World Journal of Medical Sciences 12 (3): 303-315, 2015ISSN 1817-3055© IDOSI Publications, 2015DOI: 10.5829/idosi.wjms.2015.12.3.9615

Corresponding Author: Ahmed Abdel Aziz Baiomy, Department of Zoology, Faculty of Science, Cairo University, Egypt.,Department of Medical Biotechnology, Faculty of Applied Medical Sciences, Taif University, KSA.E-mail: aabdelaziz.baiomy@gmail.com.

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Histopathological and Immunohistochemical Studies ofAntiseptic Effect of Sepia officinalis Against Induced Sepsis in

Male Albino Rats (Rattus norvegicus)

Ahmed Abdel Aziz Baiomy and Dalia Yossri Saad1,2 1,3

Department of Zoology, Faculty of Science, Cairo University, Egypt1

Department of Medical Biotechnology, Faculty of Applied Medical Sciences, Taif University, KSA2

Department of Medical Laboratory, Faculty of Applied Medical Sciences, Taif University, KSA3

Abstract: In the present study we investigated the effect of both methanolic extract and cuttlebone ofSepia officinalis against sepsis induced by cecal ligation and puncture CLP in rats. 48 male albino rats weredivided into four groups. Group I Sham control rats. Group II CLP model (septic rats). Group III Septic ratstreated with body tissue extract of sepia 200mg/kg body weight, one dose daily for seven days. Group IV Septicrats treated with cuttlebone extract 200mg/kg body weight, one dose daily for seven days. It was found thatCLP models lead to histopathological changes in the lung characterized by interstitial pneumonia, the kidneyshowed atrophy of glomerular tuft and congestion of renal blood vessel, the testis showed degeneration ofspermatogoneal cells. Group III showed mild histopathological changes. Group I and group IV rats showed nohistopathological changes.The immunohistochemical staining of caspase-3 in studied organs showed strongimmunopositive reaction in CLP group, mild immunopositivity in Group III, while group I and group IV ratsshowed no expression of caspase-3. In conclusion, the current study indicated that the body tissue extract ofSepia had a mild antiseptic effect against sepsis while its cuttlebone showed strong antiseptic effect.

Key words: Histopathology Caspase-3 Sepia officinalis Sepsis Cecal Ligation and Puncture

INTRODUCTION permeability at 2 hours and a decrease in renal capillary

Sepsis is a critical condition often caused by bacterial infections, either localized or systemic, can lead to maleinfection and is considered a common cause of morbidity infertility [6]. Apoptosis is a second prominent feature ofand mortality [1]. Sepsis is the biggest obstacle sepsis. This process is a mechanism of tightly regulatedpreventing improvement of the success rate in curing disassembly of cells caused by activation of certaincritical illnesses. It is a systemic inflammatory response specialized proteases called caspases. Parenchymal cells,syndrome (SIRS) induced by infection, is accompanied by including intestinal and lung epithelial cells, also havethe presence of bacteria or a highly suspicious focus of increased apoptotic cell death in animal models of sepsisinfection leading to a massive, uncontrollable release of [7, 8]. Caspases have been recognized to play aninflammatory factors [2]. High levels of inflammatory important role in inflammation and cell death. The rolemediators can lead to the increase of blood capillary of apoptosis in the development of multiple organpermeability and pulmonary oedema, resulting in acute dysfunction syndrome (MODS) and compensatory anti-respiratory distress syndrome, multiple organ failure, inflammatory response syndrome (CARS) is not wellhigh mortality and other clinical disorders requiring established [9, 10, 11]. Cytosolic caspase-3 activation ishospitalization. Cecum ligation and puncture CLP , is regulated by both TNF- receptor mediated exrtinsic andcurrently the most widely used animal model of sepsis intrinsic apoptotic cascades [12]. Additionally, in situ[3, 4]. CLP caused an increase in renal capillary localization of cleaved caspase-3 has found some favor

