Research Article Protective Effects of the Mushroom ...Protective Effects of the Mushroom Lactarius deterrimus Extract on Systemic Oxidative Stress and Pancreatic Islets in Streptozotocin-Induced

Research ArticleProtective Effects of the Mushroom Lactarius deterrimusExtract on Systemic Oxidative Stress and Pancreatic Islets inStreptozotocin-Induced Diabetic Rats

Mirjana MihailoviT1 Jelena ArambašiT NovanoviT1

Aleksandra UskokoviT1 Nevena GrdoviT1 Svetlana DiniT1 Senka VidoviT2

Goran PoznanoviT1 Ibrahim MujiT3 and Melita VidakoviT1

1 Department of Molecular Biology Institute for Biological Research University of Belgrade Bulevar Despota Stefana 14211060 Belgrade Serbia

2Department of Biotechnology and Pharmaceutical Engineering Faculty of Technology University of Novi SadBulevar Cara Lazara 1 21000 Novi Sad Serbia

3 Biotechnical Faculty University of Bihac Kulina Bana 2 77000 Bihac Bosnia and Herzegovina

Correspondence should be addressed to Melita Vidakovic melitaibissbgacrs

Received 2 July 2014 Revised 2 September 2014 Accepted 14 September 2014

Academic Editor Dimitrios Papazoglou

Copyright copy 2015 Mirjana Mihailovic et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to assess the in vivo effects of the extract of the medicinal mushroom Lactarius deterrimus whenadministered (60mgkg ip) daily for four weeks to streptozotocin- (STZ-) induced diabetic rats Diabetic rats treated with the Ldeterrimus extract displayed several improved biochemical parameters in the circulation reduced hyperglycemia lower triglycerideconcentration and reduced glycated hemoglobin glycated serum protein and advanced glycation end product (AGE) levels Thistreatment also adjusted the diabetes-induced redox imbalance Thus higher activities of the antioxidative enzymes superoxidedismutase and catalase in the circulation were accompanied by increased levels of free intracellular thiols and glutathionylatedproteins after treatment with the L deterrimus extract In addition to a systemic antioxidant effect the administration of theextract to diabetic rats also had a positive localized effect on pancreatic islets where it decreased AGE formation and increasedthe expression of chemokine CXCL12 protein that mediates the restoration of 120573-cell population through the activation of theserinethreonine-specific Akt protein kinase prosurvival pathway As a result the numbers of proliferating cell nuclear antigen-(PCNA-) and insulin-positive 120573-cells were increased These results show that the ability of the L deterrimus extract to alleviateoxidative stress and increase 120573-cell mass represents a therapeutic potential for diabetes management

1 Introduction

Diabetes mellitus (DM) is a metabolic disorder caused byabsolute insulin deficiency or insufficient insulin secretionandor insulin sensitivity [1] and is characterized by hyper-glycemia Pancreatic 120573-cells are responsible for insulin pro-duction and maintenance of blood glucose concentrations[2] 120573-cell dysfunction and declining 120573-cell numbers areresponsible for the loss of endocrine pancreas function inboth type 1 (T1D) and type 2 diabetes albeit loss of 120573-cells is more rapid in autoimmune destruction in T1DHyperglycemia is at the root of the significant increase

in the formation of toxic reactive oxygen species (ROS)and establishment of oxidative stress that is responsible forthe progression of diabetes and its complications [1] withvascular injury hypertension nephropathy retinopathy andneuropathy as major outcomes

Despite many strategies and agents DM managementrequires constant improvement Considering that syntheticdrugs have specific limitations in treating diabetic complica-tions it is important to refine approaches based on novel nat-ural compounds that could support the restoration andmain-tenance of pancreatic 120573-cell numbers and assist in controllingdiabetic complications [3ndash5] Since oxidative stress is an

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2015 Article ID 576726 10 pageshttpdxdoiorg1011552015576726

2 Journal of Diabetes Research

essential contributor to the development and progression ofdiabetes and related complications the therapeutic approachto DM is based on improving hyperglycemia and the organ-ismrsquos endogenous antioxidant activities Recent investigationsthat have provided evidence for the antidiabetic effectivenessof mushrooms provide novel approaches for controlling DMand its complications [6] Edible mushrooms are rich invitamins (B D A C K) contain high protein contents andminerals and are low in saturated fats Many of the biologicalcharacteristics of mushrooms are mainly due to the presenceof dietary fiber in particular chitin and the 120572 and 120573 glucansthat have significant nonspecific immunostimulatory effects[7 8] Mushrooms have also proven to be effective ROSscavengers their antioxidant properties correlatingwith theirtotal content of phenolics [4 9 10]

