The Protective Impacts of Spriulina Platensis Against Cisplatin-Induced Renal Injury Through The Regulation of Oxidative Stress, Pro-Inammatory Cytokines and Bax/Bcl2 Expression Cascade

One of the main antineoplastic chemotherapy medications is cisplatin; nephropathy is a major side effect of cisplatin. The current study investigates the molecular protective effect of Spirulina Platensis (SP) on cisplatin-induced nephrotoxicity. Forty eight healthy male albino rats were allocated into 4 groups. Group 1 received saline intraperitoneally (IP) twice per week (normal rats). Group 2, received SP (100 mg/kg bw orally). Group 3 injected cisplatin (1.5mg/kg IP) twice per week. Group 4 received SP and at 4 th day received cisplatin (1.5mg/kg IP) for 21 days. After 3 weeks of experimentation, blood and renal tissues were taken for serum analysis, gene expression using qRT-PCR and renal histopathology. SP signi�cantly ameliorated the alterations in the body weight, relative kidney weight, and the disturbance in examined renal markers. Furthermore oxidative stress biomarkers (MDA, NO, SOD, and GSH) induced by cisplatin were recovered and restored by SP. Cisplatin induced upregulation in the gene expression of TNF-α, iNOS, TGF1-β, IL-1β and IL-6 that were ameliorated by pre-administration of SP. Finally, cisplatin upregulated pro-apoptotic gene; Bax and downregulated anti-apoptotic gene; Bcl2. Of interest, SP mitigated this alteration in apoptosis and anti-apoptosis associated genes. Renal histopathology revealed the protective impacts of SP against cisplatin induced severe glomerular congestion, hemorrhage, in�ammatory cell in�ltration, degeneration and sever necrosis in renal glomeruli and tubules. In conclusion, SP has protective impact against cisplatin induced renal damage through the modulation of oxidative stress, anti-inammatory, anti-necrotic and anti-apoptotic associated genes.