perfusion at 4 hours in murine sepsis [5]. Microbial

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for histological labeling of cells in apoptosis (12). small pieces, homogenized and extracted with methanolApoptotic renal tubular cell death was increased at room temperature for 24-48h. Then, the methanolicsignificantly 6 hours after CLP [13]. Despite huge extract was centrifuged to collect the supernatant andexpenditure of time and resources on the evaluation of concentrated under vacuum in a rotary evaporator atnew drugs for the treatment of sepsis, little success has low temperature. This method will perform according tobeen achieved [14]. In recent years, human pathogenic Ely et al. [23].microorganisms have developed resistance in response tothe indiscriminate use of commercial antimicrobial drugs Preparationof Polysaccharide Extract from Cuttlebone:commonly employed in the treatment of infectious The polysaccharide extract was prepared using thediseases. The undesirable side effects of certain method described by Okutani and Morikawa [24]. The airantibiotics and the emergence of previously uncommon dried cuttlebones were pulverized and washed withinfections has forced scientists to look for new acetone. The powder was extracted with hot 10mM EDTAantimicrobial substitutions from various sources such as solution and filtered using Whatman No. 1filter paper.from plant and animal origin [15]. Lately, Mohanraju et al. Then saturated barium hydroxide solution was added to[16] reported that molluscs have good antibiotic the filtrate. The precipitate obtained after standingproperties. Cephalopods are important as a food resource overnight was collected on Whatman No. 1 paper andas well as animal models in scientific investigations [17]. washed with water. The precipitate was dissolved inSepia officinalis is one of the most common cephalopods 10mM EDTA solution and was dialyzed against deionized[18]. The present study select cuttlefish (Sepia officinalis) water. The dialysate was freeze-dried which was thenmember of class cephalopoda due to its availability and its used in the present investigation.own bioactive compounds. Good antimicrobial activitywas seen in the extracts of O. dollfusi, S. lessoniana and Animals: Adult male albino rats (Rattus norvegicus)S. inermis species, which indicate the presence of potent weighing 150 -170 g were obtained from the King Fahdantimicrobial compounds in them [19]. Methanolic extracts Research Unit in King Abdulaziz University, Jeddah,of six species of cephalopods (Sepia kobiensis, Sepiella KSA. Rats were housed in polypropylene cages in an airinermis, Sepioteuthis lessoniana, Octopus aegina, conditioned room at a temperature of 25 ± 2°C and underOctopus aerolatus, Octopus dollfusi) showed in vitro natural day and night cycle. They were fed standard chowantimicrobial activity [19]. The antimicrobial activity of pellets and drinking water ad libitum. The rats were keptpolysaccharides extracted from cephalopods such as for one week before the commencement of the experimentSepia aculeata and Sepia brevimana and heparin – like for acclimatization. glycosaminoglycans (GAGs) from the cephalopodEuprymna berryi was reported against the human Experimental Design: 48 male albino rats were dividedpathogenic microorganism [20, 21]. Cuttlebone has into four groups. Group I Sham control rats, group IIantibacterial and antifungal activity against some of the CLP model (septic rats) both fed with drinking waterhuman pathogenic microorganisms [22]. Therefore, the ad libitum, while group III Septic rats treated withpresent study aims to assess the antiseptic and body tissue extract of sepia fed with cuttlefishantiapoptotic effect of the body tissue and cuttlebone methanolic extract at a limit test dose of 200mg/kg bodyextract of Sepia officinalis against induced sepsis in male weight, one dose daily for seven days. Group IV Septicalbino rats, using histopathological and rats treated with cuttlebone extract fed withImmunohistochemical studies. polysaccharide extract at a limit test dose of 200mg/kg

MATERIALS AND METHODS will observe at 0 min, 30 min, 1 , 2 , 4 , 6 hrs and thereafter

Sample Collection: Fresh cuttlefish (Sepia officinalis) animals were euthanized by ether anesthesia andwere collected from Thuwal area, Saudi Arabia. The dissected for examination of lung, kidney and testis foranimals were transported to our laboratory in an ice box pathological changes [25, 26].where they were dissected and the cuttlebones werecollected and air dried. Rat Septic Model: Sepsis is achieved in rats by operating

Preparation of Methanolic Extract from the Body Tissue: considered to be the most clinically relevant models ofThe body tissues of the Sepia officinalis will cut into sepsis [27].

body weight, one dose daily for seven days. The animals

every day for 7 days. At the end of the 7 day theth

of the cecal ligation and puncture (CLP) process which is

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Histopathological Examination: Samples for hyperplasia, vacuolations of bronchial epitheliumhistopathological examination were taken from lung, associated with focal desquamation of epithelial liningkidney and testis. Samples were fixed in 10% neutral and hemorrhage with perivascular round cells infiltrationbuffer formalin solution then washed in tap water and (Fig. 2B, C and D). The septic group treated withdehydrated by different grades of alcohol and cleared by methanolic body tissue extract of sepia showed mildxylene then embedded in paraffin. The paraffin embedding interstitial pneumonia (Fig. 2E). The septic group treatedblocks were cut at 5 µm thick. The sections were routinely with cuttlebone extract showed apparent normalstained with haematoxylin and eosin [28]. pulmonary parenchyma (Fig. 2F).