Lactarius deterrimus also known as false saffronmilkcapis an edible mushroom from the family Russulaceae thatmainly grows in coniferous woods in northern northeast-ern and Central Europe L deterrimus and other Lactariusspecies such as Lactarius salmonicolor Lactarius deliciosusand Lactarius sanguifluus possess potentmedicinal activitiesThe antimicrobial activities against several Gram (+) andGram (minus) bacteria and the anticancer and antiviral activi-ties of their antioxidant constituents have been describedin addition it has been suggested that these mushroomsrepresent a potential source of natural immunostimulatorysubstances [11ndash13] Previously we described the in vitroantioxidant and scavenging properties of the L deterrimusextract especially NO-scavenging and metal-chelating activ-ities which correlatedwith the ability of the extract to preventlipid peroxidation and DNA damage during streptozotocin-induced oxidative stress in Rin-5F cells [14]

The aim of this study was to examine the potential invivo beneficial effect of the extract obtained from the ediblemushroom Lactarius deterrimus on the systemic antioxidantstatus and control of pancreatic damage in streptozotocin-(STZ-) induced diabetic rats

2 Materials and Methods

21 Mushroom Collection and Extract Preparation ThemushroomLactarius deterrimuswas collected near the villageMune from the Istra region in Croatia in the summer of 2008Fruiting bodies were gently cleansed of any residual compostFreshmushroomswere air-dried and stored in airtight plasticbags at room temperature The dried mushroom sampleswere broken up in a blender before extraction with 50ethanol at a sample solvent ratio of 1 10 (wv)The extractionprocess was carried out using an ultrasonic bath (B-220Branson and SmithKline Company) at 45∘C for 40min Afterfiltration the extraction solvent was removed by a rotaryevaporator (Devarot Elektromedicina) under vacuum Theobtained L deterrimus extracts (Ld) were dried at 60∘C to aconstant mass and stored in glass bottles at minus80∘C to preventoxidative damage

22 Phytochemical Analysis of the L deterrimus Extract Thetotal phenolic compounds and other oxidation substrates

Table 1 Phytochemical constituents of the L deterrimus extract

Phenolic content(mgGAEg extract)a

Flavonoid content(mgQEg extract)b

Lactarius deterrimus 148 plusmn 223 507 plusmn 197Total phenolic and flavonoid contents were determined by the Folin Cio-calteu and aluminium-chloride colorimetric methods respectively and areexpressed as milligrams of gallic acid (GA) per gram of dry mushroommateriala and as milligrams of quercetin (QE) per gram of dry mushroommaterialb Data are presented as means plusmn SD

contained in dry mushroom extracts were determined by theFolin-Ciocalteu colorimetric method based on absorbanceat 765 nm [15] and are shown in Table 1 The values areexpressed as g of gallic acid equivalents (GAE) per 100 gof the dry mushroom extract sample The total flavonoidcontent was analyzed by the aluminum-chloride colorimetricassay at 510 nm [16] and is expressed as g of quercetinequivalents (QE) per 100 g of the dry extract sample Previousdetailed qualitative and quantitative analyses of the extract byhigh-performance liquid chromatography with diode-arraydetection (HPLCDAD) revealed the presence of tryptophanp-hydroxybenzoic acid and unsaturated oxy(hydroxyl- orepoxy-) fatty acids [14]

23 Animals Experiments were performed on 25-month-old adult albino Wistar rats weighing 220ndash250 g All animalprocedures were in compliance with the EEC Directive(86609EEC) on the protection of animals used for experi-mental and other scientific purposes and were approved bythe Ethical Committee for the Use of Laboratory Animalsof the Institute for Biological Research ldquoSinisa StankovicrdquoUniversity of Belgrade

24 Experimental Design The experimental model of multi-ple low dose (MLDS) STZ-induced diabetes was used Dia-betes was induced by injection (ip) of STZ (40mgkgday)to Wistar rats for five consecutive days STZ was dissolvedbefore use in sodium citrate buffer (01M pH 45) Bloodglucose was measured 24 h after the last STZ injectionBlood samples were obtained from the tail vein of rats thatfasted overnight and glucose was measured with a bloodglucose meter (Accu-Chech Active) Rats were considered tohave diabetes when the fasting blood glucose level exceeded20mmolL (the baseline glucose level measured before dia-betes induction by STZ was 62 plusmn 03) Male albino Wistarrats were randomly divided into four groups (i) NDMthe nondiabetic group (119899 = 7) also referred to as thenegative control received citrate buffer (ip) equivalent tothe STZ injection for 5 consecutive days (ii) NDM + Ldthe nondiabetic group (119899 = 7) also referred to as thepositive control was administered the mushroom extract(Ld 60mgkg ip) daily for four weeks (iii) DM the diabeticgroup (119899 = 8) was injected STZ and left untreated throughoutthe fourweek period (iv)DM+Ld the diabetic group (119899 = 8)was treated with the Ld daily for four weeks starting from thelast day of STZ administration

