Nephroprotective Effect of Nigella sativa and Matricaria chamomilla in Cisplatin Induced Renal Injury — Supportive Treatments in Cisplatin Nephrotoxicity

Nigella sativa and Matricaria chamomilla are extensively consumed as tea or tonic. Despite their widespread use as a home remedy, relatively few trials evaluated their benefits in nephroprotection. Hence, this study evaluates the nephroprotective effects of supportive treatments (N. sativa, M. chamomilla and vitamin E) in cisplatin nephrotoxicity rat model. Eighty rats divided into 10 groups, of 8 animals each. The first group (G1) injected with saline intrapretoneal (I.P). G2 injected with 5 mg/kg cisplatin I.P on zero day of experiment and repeated 4 times, with 5 days free interval. G3 G10 received daily supportive treatments, started 5 days before the experiment (–5 day). Concomitantly G4, G6, G8 and G10 injected with 5 mg/kg cisplatin I.P like G2. On day sixteen, animal scarified, serum and/or kidney tissue were used to determine kidney function tests (serum urea, creatinine, NAG, β-GAL), oxidative stress indices (NO, LPO), antioxidant activities (SOD), sulphur compounds (GGT, GSH, total thiols), apoptotic indices (cathepsin D, DNA fragmentation), two minerals (Ca and Zn). Cisplatin caused marked elevation in serum GGT that reduced significantly in group received M. chamomilla with cisplatin (P < 0.001). There is a correlation between GGT and NAG in cisplatin group (r = 0.731, P < 0.05) that may suggest one of possible mechanisms of renal injury by cisplatin. M. chamomilla followed by N. sativa and vitamin E improved the biochemical and pathological renal injury, as determined by increasing the body weight, normalizing the kidney functions, decreasing the oxidative stress markers, improving the apoptotic markers, minimizing the pathological changes. Hence, N. sativa and M. chamomilla will be promising nephroprotective agents for reducing cisplatin nephrotoxicity, most probably, by antioxidants effects and inhibition GGT production, respectively.


Introduction
Cisplatin is a major antineoplastic drug for the treatment of solid tumors, but it has dose-dependent renal toxicity.It has multiple intracellular effects, causing direct cytotoxicity with reactive oxygen species as apoptosis, inflammation and fibrogenesis [1].
Despite all the marvelous advancements in modern medicine, traditional herbal medicine has always been practiced [2].N. sativa is the most common herbal medicine all over the world for the treatment and prevention of a number of diseases.Their seeds/oil has anti-inflammatory, analgesic, antipyretic, antimicrobial, hypoglycemic and antineoplastic activity without significant adverse effects.This may be related to their cytoprotective and antioxidant actions [3].M. chamomilla is a well-known medicinal plant as carminative, analgesic, and anticonvulsant in traditional medicine [4].It has moderate antioxidant, antimicrobial activities and significant antiplatelet activity in vitro.Animal model studies indicate potent anti-inflammatory action, some antimutagenic and cholesterol-lowering activities, as well as antispasmotic and anxiolytic effects [5].Its methanolic extract showed potent neuroprotective activity against global cerebral ischemia/reperfusion injury-induced oxidative stress in rats [6].There are numerous varieties of Chamomile but the most two popular are Roman Chamomile and German Chamomile.German Chamomile which is called Matricaria chamomilla, considered the more potent of the two and received more scientific evaluation.The traditional drug of Matricaria recutita is the dried flower heads [7].
Combination of two or more herbs may be used with chemotherapy to decrease toxic adverse effects, elevate the immune function, and improve the quality of life in the patients.However, this required further studies in human.
The aim of the present study is to design cisplatin nephrotoxicity rat model mimics human cycles of cisplatin chemotherapy, then, evaluates the nephroprotective effects of N. sativa, M. chamomilla and vitamin E in this model.

Preparation of Supportive Treatments and Cisplatin
N. sativa (seeds) and M. chamomilla (dry leaves and flowers) were selected with a fair degree of quality assurance from faculty of Pharmacy, Pharmacognosy department, Assiut University, Egypt.The identity of the planets was verified by the Center of Medicinal, Aromatic and Poisonous Plants and a voucher specimen were kept on record in the herbarium of the Faculty of Pharmacy.The dry leaves crushed to powder and extracted according to method described by El-Daly [8].Twenty five grams of either powder extracted by 95% ethanol (500 ml) with continuous stirring at 4˚C, overnight, this was repeated for three successive days.The pooled extracts (1500 ml), evaporated under reduced pressure using Duché rotary flash evaporator rendering the product alcohol-free.Then, one gram of the product, reconstituted with 20 ml saline so, final concentration was 50 mg/ml and stored at 4˚C.N. sativa oil and Vitamin E purchased from Pharco-Company, Egypt.Eight grams of N. sativa oil (w/v) emulsified with 100 ml 2% polyethylene glycol-400 w/v (final concentration was 80 mg/ml).Vitamin E (90 mg, w/v) dissolved in 30 ml corn oil, so, final concentration was 3 mg/ml as described by El-Daly [8].Vial of 50 mg Cisplatin, Bristol-Myers Squibb Company, USA, reconstituted in 20 ml saline immediately before injection with final concentration 2.5 mg/ml.