Immunohistochemistry: The samples embedded in structure with intact Boman's capsule and normal renalparaffin were cut into 3µm thick sections and mounted on tubular structures (Fig. 4A). The septic group showed apositively charged slides for caspase-3 marked atrophy of glomerular tuft, intra-tubular proteinimmunohistochemistry. Sections were dewaxed, casts, dilation and congestion of renal blood vessel withrehydrated and incubated in antigen retrieval buffer perivascular inflammatory cells infiltration and edema(boiling the sections at 98°C for 20 min in 10 mmol/L (Fig. 4B, C and D). The septic group treated withsodium citrate buffer), treated with 3% H2O2 to block methanolic extract of sepia showed protein casts in theendogenous peroxidase. Primary rat-specific antibody for lumen of renal tubules (Fig. 4E). The septic group treatedcaspase-3 (Thermo Fisher Scientific Co., USA), was added with cuttlebone extract showed normal glomerularafter dilution by PBS (1:100) and incubated for 30 min. structure with intact Boman's capsule and normal renalThe slides were washed three times for 3 min each with tubular structures (Fig. 4F).PBS. Biotinylated polyvalent secondary antibody(Cat. No. The testis of sham control group showed normal32230, Thermo Scientific Co., UK) was applied to tissue histological structure of active mature functioningsections and co-incubated for 30 min. The slides were seminiferous tubules composed of spermatogenic cellswashed three times for 3 min each with wash buffer. (spermatogonia, spermatocytes, spermatids) (Fig. 6A).The reaction was visualized by adding Metal Enhanced The septic group showed small diameter of seminiferousDAB Substrate Working Solution to the tissue and tubules and degeneration of spermatogoneal cellsincubated 10 min. The slides were washed two times for (Fig. 6B). The septic group treated with methanolic extract3 min each with wash buffer. Counterstaining was of sepia improved histological structure of seminiferousperformed by adding adequate amount of haematoxylin tubules with accumulation of germ cells in the lumenstain to the slide to cover the entire tissue surface [29]. (Fig. 6C). The septic group treated with cuttlebone

RESULTS (Fig. 6D).

Mortality Description: The mortality rate was Immunohistochemical Staining of Caspase-3: Thesignificantly higher in CLP group than the other groups. immunohistochemical staining of caspase-3 was localized24 h after CLP, mortality was 16.66% of rats died and it in the cytoplasm and the nucleus of pulmonary cells.became 8.33% after 2 days and it became 33.32% after The lung of sham control group and the septic group7 days in septic group. Mortality rate was 8.33% of rats treated with cuttlebone extract showed no expression ofdied and it became 8.33% after 2 days and it became zero caspase-3 (Fig. 3A and D). The Septic group showedafter 7 days in the septic group treated with methanolic strong immunopositive reaction (Fig. 3B). The septicbody tissue extract. The septic group treated with group treated with methanolic extract of sepia showedcuttlebone extract showed mortality rate 8.33% after 24 h mild immunopositive reaction (Fig. 3C). and it became zero after 2 days and 7 days. Mortality rate The immunohistochemical staining of caspase-3 waswas zero in sham group during the experimental period localized in the nuclei of renal tubular epithelial cells.(Fig. 1). The kidney of sham control group and the septic group

Histopathology: The lung of sham control group showed caspase-3 (Fig. 5A and D). The Septic group showeda normal structure of alveoli and alveolar sacs (Fig. 2A). strong immunopositive reaction (Fig. 5B). The septicIn contrast, the Septic group showed interstitial group treated with methanolic extract of sepia showedpneumonia with edema, leucocytic infiltration, mild immunopositive reaction (Fig. 5C).

The kidney of sham group showed normal glomerular

extract showed normal structure of seminiferous tubules

treated with cuttlebone extract showed no expression of

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Fig. 1: Mortality rate of group I sham control rats, group II septic rats, group III septic rats treated with methanolic extractand group IV septic rats treated with cuttlebone extract of Sepia officinalis.