Journal of Diabetes Research 3

25 Serum and Hemolysate Preparation Rat blood serumwas collected after blood clotting and centrifugation at2000timesg for 10min The serum was used for determi-nation of glycated proteins and the amounts of proteinsulfhydryl groups (ndashSH) For the preparation of red blood cell(RBC) hemolysates blood was collected in heparinized tubes(1 000 IU heparin) and centrifuged at 2000timesg for 10minRBCs were washed twice with 09 NaCl and centrifugedThe washed RBCs were lysed in 3 volumes of cold water for30min on ice Hemolysates were used for the determinationof catalase (CAT) superoxide dismutase (SOD) glutathiony-lated proteins (GSSP) hemoglobin (Hb) and glycated (Gly)Hb

26 Determination of Biochemical Parameters of DiabetesBlood glucose levels were measured with a commercialkit (Gluco-quant GlucoseHK Boehringer Mannheim Ger-many) based on the hexokinaseG6P-DH enzymatic methodGlyHb was determined by the colorimetric assay accordingto Parker et al [17] Serum triglycerides were measured bythe glycerol-3-phosphate (GPO) oxidase-p-aminophenazone(PAP) method with an enzymatic kit (Randox LaboratoriesUK) Detection of glycated serum proteins was by thefructosamine assay The glycation of serum proteins wasmeasured according to Johnson et al [18] Aliquots of sera(50120583L) were added to 450 120583L of 100mM carbonate buffer(pH 108) containing 05mM nitro blue tetrazolium (NBT)The samples were incubated for 1 h at 37∘C Absorbance wasmeasured at 595 nm

27 Determination of GSSP in the Circulation Acid-precip-itated proteins in hemolysates were thoroughly washed withthe precipitating solution until no trace of soluble reducedglutathione (GSH) or glutathione disulfide (GSSG) wasdetected The pellets were then resuspended and broughtto an alkaline pH (pH 75ndash8) for 5ndash30min Under theseconditions GSH is released via an ndashSHdisulfide (ndashSndashSndash)exchange reaction The reaction was stopped by the additionof trichloroacetic acid to a final 5 concentration Theamount of releasedGSHwas determined enzymatically in thesupernatants after centrifugation

28 Determination of Protein Sulfhydryl Groups inCirculationProtein ndashSH groups were determined by Ellmanrsquos method[19] Briefly 05mL of serum was added to a cuvette con-taining 01M phosphate buffer pH 74 (05mL) 02mL of3mM 551015840-dithiobis (2-nitrobenzoic acid) was then added tostart the reaction Absorbance was measured at 412 nm after10min The number of ndashSH groups was calculated accordingto the following formula

mol ndashSHg proteins

= [119860 sample14150

times dilution factor] g proteins(1)

29 Determination of SOD and CAT Activities in the Circu-lation CAT activity was determined according to Beutler

[20] as the rate of hydrogen peroxide decomposition andexpressed as 120583mol H

2O2ming Hb The Hb was removed

from hemolysates prior to measurement of SOD activity bythe epinephrine method [21] One unit of SOD activity wasdefined as the amount of Hb that causes 50 inhibition ofadrenaline autoxidation

210 Determination of AGE in Circulation The fluorescentproducts of AGE were detected according to Munch et al[22] Henle et al [23] and Kalousova et al [24] Blood serumwas diluted 1 50 with phosphate buffered saline (PBS) pH74 and fluorescence intensity was measured at an excitationwavelength of 350 nm and an emission wavelength of 440 nmwith a luminescence spectrometer (LS50B Perkin-ElmerLtd Buckinghamshire England) The fluorescence intensitywas expressed in arbitrary units

211 Histological Analysis and Immunostaining The pancre-ata from all experimental groups were removed and fixedin 10 buffered formalin for histological and immunohis-tological examination Pancreatic tissues from all examinedgroups were blocked in paraffin and sectioned at 5120583m thick-ness for histological and immunohistochemical examination(Leica DMLB objective magnification 40x) For histologicalanalysis tissue sections were stained with hematoxylin andeosin and observed under a light microscope For immuno-histochemical analysis deparaffinized sections were passedthrough xylene and rehydrated in sequentially graduatedethyl alcohol Slides were incubated in 03 hydrogen perox-idemethanol for 20min to reduce nonspecific backgroundstaining due to endogenous peroxidase After washing inPBS the sections were treated with 001M sodium citratebuffer at 98∘C for antigen retrieval The cooled tissueswere washed four times in PBS prior to application of theblocking serum for 5min (005 Tween 20 5 bovineserumalbumin)Theprimary antibodywas applied overnightat room temperature (RT) Polyclonal antibodies raisedagainst insulin CXCL12 C-X-C chemokine receptor type 4(CXCR4) N(120576)-(carboxymethyl)lysine (CML) an advancedglycation end product (AGE) receptor for AGE (RAGE)phosphorylated protein kinase B (pAkt) andproliferating cellnuclear antigen (PCNA) (Santa Cruz Biotechnology SantaCruz CA USA) were diluted 1 50 in PBS with 2 dryskimmedmilk Afterwashing in PBS sectionswere incubatedat RT for 1 h with secondary antibody horseradish peroxidase(1 100) (Santa Cruz Biotechnology Santa Cruz CA USA)Tissues were incubated for 20min at RT in a solution of331015840-diaminobenzidine (DAB) After washing with PBS thetissueswere counterstainedwith haematoxylin andwashed inwater and the coverslips were applied with mounting mediaFor the negative control the primary antibody was not addedto the sections