Introduction
Cisplatin is commonly used for chemotherapy to treat different cancers effectively 1 .The use of cisplatin in treatment of various diseases is restricted due to its serious side effects 2 .Nephrotoxicity, neurotoxicity and ototoxicity are common cisplatin side-effects.Nephrotoxicity occurs predominantly in kidney proximal tubule epithelial cells 3 , The cisplatin is less toxic in blood level compared to the kidneys, as cisplatin concentration in epithelial tubular cells is ve times higher than in the blood 4 Renal dysfunction occurs within days from the usage of cisplatin (50-120 mg/m2) in treatment 5 .Proximal and distal convoluted tubules are the key targets of cisplatin effects in the kidneys, where it promotes cellular damage, oxidative stress, DNA damage, apoptosis and nephritis 6,7 .
Nephrotoxicity and ototoxicity induced by cisplatin are due to toxic generation of reactive oxygen species (ROS) and imbalance of antioxidant glutathione system, as cisplatin is highly reactive with molecules contain thiol such as including glutathione 8 .In parallel, cisplatin inhibits various antioxidant enzymes such as glutathione peroxidase (GPX), glutathione-S-transferase (GST), and superoxide dismutase (SOD) 9 .Conjugation of glutathione with cisplatin contribute to mitochondrial lysis, oxidative stress and lipid peroxidation causing ROS generation in the cells 10 .In addition to ROS, cisplatin increases protein expression of renal inducible nitric oxide synthase (iNOS) and the formation of nitrotyrosine that are associated with the stress response 11 .
Generation of ROS triggers cascades of signal transduction that results in apoptosis, necrosis.Till now, the mechanism of ROS induces cisplatin signaling pathways still unclear.Lipid peroxidation due to production of highly toxic 4-hydroxynonenal (4-HNE) is one possible pathway.Increased 4-HNE levels cause an increase in Ca 2+ in ux, and apoptosis 12 .Mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 are also activated by Cisplatin.It is uncertain the pathway of cisplatin to activate MAPKs, but inhibition of MAPKs decreases cisplatin nephrotoxicity.Cisplatin activates EGFR/Src, inducing ERK and caspase-3, leading to apoptosis and nephrotoxicity 13,14 .A number of cellular stress pathways, including oxidative stress and in ammation leading to activation of p38 and JNK contribute to TNF-alpha transcriptional induction that cause nephrotoxicity 15 .
Spirulina platensis (SP) is a blue-green freshwater algae, naturally grows in warm climate countries and has been regarded as a human and animal food supplement. 16.Recent studies demonstrated the protective impacts for human health 17 .SP is a unicellular lamentous blue-green cyanobacterium, because of its high nutritional value.It is very rich in minerals, essential amino acids, essential fatty acids (ω-3 and ω-6), vitamins A, C, and E, and accessory pigments such as phycobiliproteins.Therefore, it is useful as food by humans and other animals 18 .In addition, several studies showed that SP species exhibit various biological activities such as anti-in ammatory 19 and antioxidant effects 20 .Therefore, current study was speculated to examine the possible protective impacts of SP against cisplatin induced renal toxicity at different cellular levels.
Lipid peroxidation (MDA), superoxide dismutase (SOD), reduced glutathione (GSH), nitric oxide (NO) kits were purchased from Biodiagnostic Company, Cairo, Egypt (Cat number 2529, 2521, GR25H, 2533, respectively).Spirulina platinsis was from Algal Biotechnology unit, National Research Center Dokki, Cairo, Egypt Animals and Experimental design.The Ethical Committee of Kafrelsheikh University has approved all used procedures and animal use for this study for project 13/2/2018.Forty eight male Wistar rats (170-200 g) were bought from Egyptian Organization for Biological Products and Vaccines (Giza, Egypt).Rats were housed Biochemistry Department, Faculty of Veterinary Medicine, Kafrelsheikh University.Rats were handled daily for 7 days.Rats lived on 12/12 hours light/dark cycle and temperature with range 24 ± 2 °C.All animals gained free access to food and water.
Animals were allotted into 4 groups.Group 1 (control group); injected intraperitoneally saline twice a week for 3 weeks.Group 2 (spirulina platinsis; SP) received SP in saline (1000 mg/kg/bw, intragastric) for consecutive 3weeks.Group 3 (cisplatin-group); rats injected intraperitoneally with cisplatin (1.5 mg/kg) twice a week for 3 weeks.Group 4 (SP + cisplatin), rats received SP as in group 2 and cisplatin as in group 3 for consecutive 3 weeks.Blood sampling.Animals were anaesthetized at the end of the experiment with thiopental sodium (50 mg/kg body weight, IP) 21 .Blood samples were obtained from rats' retro-orbital venous plexus and obtained in clean and dry centrifuge tubes and held in an ice box, centrifuged for 20 minutes at 3000 rpm after coagulation.Sera was transferred to a clean dry Eppendorf tubes and processed for biochemical analysis at -80 °C.Tissue sampling.Animals were subjected to mild anesthesia using thiopentone sodium of 50 mg/kg of body weight by IP injection at the end of the experiment 21 , and were sacri ced by decapitation.Kidney tissue was excised and weighed.The right kidney was split into two different sections and rapidly immersed in liquid nitrogen and deposited in 80 °C for RNA extraction quantitative real-time PCR.Part from kidney tissues was used for antioxidant estimation.For histopathology examination, left kidney were immersed in 10 % neutral buffer formalin (NBF).Biochemical assays.All kidney markers such as serum creatinine was estimated utilizing using commercially available kit (Spinreat, S.A # 1001110).Serum urea was measured using commercial available urea kit (Spinreat, S.A # 1001323).Uric acid was estimated using uric acid kit (Spinreat, S.A # 1001010).Albumin was measured using albumin kit (Spinreat S.A# 1001020).MDA, SOD, GSH and NO kits were purchased from (Biodiagnostic Co, Cairo, Egypt) All methods were carried out according to the instructions provided by the manufacturer.Histopathology analysis.Left kidney specimens were sectioned at a thickness of 5 μm and cleared in xylene and stained with H&E 22 .Microscopic histopathological changes were examined (Leitz DMRBE, Germany).Pictures were taken on the X200 for H&E ampli cation with an advanced camera (Leica DFC 295).