Fig. 2: Photomicrograph of rat's lung. (A) Sham control group showed normal alveolar structure (a), alveolar sacs (s) andnormal thin interalveolar septum (arrows): H&E. (bar=14.83µm). (B) Septic group showed interstitial pneumoniawith edema and leucocytic infiltration (arrow). Alveolus (a): H&E. (bar=14.20µm). (C) Septic group showeddesquamation of superficial lining epithelium (upper long arrow), hyperplasia (lower long arrows) andvacuolations of bronchial epithelium (short arrow) associated with focal desquamation of epithelial lining.Bronchiole (b): H&E. (bar=14.52µm). (D) Septic group showed hemorrhage (long arrow) , edema (o) withperivascular round cells infiltration (short arrows). Alveolus (a): H&E. (bar=14.19µm). (E) Septic group treated withbody tissue extract of sepia showed mild interstitial pneumonia (arrow). Alveolus (a), alveolar sac (s): H&E.(bar=14.51µm). (f) Septic group treated with cuttlebone extract showed apparent normal pulmonary parenchymaand normal thin interalveolar septum (arrow). Alveolus (a), alveolar sac (s): H&E. (bar=14.51µm)

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Fig. 3: Photomicrograph of immunohistochemical staining of caspase-3 in rat's lung. (A) Sham control group showed noexpression of caspase. Alveolus (a), Alveolar sacs (s): (bar=14.51µm). (B) Septic group showed strongimmunopositivity in pneumocytes and round cells (arrows). Alveolus (a): (bar=15.14µm). (C) Septic group treatedwith body tissue extract of sepia showed mild immunopositivity in pneumocytes and round cells (arrows):(bar=14.52µm). (D) Septic group treated with cuttlebone extract showed no expression of caspase. alveolus (a):(bar=14.52µm)

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Fig. 4: Photomicrograph of rat's kidney. (A) Sham control group showed normal glomerular structure (g) with intactBoman's capsule (b) and normal renal tubular structures (t): H&E. (bar=11.05µm). (B) Septic group showed atrophyof glomerular tuft (short arrow) and intra-tubular protein casts (long arrows): H&E. (bar=11.68µm). (C) Septic groupshowed dilation and congestion of renal blood vessel(short arrow) with perivascular inflammatory cells infiltrationand edema (long arrow) and intra-tubular protein cast (c): H&E. (bar=12.31µm). (D) Septic group showed severcongestion of renal blood vessel(arrows): H&E. (bar=15.14µm). (E) Septic group treated with body tissue extractof sepia showed protein casts in the lumen of renal tubules (arrows): H&E. (bar=14.19µm). (f) Septic group treatedwith cuttlebone extract showed normal glomerular structure (g) with intact Boman's capsule (b) and normal renaltubular structures (t): H&E. (bar=13.69µm)

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Fig. 5: Photomicrograph of immunohistochemical staining of caspase-3 in rat's kidney. (A) Sham control group showedno expression of caspase: (bar=13.25µm). (B) Septic group showed strong immunopositivity in the nuclei of renaltubular epithelial cells (arrows): (bar=13.57µm). (C) Septic group treated with body tissue extract of sepia showedmild immunopositivity (arrows): (bar=14.52µm). (D) Septic group treated with cuttlebone extract showed noexpression of caspase: (bar=14.52µm)

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Fig. 6: Photomicrograph of rat's testis. (A) Sham control group showed normal histological structure of active maturefunctioning seminiferous tubules (arrow) composed of spermatogonia (g) followed by spermatocytes (c): H&E.(bar=11.04µm). (B) Septic group showed small diameter of seminiferous tubules and degeneration ofspermatogoneal cells (arrows): H&E. (bar=11.66µm). (C) Septic group treated with body tissue extract of sepiashowed desquamated germ cells and accumulated in the lumen (arrow): H&E. (bar=14.51µm). (D) Septic grouptreated with cuttlebone extract showed normal structure of active mature functioning seminiferous tubulescomposed of spermatogonia (g) followed by spermatocytes (c) and spermatozoa (z): H&E. (bar=15.48µm)

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Fig. 7: Photomicrograph of immunohistochemical staining of caspase-3 in rat's testis. (A) Sham control group showedno expression of caspase: (bar=13.57µm). (B) Septic group showed strong immunopositive reaction in the nucleiof spermatogenic cells (arrows): (bar=14.52µm). (C) Septic group treated with body tissue extract of sepia showedmild immunopositive reaction (arrows): (bar=13.56µm). (D) Septic group treated with cuttlebone extract showedno expression of caspase: (bar=14.21µm).

The immunohistochemical staining of caspase-3 was tissue extract of sepia showed mild immunopositivitylocalized in the nuclei of spermatogenic cells. The testis of (Fig. 7C). Compared to The septic group treated withsham control group and the septic group treated with methanolic extract of sepia the septic group showed acuttlebone extract showed no expression of caspase-3 significant increase in the number of caspase-3 positive(Fig. 7A and D). The septic group treated with body cells observed in the seminiferous epithelium (Fig. 7B).