212 Statistical Analysis Thedatawere expressed as themeanplusmn SEM (standard error of mean) Statistical differencesbetween groups were analyzed using one-way analysis ofvariance (ANOVA) followed byDuncanrsquosmultiple range test


NDM DMGlucose (mmolL)

Triglycerides (mmolL)Total glycated Hb (120583mol fructosegHb)

62 plusmn 02a

65 plusmn 03a

075 plusmn 003a

65 plusmn 03a

67 plusmn 03a

05 plusmn 002a

301 plusmn 11b

116 plusmn 05b

14 plusmn 006b

227 plusmn 09c

91 plusmn 04c

1 plusmn 003c

NDM + Ld DM + Ld

(a)

Serum protein glycation level

0010203040506070809

1

NDM DM

Abso

rban

ce a a

b

c

NDM + Ld DM + Ld

(b)

Figure 1 The effect of L deterrimus extract administration on the biochemical parameters in sera (a) and glycation levels of serum proteins(b) NDM control rats NDM + Ld control rats treated daily with L deterrimus extract for four weeks DM STZ-induced diabetic rats DM+ Ld STZ-induced diabetic rats treated with L deterrimus extract for four weeks Hb haemoglobin The values are presented as the mean plusmnSEM values not sharing a common superscript letter differ significantly at 119875 lt 005

The difference was considered statistically significant at 119875 lt005

3 Results

31 Biochemical Parameters of Diabetes in Rats Treated withthe L deterrimus Extract Administration of Ld led to anoverall improvement of the main biochemical parameters ofdiabetes albeit short of their restoration to their respectivephysiological levels As can be seen in Figure 1(a) treatmentof diabetic animals with Ld reduced the glucose concentra-tion by almost 25 and lowered the level of triglyceridesby about 28 and GlyHb by about 21 The treatment alsolowered the level of serum protein nonenzymatic glycationto the level measured in control rats (Figure 1(b)) Ld-treated nondiabetic rats did not exhibit any changes of theseparameters when compared to the control group

32 Oxidative Stress and Antioxidative Protection in theCirculation of Rats Treatedwith the L deterrimus Extract Theredox status in experimental rats was estimated bymeasuringthe levels of free intracellular thiols (Figure 2(a)) and GSSP(Figure 2(b)) that are directly linked to the redox state of thecell [25 26] These experiments revealed that the treatmentwith Ld significantly improved the redox parameters in thecirculation While the level of serum protein ndashSH groups wassignificantly decreased in diabetic rats the treatment with Ldrestored the ndashSH content almost to the control levelThe ndashSHcontent was not significantly changed when control rats wereadministered the extract As can be observed in Figure 2(b)the 13-fold increase in the level of GSSP in diabetic rats ascompared to the control level was brought to the controllevel by the administration of Ld The GSSP level was not

significantly changed when healthy rats were administeredLd

The activities of the major antioxidant enzymes SODand CAT provide the first line of antioxidant defenses andprotect cells from ROS damage [27] As can be seen inFigure 2(c) the decreased levels of SOD and CAT activitiesin the circulation of diabetic rats were improved after Ldadministration the effect of Ld being more pronouncedon CAT activity In addition a significant increase in theSODCAT ratio was observed under diabetic conditionswhile the Ld treatment reduced this ratio to the control levelin diabetic animals SOD and CAT activities did not changeafter Ld administration to control rats

33 L deterrimus Extract Administration Induces Histolog-ical Changes and Stimulates the Production of Insulin andCXCL12 in Pancreatic Islets Hematoxylin and eosin sections(Figure 3) show that the pancreas of control rats is comprisedof numerous compactly arranged cells in the islets of Langer-hans that appear as dense cords The pancreas of diabeticrats had smaller pancreatic islets with lower numbers of 120573-cells displaying increased vacuolation and clumped cells Ld-treated diabetic rats resembled more closely normal islet cellarchitecture which is suggestive of a protective role of Ldon the pancreas of diabetic rats (Figure 3 HE) Immunohis-tochemical staining with insulin revealed disorganized isletsin diabetic rats with unevenly distributed insulin-positivecells in comparison to control islets that displayed stronginsulin immunostaining (Figure 3 insulin)The Ld treatmentincreased the number of insulin-positive cells in the islets ofdiabetic rats (Figure 3 insulin)

The chemokine CXCL12 and its receptor CXCR4 havebeen shown to mediate 120573-cell repair [28] Immunohisto-chemical staining revealed the presence of CXCL12-positive


