase-3. These results indicate that crocin could inhibit hepatic cell apoptosis via preventing caspase-3 activation.

In summary, the results of biochemical analysis corroborated with those of histological examination to demonstrate the protective effects of crocin against CDDP-induced liver damage. The precise mechanism of crocin action against CDDP is still not completely understood, but our data suggest that crocin exhibits protective effects against CDDP-induced hepatotoxicity probably through the attenuation of CDDP-induced oxidative stress and apoptotic tissue damage. Crocin could be used as a combinational therapy with CDDP to prevent the side effects of CDDP.