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DISCUSSION During sepsis the most common injury to the kidney

The present study demonstrated by dilation and congestion of renal blood vessel withhistopathological and immunohistochemical examinations, perivascular inflammatory cells infiltration and thesethat sepia tissue extract and polysaccharides attenuate results agreed with [39, 41, 33]. Activated macrophagessepsis-induced lung, kidney and testis injuries in the rat produce oxygen radicals are responsible for cellular lipidmodel of sepsis. peroxidation, protein oxidation and mitochondrial

We have used surgical operation similar to that impairment function, which cause further damage todescribed by [27] as a sepsis model by cecal ligation and tissues and can induce cell death [42]. Also, Wang et al.puncture (CLP) , which led to persistent fecal leakage from [5] demonstrated that CLP caused an increase in renalthe cecum into the abdomen, thus providing a capillary permeability. The cuttlebone extract showedpolymicrobial source of infection in rats and sudden normal glomerular structure with intact Boman's capsuleenterance of large numbers of pathogens and its toxins and normal renal tubular structures. These results wereinto the peritoneal cavity [30]. The host response towards supported by the findings of Subhapradha et al. [43] thatthese invading pathogens is characterized by systemic cuttlebone extract of Sepia aculeate showed goodpro–inflammatory response that is primarily mediated by antioxidant activity.cytokines , which can lead to fatal multiorgan failure and The testis of sham and cuttlebone extract treatedseptic shock [31]. The histopathological lesions , that groups showed normal histological structure of activewere observed in the Lung , kidney and testis in rats with mature functioning seminiferous tubules, the septic groupsepsis are due to sever bacteraemia and septicaemia that treated with methanolic extract of sepia improvedresult from rapid transfer of septic pathogens and its histological structure of seminiferous tubules, while thetoxins from the peritoneal cavity into the systemic septic group showed degeneration of spermatogonealcirculation . Gram negative aerobes and Facultative cells [44]. Also, it has been documented that ROSanaerobes such as E. coli which considers the most overproduction associated with inflammatory reactionscommon pathogen agents that identified in circulation in may be primarily caused by pathological bacterial strainsman and laboratory animals [32, 33]. that colonize or infect the reproductive tract [45].

The lung is one of the most common organs affected Caspases inactivate proteins that protect living cells fromin sepsis and cellular infiltration, together with the release apoptosis and they contribute to cell death by directof proinflammatory mediators, leads to the development disassembly of cell structures [46]. Cytosolic caspase-3of acute lung injury, characterized by oedema, activation is regulated by both TNF- receptor mediatedhemorrhage, destruction of alveolar wall with severe extrinsic and intrinsic apoptotic cascades (12). In theinfiltration of inflammatory cells and these results agreed different pathways inducing apoptosis, caspase-3 appearswith [34, 35, 36]. Loss of endothelial barrier integrity to play a central role as most pathways result in theresults in increased capillary permeability and the activation of caspase-3 [47]. Additionally, in situproduction of interstitial and alveolar edema [37]. It is now localization of cleaved caspase-3 has found some favorwidely accepted that the formation of inflammatory for histological labeling of cells in apoptosis [12, 33].mediators plays an important role in the pathophysiology Based on these data, we used caspase- 3of inflammation in acute lung injury [38]. Apoptotic cells immunohistochemical staining for determining theand bodies are recognized by specific surface receptors of apoptosis in pulmonary, renal and testicular tissues.tissue macrophages and digested [39]. Based on these The cytoplasm and the nucleus of pulmonary and rounddata, we could conclude that the increased mononuclear cells in the lung of the Septic group showed strongcell infiltration in lung tissue in sepsis group compared caspase-3 immunopositive reaction, this result agreedwith the other groups in the present study may be with Ozdulger et al. [26], they demonstrated septic lunginvolved in sepsis induced tissue inflammation and high injury through strong caspase-3 immunopositive reactionapoptotic activity. Another recent area of interest in the in rats. The nuclei of renal tubular epithelial cells of thepathogenesis of lung injury is the role of reactive oxygen Septic group showed strong immunopositive reaction,species . Apart from direct cytotoxic effects, reactive agreed with Messaris et al. [13] they investigated theoxygen species have important effects on the apoptotic death of renal tubular cells in rat CLP model ofinflammatory response mediated via changes in sepsis and Koca et al. [33] they observed high apoptoticoxidant/antioxidant balance [40]. activity and renal tissue inflammation in septic induced

is atrophy of glomerular tuft, intra-tubular protein cast,

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rats. The nuclei of spermatogenic cells of the Septic group 6. Pacey, A.A. and A.A. Eley, 2004. Chlamydiashowed strong caspase-3 immunopositivity, agreed with trachomatis and male fertility. Hum. Fertil., 7: 271-6.Karaca et al. [48], they observed a significant increase in 7. Coopersmith, C.M., K.C. Chang, P.E. Swanson,the number of caspase-3– positive cells in the K.W. Tinsley, P.E. Stromberg, T.G. Buchman, I.E. Karlseminiferous epithelium of the streptozotocin-induced and R.S. Hotchkiss, 2002. Overexpression of Bcl-2 indiabetic rats and Lee et al. [49] in testicular germ cells of the intestinal epithelium improves survival in septicvaricocela induced rats. mice. Crit. Care Med., 30: 195-201.