RNA extraction and Real-time polymerase chain reaction (PCR).
Prior to RNA extraction, the right kidney tissues were snap-frozen in liquid nitrogen and stored at -80 °C.Following the manufacturer's protocol (GeneZOlTM RNA extraction reagent), complete RNA was extracted using Trizol reagent.In RNase free water (Nanodrop 2000c, Thermo Scienti c, USA).RNA was dissolved and the integrity was examined in denatured gel.Complementary DNA was blended using the HiSenScriptTM[-] cDNA synthesis kit (iNtRON Biotechnology).By mixing 10μl of 2X RT reaction solution, 1μl of enzyme mix solution, 1μg of RNA and completion of RNase free water to 20μl total amount, complementary DNA was added.The mixture was incubated for 30 minutes at 50 °C and 10 minutes at 85 °C.Using real time PCR with SYBR green qRT-PCR, the gene expression levels in the kidney tissue were investigated.The primers were synthesized by Macrogen Co. Seoul, Korea, and were listed in (Table 1).
The gene expression levels in the kidney tissue were determined using real-time PCR using SYBR green.Introductory denaturation at 92 °C for 10 min, accompanied by 40 cycles of 15 seconds at 92 ° C, 30 seconds at 60 °C and 30 seconds at 72 °C, according to the PCR thermal cycler machine program.The differences between the groups in gene expression were determined using the method of △△ Ct (cycle time, Ct), normalized to β-actin, and expressed as relative levels of mRNA compared to placebo.Data analysis.Prism software (GraphPad Software Inc., San Diego, CA, USA) was used to perform the data analysis.One-way variance analysis (ANOVA) was carried out, followed by a Bonferroni test to assess the statistically relevant differences between groups.Values of p ≤ 0.05 have been deemed statistically signi cant.