SH groups

0105011

0115012

0125013

0135

NDM DM

(mol

SHg

pro

tein

s)a a

b

c

NDM + Ld DM + Ld

(a)

GSSP

0010203040506070809

1

a a

b

a

(120583m

olg

Hb)

NDM DMNDM + Ld DM + Ld

(b)

SOD and CAT activity

0

5

10

15

20

25

30

35

(Ug

Hb)

SODx100CATSODCAT ratio x 10

a

a

a

a

a

a

b

b

b

c

c

a


(c)

Figure 2The effect of L deterrimus extract administration on the content of free ndashSH (a) level of protein bound glutathione (GSSP) (b) andactivities of antioxidative enzymes (c) in the circulation NDM control rats NDM + Ld control rats treated daily with L deterrimus extractfor four weeks DM STZ-induced diabetic rats DM + Ld STZ-induced diabetic rats treated with L deterrimus extract for four weeks Thevalues are presented as the mean plusmn SEM values not sharing a common superscript letter differ significantly at 119875 lt 005

cells only in diabetic islets and their increased presencein islets from Ld-treated rats As can be seen in Figure 3CXCR4-positive immunostaining was only detected in theislets of diabetic rats

34 The Effects of the L deterrimus Extract Administration onCML-Modified Proteins in Pancreatic Islets Chronic hyper-glycemia causes tissue damage that is mediated in part bythe nonenzymatic glycation and oxidation of proteins andlipids and the formation of AGE of which CML is one of themost often usedmarkers [29] AGE exert their effects throughinteractions with their receptor RAGE that is normallyexpressed at low levels on the surface of most cell types Inaddition to the circulation AGE accumulate in tissues wherethey contribute to the development of diabetic complicationsIn the pancreas they play a part in progressive 120573-cell loss[30] Determination of the fluorescent products of AGE in theserum revealed a 15-fold increase in AGE in diabetic rats Indiabetic rats administered Ld the increase in AGE was at thecontrol level (Figure 4(a)) Immunohistochemical stainingrevealed an extensive distribution ofCML-positive cells in theislets of diabetic rats In Ld-treated diabetic rats the CML-positive cells were more disperse (Figure 4(b) CML) RAGEwas observed only in the islets of diabetic rats (Figure 4(b)RAGE)

35 The Effects of L deterrimus Extract Administration onthe Prosurvival and Proliferative Pathways in Pancreatic IsletsPhosphoinositide 3-kinases (PI3Ks) and its downstreameffector protein kinase Akt mediate cellular survival signalsthat have an essential function in pancreatic 120573-cell survival[31] Immunohistochemical staining with activated pAktshown on Figure 5 revealed its presence in the islets ofboth control groups whereas in diabetic islets pAkt-positivestaining was weaker Treatment of diabetic animals with Ldcaused a considerable increase in pAkt staining that points tothe stimulation of the prosurvival pathway in islets

Immunohistochemical staining with PCNA (Figure 5)which assumes an essential function in DNA replicationand repair revealed its extensive distribution in the nucleiof islets of both control groups However in the islets ofdiabetic animals no PCNA immunostaining was detectedThe Ld-treated diabetic rats exhibited PCNA staining intheir islets (Figure 5 PCNA) The increased presence ofPCNA in Ld-treated diabetic rats points to the activationof proliferative mechanisms which is in agreement with thedescribed stimulation of the prosurvival pathway

4 Discussion

In this report we have presented evidence for the beneficialeffect of the daily ip administration for four weeks of the


NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld

Figure 3 The effect of administration of the L deterrimus extract on histological changes and immunohistochemical localization of insulinand CXCL12 in pancreatic islets HE hematoxylin and eosin staining of pancreatic sections light photomicrographs of insulin CXCL12 andCXCR4 immunohistochemical staining of pancreatic sections within islets (magnification 40x) NDM control rats NDM + Ld control ratstreated daily with L deterrimus extract for four weeks DM STZ-induced diabetic rats DM + Ld STZ-induced diabetic rats treated with Ldeterrimus extract for four weeks

extract of Lactarius deterrimus (Ld) to STZ-induced diabeticrats This was manifested as improved hyperglycemia anda resulting decline in GlyHb serum protein glycation anddecreased formation of AGE in the circulation as well aslower triglyceride concentrations We interpret these globalchanges as part of a systemic antioxidant effect that impactedpancreatic islets in diabetic rats By suppressing the formationand accumulation of potential inducers of 120573-cell damagein diabetic rats (detected as a decrease in CML-containingspecies in the pancreas) Ld administration activated prosur-vival CXCL12Akt signaling and the proliferative pathwayobserved as the increased presence of PCNA-containing 120573-cellsTherefore by shifting the balance of120573-cell death in favorof 120573-cell survival the net result of Ld administration was anincrease in the number of functional insulin-positive 120573-cells