In lung, kidney and testis from CLP rats caspase-3 8. Coopersmith, C.M., P.E. Stromberg, W.M. Dunne,positive staining cells were decreased sharply, providing C.G. Davis, D.M. Amiot, T.G. Buchman, I.E. Karlthe protective effect of methanol extract treatment on cell and R.S. Hotchkiss, 2002. Inhibition of intestinalapoptosis mediated by sepsis. While, the septic group epithelial apoptosis and survival in a murinetreated with cuttlebone extract showed no expression of model of pneumonia-induced sepsis. J.A.M.A.,caspase-3 in studied organs, providing the significant 287: 1716-1721.antiapoptotic effect of cuttlebone extract. Our results 9. Oberholzer, C., A. Oberholzer, M. Clare-Salzler andmight support earlier studies that the polysaccharide L.L. Moldawer, 2001. Apoptosis in sepsis: a newextract from Sepia aculeate cuttlebone showed good target for therapeutic exploration. FASEB Journal,antioxidant and antiradical activities, so the 15(6): 879-892. polysaccharide may be used as a source of natural 10. Hotchkiss, R.S., P.E. Swanson, J.P. Cobb,antioxidant or ingredient in the pharmaceutical industries A. Jacobson, T.G. Buchman and I.E. Karl, 1997.[43]. The methanolic extract of Sepia showed antibacterial Apoptosis in lymphoid and parenchymal cells duringand antifungal activity [19, 16]. sepsis: findings in normal and T- and B-celldeficient

In conclusion, the present study showed the mice. Critical Care Medicine, 25(8): 1298-1307. significant antiseptic and antiapoptotic effect of the 11. Hotchkiss, R.S., P.E. Swanson, B.D. Freeman,cuttlebone of Sepia and mild effect of the methanolic K.W. Tinsley, J.P. Cobb, G.M. Matuschak,extract against induced sepsis in rats. In our opinion, T.G. Buchman and I.E. Karl, 1999. Apoptotic cellSepia officinalis is a good food source being important in death in patients with sepsis, shock and multiplethe food menu. organ dysfunction. Critical Care Medicine,

REFERENCES 12. Oliver, L. and F.M. Vallette, 2005. The role of

1. Raoofi, R., Z. Salmani and F. Moradi, 2008. Resistance Updates, 8(3): 163-170.Procalcitonin as a marker for Early Diagnosis of 13. Messaris, E., N. Memos, E. Chatzigianni, A. Kataki,Sepsis. American Journal of Infectious diseases, M. Nikolopoulou, A. Manouras, K. Albanopoulos,10(1): 15-20. M.M. Konstadoulakis and J. Bramis, 2008. Apoptotic

2. Zhao, L., J.Y. Tao, SL. Zhang, F. Jin, R. Pang and death of renal tubular cells in experimental sepsis.J.H. Dong, 2010. N-butanol Extract from Melilotus Surgical Infections, 9(3): 377-388. Suaveolens Ledeb Affects Pro- and Anti- 14. Zanotti-Cavazzoni, S.L. and R.D. Goldfarb, 2009.Inflammatory Cytokines and Mediators. Evid. Based Animal models of sepsis. Crit. Care Clin.,Complement. Alternat. Med., 7: 97-106. 25(4): 703-719.

3. Alejandra, G., P.D. Luiz Francisco and R.S. Maurício, 15. Ramachandran, M., T. Thirumalai and P.2004. Experimental models of sepsis and septic shock: Vinothkumar, 2011. Antibacterial activity of variousan overview. Acta Cirúrgica Brasileira, 19(2): 82-88. leaf extracts of Merremia emarginata EK Elumalai.