Results
Effect of spirulinaplatensis on Body weight and kidney/Body weight.The body weight was signi cantly decreased (P<0.05) in cisplatin-treated rats by 31.1% comparison with normal rats, and by 29.9% compared to SP alone treated rats.Interestingly, spirulina treatment to cisplatin-treated rats was signi cantly increase (P < 0.05) by 36.8% compared to cisplatin-treated rats as shown in (Figure 1A).Furthermore, rats treated by cisplatin alone showing signi cantly increase (P<0.05)kidney/body weight by 111.5% compared to normal rats and by 96.4% compared to group received spirulina alone.Interestingly, administration of spirulina to cisplatin-treated rats signi cantly decreased (P<0.05)kidney/body weight by 41.5% compared to cisplatin-treated rats (Figure 1B) Effect of spirulina platensis on renal biochemical parameters.Kidney function markers were shown in table (2).Cisplatin injected rats showed an increase in creatinine (P < 0.05), which was 214% verse to normal rats and 209.2% verse to rats receiving Spirulina alone.Administration of Spirulina in cisplatintreated rats decreased the serum creatinine level substantially (P < 0.05) by 48.8 % compared with cisplatin group.Serum urea levels in cisplatin-treated rats were decreased signi cantly (P < 0.05) by 219.8 % compared to normal rats, and by 207.5% compared to rats treated by spirulina alone.Spirulina pre-administration to cisplatin-treated rats signi cantly decreased serum urea by 43.5 % relative to rats treated with cisplatin group.
The level of serum uric acid was signi cantly increased by 114.4 % compared to normal rats, and by 108 % compared to rats treated with spirulina alone.Serum uric acid was signi cantly reduced by 45.6 % compared to cisplatin-treated rats by spirulina pre-administration.In cisplatin-treated rats, serum albumin decreased signi cantly by 57.2 % relative to normal rats, and by 59.4 % compared with group received spirulina alone.Serum albumin levels were increased (73.3%) when spirulina pre-administered to cisplatin group.
Effect of spirulina platensis on antioxidant enzyme activity.There was a substantial increase in lipid peroxidation in kidney tissue of cisplatin-treated rats (278.5 %) compared to normal rats and 303.9 % compared to rats treated with spirulina alone.Pre-administration of spirulina to cisplatin group, a substantial reduction in MDA by 60.6 % was reported.In parallel, NO was a signi cantly increased in cisplatin-treated rats by 429.7 % compared to normal rats, and by 625.9 % compared to spirulina-treated rats.Spirulina pre-administration to cisplatin-treated rats decreased NO by 62.7 %.Interestingly, spirulina administration induced signi cant increase in SOD, and GSH activity by 74.1%, and 104.7% respectively compared to cisplatin-treated rats as listed in Table 3.
Renal histopathological analysis.Histopathological analysis of the kidney sections of normal and spirulina treated rats showed normal glomerular and tubular structures as shown in (Figure 2A-B).Cisplatin treated rats showed severe glomerular congestion, in ammatory cell in ltration inside the interstitium and serious hemorrhage occurred in the medullar and cortical regions in addition to presence of hyaline cast, sever degeneration and severe necrosis in the tubule as shown in (Figure 2C).
Interestingly, the spirulina pre-administration to cisplatin treated rats showed marked decrease in all pathological changes induced by cisplatin ( gure 2 D).
Impacts of Spirulina platensis on gene expression analysis.In order to investigate the impact of Spirulina on genes of oxidative stress biomarkers, TNF-a, iNOS and TGF1b were examined using qRT-PCR.TNF-a, iNOS and TGF1b expressions were up-regulated in cisplatin-treated rats compared to normal and Spirulina treated rats.Interestingly, reduction in TNF-a, iNOS and TGF1b expression were seen in cisplatintreated rats pre-administered with Spirulina (Figure 3A-C).
In addition, IL-1β, IL-6 mRNA expressions in cisplatin-treated rats were signi cantly up-regulated compared to normal and Spirulina treated rats respectively.Pre-administration of Spirulina to cisplatin injected rats showed restoration and recovery for alter cytokines (Figure 4 A-B).Furthermore, we investigated the effect of spirulina administration on apoptotic and anti-apoptotic genes.
The result revealed that the expression level of Bax was signi cantly upregulated in cisplatin-treated animals compared to normal control and spirulina treated rats.Spirulina pre-administration for cisplatintreated rats counteracted levels such upregulation and restored it to nearly control levels (Figure 5A).In contrast to Bax ndings, Bcl2 mRNA expression was signi cantly downregulated in cisplatin-treated rats compared to normal control and Spirulina treated rats.Pre-treatment of cisplatin injected rats by Spirulina signi cantly restored and upregulated Bcl2 expression ( gure 5B).Such ndings con rmed that Spirulina Platinsis has anti-necrotic, anti-in ammatory, and anti-apoptotic properties, and con rmed the protective effect of Spirulina on renal damage induced by cisplatin (Figure 6).