High concentrations of ROS are involved in pathologicalchanges of cellular functions and disruptions of cellular

homoeostasis [1] In diabetes persistent hyperglycemiacauses enhanced glucose autooxidation and protein glycosy-lation that increase the production of ROS that are importantagents in the development of diabetic complications Thelower activities of the antioxidant enzymes CAT and SODalong with the decreased protein ndashSH content and increasedamounts of GSSP moieties serum protein glycation andGlyHb revealed the presence of oxidative stress in thecirculation of diabetic rats After the four-week treatmentof diabetic rats with Ld these parameters were practicallyrestored to their physiological values Ld administrationcaused a significant decrease in the SODCAT ratio that wasassociated with lower hydrogen peroxide levels decreasedoxidative stress This shows that the administration of Ldunder diabetic conditions attenuated the oxidative stress-induced harmful processes by improving hyperglycemia andpromoting increased activities of antioxidative enzymes


AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)

Figure 4 The effect of L deterrimus administration on AGE in the circulation (a) and presence of CML-modified proteins in pancreaticislets (b) Fluorescent products of AGE in the serum (a) Light photomicrographs of immunohistochemical staining for CML and RAGEof pancreatic sections within the islet (b) (magnification 40x) NDM control rats NDM + Ld control rats treated daily with L deterrimusextract for four weeks DM STZ-induced diabetic rats DM + Ld STZ-induced diabetic rats treated with L deterrimus extract for four weeksThe values are presented as the mean plusmn SEM values not sharing a common superscript letter differ significantly at 119875 lt 005

pAkt

PCN

A


Figure 5 The effect of L deterrimus extract administration on the prosurvival pathway and 120573-cell proliferation in pancreatic islets Lightphotomicrographs of immunohistochemical staining for pAkt and PCNA of pancreatic sections within the islet (magnification 40x) NDM+Ld control rats treated daily with L deterrimus extract for four weeks DM STZ-induced diabetic rats DM + Ld STZ-induced diabetic ratstreated with L deterrimus extract for four weeks


These results are consonant with literature data showingthat edible mushrooms possess antioxidant and free radicalscavenging properties [3] The Ld applied herein is enrichedin both phenolics and flavonoids that contributed to its free-radical scavenging activity (Table 1) [14] This assumption isin agreement with the generally held view that the antiox-idant properties of mushroom extracts correlate with theirtotal content of phenolics [10] and with Wang and Xu [5]who recently provided additional evidence that phenols andpolyphenols are the major naturally occurring antioxidantcompounds in wild edible mushroomsWe speculate that thefree radical scavenging activity of Ld could also be attributedto 120573-glucan a principal component of mushrooms knownto play important roles in the activation of the nonspecificimmune response reduction of blood cholesterol and bloodglucose concentrations protecting blood macrophages fromionizing radiation and restoring bone marrow production[5 32 33] Results fromour previousworkwith plant-sourced120573 glucan revealed its ability to cause a systemic adjustmentof redox disturbance and to exert a beneficial effect underdiabetic conditions via its antioxidant and anti-inflammatoryactivities [34 35]

AGE also play a role in toxic signaling in diabetic pathol-ogy by contributing to unbalanced free-radical formationand activating stress signaling in cells through interactionswith RAGE AGE are caused by glycation that involvesa series of nonenzymatic reactions between the carbonylgroup on reducing sugars and the amino group on proteins[36] The classical pathway of AGE formation involves aglucose-protein condensation reaction to form Schiff baseadducts that undergo Amadori rearrangement [37]The earlyglycosylation products accumulate predominantly on long-lived proteins that undergo a series of in vivo rearrangementsto form irreversible compounds and a number of reactiveintermediates that enhance oxidative stress [38] Our resultsdescribe a significant increase in serum AGE in diabetic ratsand their reversion to the control level after the Ld treatmentThrough the generation of ROS and reactive nitrogen speciesAGE contribute to tissue injury by changing the structureand function of proteins thereby affecting important cellularfunctions either directly or via RAGE-activated pathways[39] In addition to AGE in the circulation we establishedthe presence of CML the most abundant AGE in vivo andof RAGE in pancreatic islets of diabetic rats Importantlyin pancreatic islets of Ld-treated diabetic animals CML wasbarely detectable and RAGE was undetectable by the experi-mental procedure applied herein AGE generally accumulatein tissues that display diabetic complications [24 40] Leeand coworkers [30] have demonstrated that the interactionbetweenAGEandRAGEcontributes to the progressive loss of120573-cells in diabetes through intracellular ROS generationThisfinding and our results indicate that the 120573-cells of Ld-treateddiabetic rats were exposed to a lower level of toxic signals than120573-cells of diabetic rats