4. Ercan, M. and S. Ozdemir, 2015. The contribution to Asian Pac. J. Tropical. Biomed., 1: 406-408.studies of the effect of -glucan on plasma viscosity 16. Mohanraju, R., D.B. Marri, P. Karthick, S. Narayana,in a rat sepsis model. Med. Sci. Disc., 2: 148-53. M.K. Narayana and C.H. Ramesh, 2013. Antibacterial

5. Wang, Z., J.H. Holthoff, K.A. Seely, E. Pathak, Activity of Certain Cephalopods from Andamans”,H.J. Spencer and P.R. Mayeux, 2012. Development of India. I.J.P.B.S., 3(2): 450-455.oxidative stress in the peritubular capillary 17. Ngoile. M.A.K., 1987. “Fishery biology of the squidmicroenvironment mediates sepsis-induced renal Loligo forbesi Steenstrup (Cephalopoda: Loliginidae)microcirculatory failure and acute kidney injury. in Scottish waters”, Ph.D thesis, University ofAmerican Journal of Pathology, 180(2): 505-516. Aberdeen, pp: 218.

27(7): 1230-1251.

caspases in cell death and differentiation. Drug

World J. Med. Sci., 12 (3): 303-315, 2015

314

18. Voss, G.L., 1973. Cephalopod Resources of the world. 27. Nemzek, J.A., K.M.S. Hugunin and M.R. Opp, 2008.Food and Agriculture Organization of the United Modeling sepsis in the laboratory, merging soundNations, USA. science with animal well-being. Comp. Med.,

19. Ramasamy, P., N. Subhapradha, A. Srinivasan, 58(2): 120-128.V. Shanmugam, J. Krishnamoorthy and A. 28. Bancroft, J. and H. Cook, 1994. Manual of histologicalShanmugam, 2011. In vitro evaluation of antimicrobial technique. 3 Ed Churchil, Livingeston, London,activity of methanolic extract from selected species of New York. Cephalopods on clinical isolates. Afric. J. Microbio. 29. Zarnescu, O., F.M. Brehar, M. Chivu and A.V. Ciurea,Res., 5(23): 3884-3889. 2008. Immunohistochemical localization of caspase-3,

20. Shanmugam, A., T.S. Mahalakshmi and A. Barwin caspase-9 and Bax in U87 glioblastoma xenografts. J.Vino, 2008. Antimicrobial Activity of Polysaccharides Mol. Hist., 39: 561-569.Isolated from the Cuttlebone of Sepia aculeata 30. Hongyan, X., S. Javed and G. Daniel, 2006.(Orbingy, 1848) and Sepia brevimana (Steenstrup, Mechanisms of Mortality in Early and Late Sepsis.1875): An approach to selected Antimicrobial Infec. and Immu., 7(9): 5227-5235.Activity for Human pathogenic Microorganisms. J. 31. Castellheim, A., O.L. Brekke, T. Espevik, M. HarboeFish. Aqua. Sci., 3(5): 268-274. and T.E. Mollnes, 2009. Innate immune responses to

21. Shanmugam, A., T. Amalraj and C. Palpandi, 2008. danger signals in systemic inflammatory responseAntimicrobial Activity of Sulfated syndrome and sepsis. Scand. J .Immunol., 69: 479-491.Mucopolysaccharides [Heparin and Heparin – Like 32. Fubini, S.L. and N.G. Ducharme, 2004. Farm animalGlycosaminoglycans (GAGs)] from Cuttlefish surgery .Saunders. St. Louis, pp: 267-281.Euprymna berryi Sasaki.Trends in App. Sci. Res., 33. Koca, U., C.G. Olguner, B.U. Ergür, M. Altekin,3(1): 97-102. A. Taþdö en, S. Duru, P. Girgin, K. Gündüz, S.C.

22. Rajaganapathi, J., 2001. Antimicrobial activities of M c l , S. Güzelda and M. Akku , 2013. The Effectsaggregates of deoxycholic acid-modified chitosan. of Dexmedetomidine on Secondary Acute Lung andmarine molluscs and purification of anti-HIV Kidney Injuries in the Rat Model of Intra-Abdominalprotein. Ph. D., Thesis, Annamalai University, India, Sepsis. Sci. Worl. J., pp: 1-11.pp: 130. 34. Mani, C., S. Jayne and C. Avadhesh, 2009. Acute

23. Ely, R., C. Tilvi Supriya and G. Naik, 2004. Lung Injury: Apoptosis and Signaling Mechanisms.Antimicrobial activity of marine organisms collected Experimental Biology and Med., 234: 361-371.off the coast of South East India. J. Exp. Mar. Biol. 35. Shrum, B., R.V. Anatha, S. Xu et al., 2014. A robustEcol., 309: 121-127. scoring system to evaluate sepsis severity in an

24. Okutani, K. and N. Morikawa, 1978. Gel filtration and animal model. BMC Res. N., 7: 233.sugar constituent of the polysaccharide extracted 36. Ates, I., N. Dogan, M. Aksory, Z. Hal c ,from the internal shell of squids. Bull. Jap. Soc. Fish, C. Gündogdu and M.S. Keles, 2015. The protective44: 369-372. effects of IgM-enriched immunoglobulin and