Experimental Design
The study carried out on 80 healthy adult male Sprague-Dawley rats, weighing 250 -350 g.Their ages ranged from 26 -28 weeks.The animals were housed conventionally in clean cages and fed with standard food and water ad libitum.The animals were housed in groups in 12-hour light/ dark cycle.The care and treatments of the animals were approved and performed according to the guidelines of Animal House and ethical standards of Faculty of Medicine, Assiut University, Egypt.The experiment carried out in 10 groups of 8 animals each (G1 to G10).The first group (G1) was the healthy reference (H.R) group, injected with 1 ml saline I.P in the same way as group 2. The second group (G2) was cisplatin nephrotoxicity rat model, injected with 5 mg/kg cisplatin I.P on the zero day of experiment.The injection repeated 4 times, with 5 days free interval in between (on the day, zero th , 5 th , 10 th , 15 th ) that mimics human regimen.By the 5 th day up to 43% of the administered cisplatin is recovered in the urine [9].G3 and G4 received N. sativa extract (50 mg/kg I.P), G5 and G6 received N. sativa oil (400 mg/kg orally by tubing), G7 and G8 received M. chamomilla extract (50 mg/kg I.P), G9 and G10 received vit.E (6 mg/kg I.P).The injection of the three compounds started 5 days before the experiment (-5 day) and continued daily till the end of the experiment.In addition, G4, G6, G8 and G10 received cisplatin injection one hour before supportive treatments in the same way like group 2. On the day sixteenth, animals weighted, blood samples obtained, then animals sacrificed and both kidneys were removed and weighted.
One kidney was homogenized in 3 ml ice cold saline using a Glas-Col homogenizer fitted with teflon plunger (Terre Haute, # 099C, USA).Then centrifuged at 4000 × g for 10 min.at 4˚C, supernatant was kept at -70˚C for further biochemical measurements.The second kidney prepared for histopathological examinations.

Histopathological Examinations
Kidney was divided into 2 parts, the 1 st part fixed in 10% phosphate buffered formalin and stained by H&E for light microscopic examination.The 2 nd part fixed in 5% gluteraldhyde, then, prepared semithin sections stained by toluden blue stain, and examined by light microscope.Representative fields of the semithin sections were selected, then ultrathin sections (70 nm) were cut with diamond knife using a Reichert OMVs ultra microtom for electron microscopic studies using lead citrate, uranyl acetate stain [18], and transmission electron microscope (Jo El JEM, Japan.Model 100 CxII) in Electron Microscope Unit of Assiut University.

Statistical Analysis
Soft ware, SPSS version 16 was used for statistical analysis.Values expressed as mean ± SE.Differences between obtained values carried out by Mann-Whitney U test using G1 as reference group.One way analysis of variance (ANOVA) followed by post hoch test (Tukey HSD) multiple comparison test using G2 as reference group.A P < 0.05 is a criterion for significant.

Results
For clinical assessment, there was increase in body weight in G1 (3.99%) and in groups received supportive treatment only (G3, G5, G7, G9).Contrary, there was reduction in body weight in all groups received cisplatin plus supportive treatment.The maximum is in G2 that received cisplatin only (7.59%), then G4 (4.55%), G6 (3.98%), G10 (3.75%) and the minimum is G8 that received combination of cisplatin and M. chamomilla In this study, Cisplatin caused elevation in all kidney function indices.Co-administration of M. chamomilla extract with cisplatin provided the best protection for the kidney by reducing the levels of urea, creatinine, NAG and β-GAL followed by co-administration of vitamin E then N. sativa orally and finally N. sativa extract as shown in Table 1.Determination of oxidative stress and antioxidant indices revealed that cisplatin caused significant elevation in NO and LPO, significant decrease in SOD and GSH as shown in Tables 2-4, respectively.The best correction achieved in group received cisplatin with vitamin E then groups treated with N.sativa (extract or oil) and cisplatin.However, M. chamomilla extract with cisplatin provided the lowest protection against oxidative stress.Determination of sulpher compounds revealed that cisplatin caused marked elevation in serum GGT.The best reduction in the level of GGT achieved in M. chamomilla with cisplatin treated group (P < 0.001) as shown in Table 4. On the other hand, cisplatin caused marked decrease in total thiols and GSH compared to control (G1), but vit.E showed the best recovery for total thiols and GSH due to its antioxidant action that reserve GSH and total thiols.Co-administration of M. chamomilla with cisplatin approved better protection from apoptosis, as shown in Figure 2(f), followed by co-administration with N. sativa oil, vitamin E and finally N. sativa extract as shown in Table 5.In this study, cisplatin caused marked decrease in serum Ca 2+ and Zn 2+ levels.Co-administration of N. sativa oil with cisplatin approved better protection from cisplatin-induced hypocalcaemia, followed by M. chamomilla, vitamin E and finally N. sativa extract as shown in Table 6.
The histopathological changes demonstrated the protective effect of co-administration of supportive treatments in different groups (Figure 2) in comparison to new cisplatin rat model (Figure 1).The intensity of histopathological lesions demonstrated the protective effect of co-administration of supportive treatments in different groups as shown in Table 7.The biochemical and histological results of the rats' kidney of groups 3, 5, 7, 9 that received supportive treatment only and served as control groups for these compounds revealed no abnormal changes (data not shown).