Discussion
Oxidative stress plays a central role in kidney dysfunction 23 ,and cisplatin increased the production of superoxide, peroxynitrite and hydrogen peroxide [24][25][26][27] , as cisplatin accumulates in proximal and distal convoluted tubules and caused oxidative stress, apoptosis and in ammation 28,29 .Current study showed that cisplatin treatment induces renal marker dysfunction as indicated by a signi cant increase in serum creatinine, urea, uric acid, and decrease in albumin levels.It has been shown that single administration of cisplatin induced signi cant renal dysfunction 30,31 .Interestingly, renal dysfunction induced by cisplatin was ameliorated by Spirulina platensis treatment.In a separate study, Spirulina dose-dependently ameliorated creatinine, urea clearance and BUN elevation levels 32 .
Signi cant increase in NO caused cellular ATP depletion that can inactivate enzymes of Krebs cycle and mitochondrial electron transport enzyme 33 .Lipids peroxidation, is a marker of tissue damage 34 , and the antioxidants play a critical role in the prevention of many diseases and protected against a large variety of nephrotoxic chemicals from toxicity 35 .Cisplatin-treatment increased lipid peroxidation and decreased the activity of antioxidant enzymes, such as SOD, catalase and glutathione peroxidase as reported in this study and in others 36 .It has been shown that cisplatin induced renal oxidative and nitrosative stress 25,26,34 .Interestingly, Spirulina pre-administration signi cantly ameliorated the increase in MDA and NO, and the decrease in SOD and GSH levels in cisplatin received rats, and agree with another study that reported the antioxidant activity of Spirulina against lead-exposed animals in the liver, lungs, brain, and kidney 20,37 .Spirulina-enriched diet raised cerebellar glutathione levels, decreased lipid peroxidation levels and controlled pro-in ammatory cytokines 38 .The use of Spirulina platensis clearly decreased NO production due to gentamicin injection that might be due to inhibition of iNOS 39 .In parallel, Spirulina platensis recovered renal damage showed by histopathology as reported by us and others for Spirulina fusiformis 32 .
ROS activates the transcription factor NF-kB induced by cisplatin, which increases the development of pro-in ammatory cytokines such asIL-1, IL-6 and TNF-α 40 .ROS induced by cisplatin mediates synthesis of TNF-α through phosphorylation of p38 MAPK 41 .Many cytokines have been implicated in cisplatininduced renal toxicity, among which is TGF-β and hemeoxygenase-1; HO-1 42 .Similarly, cisplatin induced up-regulation of several cytokines, such as TNF-alpha, TGF-β and IL-1b in kidney 43 that coincided with reported results.Interestingly, all altered cytokines were ameliorated by Spirulina administration due to its antioxidant and anti-in ammatory properties 38 Cisplatin caused an increase in the proportion of Bax/Bcl-2 protein expression.Increase in Bax (proapoptotic protein (Bax)/Bcl2 (anti-apoptotic protein) increases the capacity of renal cells for apoptosis 44,45 .Cisplatin therapy up regulated Bax and down regulated Bcl2 expression, and Spirulina administration down regulated Bax and up-regulates Bcl2 expression 46 .Collectively, SP showed the potential to attenuate the cisplatin nephrotoxicity.SP regulated oxidative stress, in ammatory cytokines, apoptosis and anti-apoptosis cascade.

Conclusion
Protective impacts of Spirulina on cisplatin-induced renal damage was con rmed.The possible mechanisms of Spirulina to protect the kidney against cisplatin effects involve antioxidant, anti-necrotic, anti-in ammatory, anti-apoptotic signaling pathways.Therefore, Spirulina administration is recommended to hold the kidney against cisplatin.The collective impacts of spirulina against cisplatin nephrotoxicity are shown in gure 6.The data represent the mean ± SE (n=6).One-way ANOVA-Bonferroni's as a post hoc test and; Different liters mean signi cant at p < 0.05.Same liters mean insigni cant at p > 0.05

Declarations
of Spirulina platensis on (A) body weight, and (B) relative kidney weight to body weight of different studied groups.Data were presented as mean ± SEM at p < 0.05 (Bonferroni's as a post hoc test) (n=12).

Figure 1 Effect
Figure 1

Figure 3 Effect
Figure 3

Figure 4 Effect
Figure 4

Figure 4 Effect
Figure 4

Figure 5 Effect
Figure 5

Figure 5 Effect
Figure 5

Table 2 .
Effect of spirulina on serum biochemical parameters of cisplatin induced nephrotoxicityThe data represent the mean ± SE (n=9).One-way ANOVA-Bonferroni's as a post hoc test and; Different liters mean signi cant at p < 0.05.Same liters mean insigni cant at p > 0.05

Table 3 :
Effect of spirulina on tissue antioxidant activity in cisplatin induced nephrotoxicity