25. OECD, 2011. OECD Guideline 425: Acute Oral erythropoietin on the lung and small intestine tissuesToxicity-Up-and-Down procedure. In: OECD of rats with induced sepsis. Biochemical andGuidelines for the Testing of Chemicals Vol.2 histopathological evaluation. Phar. Bio., 53: 78-84.Organization for Economic Coorporation and 37. Kaynar, G., G. Yurdakan, F. Comert and E. Yilmaz-Development. Paris, France. “ In- Vitro and - Vivo Sipahi, 2012. Effects of peripheral benzodiazepineantioxidant activities of aqeous extract of Strychnos receptor ligand Ro5-4864 in four animal models ofhenningsii Glig”, African Journal of Pharmacy and acute lung injury. J. Surg. Res., pp: 1-8.Pharmacology, 4(2): 070-078. 38. Charles, A.D., 1997. Pathogenesis of Septic Shock

26. Ozdulger, A., I. Cinel, O. Koksel, L. Cinel, D. Avlan, Cytokines as Mediators in the Proinflammatory andA. Unlu, H. Okcu, M. Dikmenqil and U. Oral, 2003. Anti-inflammatory. Chest, 112: 321-329.The Protective Effect of N-ACETYLCYSTEINE on 39. Martin, T.R., N. Hagimato, M. Nakamura andApoptotic Lung injury in Cecal ligation and G. Matute-Bello, 2005. Apoptosis and epithelial injuryPuncture-Induced Sepsis Model. SHOCK, in the lungs. Proceedings of the American Thoracic19(4): 366-372. Society, 2: 214-220.

rd

World J. Med. Sci., 12 (3): 303-315, 2015

315

40. Galley, H.F., 2011. Oxidative stress and mitochondrial 46. Takahashi, A., E.S. Alnemri, Y.A. Lazebnik,dysfunction in sepsis. British Journal of Anaesthesia, T. Fernandes- Alnemri, G. Litwack, R.D. Moir,107(1): 57-64. R.D. Goldman, G.G. Poirier, S.H. Kaufmann and

41. Kenti, D., L. Asada, S.T. Peter and A.S. Robert, 2009. W.C. Earnshaw, 1996. Cleavage of lamin A by Mch2Animal models of sepsis and sepsis-induced kidney alpha but not CPP32: multiple interleukin 1 beta-injury. J. Clin. Invest., 119: 2868-2878. converting enzyme-related proteases with distinct

42. Zachary, J.F. and M.D. McGavin, 2009. Pathologic substrate recognition properties are active inbasis of Veterinary disease. 4 ed. apoptosis. Proc. Natl. Acad. Sci., USA, 93: 8395-8400.th

43. Subhapradha, N., P. Ramasamy, P. Seedevi, 47. Mancini, M., D.W. Nicholson, S. Roy,V. Shanmugam, A. Srinivasan and A. Shanmugam, N.A. Thornberry, E.P. LA. Peterson, Casciola-Rosen2014. Extraction, characterization and its and A. Rosen, 1998. The caspase-3 precursor has aantioxidant efficacy of polysaccharides from Sepia cytosolic and mitochondrial distribution: implicationsaculeata (Orbigny, 1848) cuttlebone. Afr. J. Biot., for apoptotic signaling. J. Cell Biol., 140: 1485-1495.13(1): 138-144. 48. Karaca, T., S. Demirtas, I. Karaboga and S. Ayvaz,

44. Potts, J.M., R. Sharma, F. Pasqualotto, D. Nelson, 2015. Protective effects of royal jelly against testicularG. Hall and A. Agarwal, 2000. Association of damage in streptozotocin-induced diabetic rats.Turk.ureaplasma urealyticum with abnormal reactive J. Med. Sci., 45: 27-32.oxygen species levels and absence of 49. Lee, J.D., S.Y. Jeng and T.H. Lee, 2006. Increasedleukocytospermia. J. Urol., 163: 1775-8. Activated Caspase-3 Expressionin Testicular Germ

45. Abd-Allah, A., G. Helal, A. Al-Yahya, A.M. Aleisa, Cells of Varicocele-Induced Rats. J.T.U.A.,S.S. Al-Rejaie and S.A. Al-Bakheet, 2009. Pro- 17(3): 81-85.inflammatory and oxidative stress pathways whichcompromise sperm motility and survival may bealtered by L-carnitine. Oximed. Cel. Long., 2: 73-81.