The efficacy of medicinal mushrooms in the treatmentof diabetes by protecting against 120573-cell damage throughenhanced antioxidant defenses reduced inflammation andincreased insulin release has been reported [6 41 42] Theresults obtained herein by immunohistological examination

of the pancreas revealed that Ld administration to diabeticrats restrained islet destruction and partially restored thenumber of insulin-positive cells The activation and directinvolvement of the chemokine CXCL12CXCR4 receptor pairin cardiomyocytes and pancreatic islets have been demon-strated after tissue injury [43 44] the latter authors showingincreased CXCR4 mRNA and protein expression in insulitisRecently it was established that injury of 120573-cells inducesCXCL12 expression that initiates the transdifferentiation of120572-cells to 120573-cells in the pancreas [45] PI 3-kinase and thedownstream effector protein Akt play key roles in mediatingsignals for cell growth cell survival cell-cycle progressionand differentiation [31 46] Activated phosphorylated Aktby insulin and growth factors is implicated in glucosemetabolism transcriptional control and mediation of anti-apoptotic and prosurvival events in 120573-cells [47 48] Yanoet al [49] described the important function of CXCL12 indiabetes attenuation in mice via promotion of 120573-cell survivalby Akt activation The functions of CXCL12 described inthe literature suggested to us that the increased presenceof CXCL12 stimulated the Akt pathway affecting elevatedexpression of PCNA protein in Ld-treated diabetic rats Thisresult indicates that Ld exerted a stimulatory effect on isletproliferation and regeneration Considering the significantcapacity of the endocrine pancreas to adjust to changesin insulin demand [50] and that the pancreas containsquiescent cells that can proliferate and replace lost cells [51]we believe that the described stimulatory activity of Ld canexert an important beneficial effect during the initial stageof diabetes development when it is potentially possible toexpand the still existing 120573-cell mass through regenerationPancreatic 120573-cell regeneration induced by 120573-cell proliferationis often mentioned as a vital goal in the development ofeffective treatments for diabetes [52] The findings describedherein point to the possibility of developing the therapeuticpotential of Lactarius deterrimus in diabetes management byemploying its ability to alleviate oxidative stress and increase120573-cell mass

5 Conclusion

In vivo antioxidant and antidiabetic effects of the extract fromthe mushroom Lactarius deterrimus were determined Ourresults show that administration of the extract to diabetic ratsrestores antioxidant enzyme activities in the circulation anddecreases oxidative stress Treatment with the extract exhibitsa potential for preserving pancreatic islet structure andincreasing 120573-cell mass through activation of the prosurvivalCXCL12Akt pathway and stimulation of 120573-cell proliferationWe believe that the effects of the mushroom extract areimportant as it is derived from an established food used forhuman consumption However as a caveat to these findingsthe described potentially positive effects have to be proven inhuman diabetics and any side effects from prolonged extractadministration as described herein need to be assessed

Conflict of Interests

The authors declared that they have no conflict of interests


Acknowledgment

This work was supported by the Ministry of EducationScience and Technological Development of the Republic ofSerbia Grant no 173020

References

[1] A C Maritim R A Sanders and J B Watkins ldquoDiabetesmellitus is a metabolicdisorder characterized by hyperglycemiaand insufficiency of secretion or action of endogenous insulinrdquoJournal of Biochemical andMolecular Toxicology vol 17 pp 24ndash38 2003

[2] A E Butler J Janson S Bonner-Weir R Ritzel R A Rizzaand P C Butler ldquo120573-cell deficit and increased 120573-cell apoptosis inhumans with type 2 diabetesrdquo Diabetes vol 52 no 1 pp 102ndash110 2003

[3] H-Y Fu D-E Shieh and C-T Ho ldquoAntioxidant and freeradical scavenging activities of edible mushroomsrdquo Journal ofFood Lipids vol 9 no 1 pp 35ndash46 2002

[4] A N Nagappa P A Thakurdesai N V Rao and J SinghldquoAntidiabetic activity ofTerminalia catappa Linn fruitsrdquo Journalof Ethnopharmacology vol 88 no 1 pp 45ndash50 2003

[5] Y Wang and B Xu ldquoDistribution of antioxidant activities andtotal phenolic contents in acetone ethanol water and hot waterextracts from 20 edible mushrooms via sequential extractionrdquoAustin Journal of Nutrition and Food Sciences vol 2 no 1 p 52014

[6] H-C Lo and S P Wasser ldquoMedicinal mushrooms for glycemiccontrol in diabetes mellitus History current status futureperspectives and unsolved problems (review)rdquo InternationalJournal ofMedicinalMushrooms vol 13 no 5 pp 401ndash426 2011

[7] P Manzi A Aguzzi and L Pizzoferrato ldquoNutritional value ofmushrooms widely consumed in Italyrdquo Food Chemistry vol 73no 3 pp 321ndash325 2001

[8] P Mattila K Konko M Eurola et al ldquoContents of vitaminsmineral elements and some phenolic compounds in cultivatedmushroomsrdquo Journal of Agricultural and Food Chemistry vol49 no 5 pp 2343ndash2348 2001