Discussion
The public received information about complementary and alternative medicines (CAMS) inaccurate or incomplete [19] that, encourage the practitioners and researchers to evaluate the actual benefits of these agents.N. sativa oil has been shown to possess 67 constituents, many of which are capable of inducing beneficial pharmacological effects in human [20].By HPLC analysis of N. sativa oil, thymoquinone (TQ), dithymquinone (DTQ), thymohydroquinone, and thymol are considered the main active ingredients [21].Bisabololoxide A is the principle constituents of some bioactivities of German chamomile such as anti-inflammatory, gastrointestinal and antipruritic actions [22].
For clinical assessment of cisplatin nephrotoxicity rat model, there was 7.59% decrease in body weight in cisplatin treated group along the period of experiment (cisplatin 5 mg/kg , 4 doses, time of experiment 21 days); compared to 14% decrease in body weights (cisplatin 15mg/kg, time of experiment 5 days) [23].In this study,    there was 1.4 times increase in the percentage of kidney weight to body weight after cisplatin-treatment.This could be explained by the marked decrease in the total body weight in cisplatin treated group.This was in agreement with another studies, where there were 1.7 and 2 times increase respectively, in percentage of kidney weight to body weight in cisplatin-treated animals [24,25].The co-administration of supportive treatment with cisplatin resulted in decrease in this percentage when compared to cisplatin treated groups; this may be due to the relatively less reduction in the total body weights in these groups.
The cut-off value for the normal range of blood urea nitrogen is (≤40 mg/dl) and serum creatinine is (≤0.2 mg/dl) based on the values obtained from normal untreated mice [23].In this study, urea and creatinine levels were 2.4 and 1.7 times increase in cisplatin-treated group when compared to control g oup.The increase in urea r Copyright © 2011 SciRes.IJCM   level by 3.7 and 2 times and creatinine level by 2.3 and 4.5 times was reported [24,25].NAG and β-GAL levels are markers of early-impaired renal function and renal tubular damage [26].Cisplatin group, in this study, had significant elevation of NAG and β-GAL levels when compared to control group (Table 1) due to dysfunction of tubular epithelial cells induced by increased traffic of proteins in the tubular lumen [27,28].This was confirmed by histopathological examinations (Figure 1(a) and (c)).These pathological changes were minimized in groups received cisplatin with supportive treatments especially in group treated with N. sativa oil (G6) or M. chamomilla (G8) (Table 1, Figure 2

(c), (d), (f) and (h)).
Oxidative stress was implicated in the pathogenesis of cisplatin-induced nephrotoxicity.Oxidative stress, associated with increased generation of reactive oxygen metabolites (ROM) caused lipid peroxidation in kidney, decreased levels of antioxidants and antioxidant enzymes [29].In this study, oxidative stress induced by cisplatin was manifested by elevation in nitric oxide (NO) and lipid peroxide levels.There was significant elevation in lipid peroxide [25,30], decrease in nitric oxide level [24], decrease in antioxidant as GSH and SOD activities was reported in cisplatin-induced nephrotoxicity [31].
The expression of Cathepsin D, the lysosomal proteases, had been shown to increase with protein degradation occurred during apoptosis [32].In this study, cisplatin induced apoptosis in the form of significant increase in cathepsin D level and DNA fragmentation that is documented by light and electron microscopy in Figures 1(b) and (f).M. chamomilla extract provided significant reduction in these parameters followed by N. sativa oil and vitamin E then, N. sativa extract as shown in Table 5.The intensity of these apoptotic changes was markedly   decreased in group treated with cisplatin and M. chamomilla as shown in Table 7 and Figure 2(f).GGT, a key enzyme of GSH metabolism, can modulate crucial redox-sensitive functions, such as antioxidant/antitoxic defenses and cellular proliferative/ apoptotic balance.The mechanisms of GGT involvement in various pathological processes suggest its potential role as therapeutic target and diagnostic/prognostic marker [33].The highest level of activity is on the luminal surface of the proximal tubule cells in the kidney.Its most common physiological substrates are glutathione and glutathione conjugates [34].The nephrotoxicity of cisplatin was the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a γ-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules [23].GGT cleaved the gamma-glutamyl group of the glutathione-conjugate, and aminopeptidase cleaved the cysteinyl-glycine bond, resulting in platinum-cysteineconjugate.Finally the cysteine conjugate was metabolized by cysteine-s-conjugate beta-lyase to reactive thiol [35].Also, they found that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice and the toxicity was specific to the proximal tubule cells.Acivicin, an inhibitor of GGT, blocks the nephrotoxicity of cisplatin in rats [36].In this study, the highest level of GGT occurred in cisplatin group and the lowest level in (G8) that received combination of cisplatin and M. chamomilla as shown in Table 4.There is a correlation between GGT and NAG in cisplatin group (r = 0.731, P < 0.05).Moreover, M. chamomilla doesn't contain glutamic or methionin [37] to help in regeneration of glutathione.Also, Chamazulene (one of constituents of M. chamomilla) affected free radical processes and inhibited lipid peroxidation in a concentration and time-dependent manner [38].On the other hand, N. sativa seeds contain glutamic acid and methionine [39]; this may explain its antioxidant effect.The beneficial effects of the use of the N. sativa seeds and thymoquinone (one of its constituent) might be related to their cytoprotective and antioxidant actions, and to their effect on some mediators of inflammation [3].
Cisplatin administration caused hypocalcaemia [24,40].Calcium released from intracellular calcium storage in the early phase of nephrotoxicity caused oxidative stress in renal tubular epithelial cells [41].The N. sativa seeds are a source of calcium, iron, and potassium [42].This can explain the improvement in cisplatin-induced hypocalcaemia in groups received N. sativa oil with cisplatin.Zinc pre-treatment caused significant protection against cisplatin enhanced mortality in rats, and reduction in lipid peroxidation and NO [43].However, zinc content had an inverse correlation with platinum incorporation owing to a positive linkage with glutathione (GSH), a zinc-dependent detoxification factor.The combined treatment with cisplatin and Zn(II)-chelator increased platinum uptake with a concomitant reduction of intracellular GSH [44].Zinc was effective factor for protection of weight loss and increased MDA levels in cisplatin hepatotoxicity [45].N. sativa and M. chamomilla has been used as tea from long time indicate their safety and minor side effects.In addition, their nephroprotective effect may encourage physicians to prescribe them after and inbetween chemotherapy.

Table 1 . Kidney functions in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8 received Matricaria chamomilla extract + cisplatin; G10 received vit.E + cisplatin.Data of groups 3, 5, 7, 9 that received only supportive treatment not shown as it is within normal range.N. B: * versus G2; ** versus G4; ***versus G6; **** versus G8.ANOVA followed by Tukey test as post ANOVA test was used to compare between group 2 and different groups.

Table 2 . Oxidative stress in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8 received Matricaria chamomilla extract + cisplatin; G10 received vit.E + cisplatin.Data of groups 3, 5, 7, 9 that received only supportive treatment not shown as it is within normal range.N. B: * versus G2; ** versus G4; ***versus G6; ****versus G8.ANOVA followed by Tukey test as post ANOVA test was used to compare between group 2 and different groups.

Table 3 . Levels of SOD in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8 received Matricaria chamomilla extract + cisplatin ; G10 received vit.E + cisplatin.Data of groups 3, 5, 7, 9 that received only supportive treatment not shown as it is within normal range.N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8.ANOVA followed by Tukey test as post ANOVA test was used to compare between group 2 and different groups.

Table 5 . Apoptotic markers in rats treated with cisplatin compared to those treated with cisplatin combinations.
G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin; G8 received Matricaria chamomilla extract + cisplatin ; G10 received vit.E + cisplatin.Data of groups 3, 5, 7, 9 that received only supportive treatment not shown as it is within normal range.N.B: * versus G2; ** versus G4; ***versus G6; **** versus G8.ANOVA followed by Tukey test as post ANOVA test was used to compare between group (2) and different groups.

Table 7 . Intensity of the histopathological lesions in kidneys of rats of different cisplatin treated groups.
Severe and involved all animals.(+++): Severe and involved most of animals.(++): Moderate and involved some animals.(+): Weak and involved few animals.(-ve ): Negative.G1 is the healthy reference group; G2 is cisplatin treated group; G4 received Nigella sativa extract + cisplatin; G6 received Nigella sativa oil + cisplatin ;G8 received Matricaria chamomilla extract + cisplatin; G10 received vit.E + cisplatin.Data of groups 3, 5, 7, 9 that received only supportive treatment not shown as it is within normal range.