[9] F Liu V E C Ooi and S T Chang ldquoFree radical scavengingactivities of mushroom polysaccharide extractsrdquo Life Sciencesvol 60 no 10 pp 763ndash771 1997

[10] S S Pekkarinen H Stockmann K Schwarz I M Heinonenand A I Hopia ldquoAntioxidant activity and partitioning ofphenolic acids in bulk and emulsified methyl linoleaterdquo Journalof Agricultural and FoodChemistry vol 47 no 8 pp 3036ndash30431999

[11] B Dulger F Yilmaz and F Gucin ldquoAntimicrobial activity ofsome Lactarius speciesrdquo Pharmaceutical Biology vol 40 no 4pp 304ndash306 2002

[12] Y Hou X Ding W Hou et al ldquoImmunostimulant activity ofa novel polysaccharide isolated from Lactarius deliciosus (l exfr) grayrdquo Indian Journal of Pharmaceutical Sciences vol 75 no4 pp 393ndash399 2013

[13] G Athanasakis N Aligiannis Z Gonou-Zagou A-L Skaltsou-nis andN Fokialakis ldquoAntioxidant properties of thewild ediblemushroom lactarius salmonicolorrdquo Journal of Medicinal Foodvol 16 no 8 pp 760ndash764 2013

[14] N Grdovic S Dinic J Arambasic et al ldquoThe protective effectof a mix of Lactarius deterrimus and Castanea sativa extracts

on streptozotocin-induced oxidative stress and pancreatic 120573-cell deathrdquo British Journal of Nutrition vol 108 no 7 pp 1163ndash1176 2012

[15] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965

[16] K R Markham ldquoFlavones flavonols and their glycosidesrdquo inMethods in Plant Biochemistry J B Harborne and P M DeyEds pp 193ndash237 Academic Press London UK 1989

[17] K M Parker J D England J da Costa R L Hess and DE Goldstein ldquoImproved colorimetric assay for glycosylatedhemoglobinrdquo Clinical Chemistry vol 27 no 5 pp 669ndash6721981

[18] R N Johnson P A Metcalf and J R Baker ldquoFructosamine anew approach to the estimation of serum glycosylprotein Anindex of diabetic controlrdquo Clinica Chimica Acta vol 127 no 1pp 87ndash95 1983

[19] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[20] E Beutler ldquoCatalaserdquo in Red Cell Metabolism A Manual ofBiochemical Methods E Beutler Ed pp 105ndash106 Grune andStratton New York NY USA 1982

[21] H PMisra and I Fridovich ldquoThe role of superoxide anion in theautoxidation of epinephrine and a simple assay for superoxidedismutaserdquoThe Journal of Biological Chemistry vol 247 no 10pp 3170ndash3175 1972

[22] G Munch R Keis A Wessels et al ldquoDetermination ofadvanced glycation end products in serum by fluorescencespectroscopy and competitive ELISA1rdquo European Journal ofClinical Chemistry and Clinical Biochemistry vol 35 no 9 pp669ndash677 1997

[23] T Henle R Deppisch W Beck O Hergesell G M Hanschand E Ritz ldquoAdvanced glycated end-products (AGE) dur-ing haemodialysis treatment discrepant results with differ-ent methodologies reflecting the heterogeneity of AGE com-poundsrdquo Nephrology Dialysis Transplantation vol 14 no 8 pp1968ndash1975 1999

[24] M Kalousova J Skrha and T Zima ldquoAdvanced glycation end-products and advanced oxidation protein products in patientswith diabetes mellitusrdquo Physiological Research vol 51 no 6 pp597ndash604 2002

[25] V Calabrese M Renis A Calderone A Russo M L Barcel-lona and V Rizza ldquoStress proteins and SH-groups in oxidant-induced cell damage after acute ethanol administration in ratrdquoFree Radical Biology and Medicine vol 20 no 3 pp 391ndash3971996

[26] A Meister and M E Anderson ldquoGlutathionerdquo Annual Reviewof Biochemistry vol 52 pp 711ndash760 1983

[27] I Fridovich ldquoSuperoxide radical and superoxide dismutasesrdquoAnnual Review of Biochemistry vol 64 pp 97ndash112 1995

[28] J Markovic N Grdovic S Dinic et al ldquoPARP-1 and YY1 areimportant novel regulators of CXCL12 gene transcription in ratpancreatic beta cellsrdquo PLoSONE vol 8 no 3 Article ID e596792013

[29] A G Huebschmann J G Regensteiner H Vlassara and J E BReusch ldquoDiabetes and advanced glycoxidation end productsrdquoDiabetes Care vol 29 no 6 pp 1420ndash1432 2006

[30] B-W Lee H Y Chae S J Kwon S Y Park J Ihm and S-HIhm ldquoRAGE ligands induce apoptotic cell death of pancreatic120573-cells via oxidative stressrdquo International Journal of MolecularMedicine vol 26 no 6 pp 813ndash818 2010


[31] R L Tuttle N S Gill W Pugh et al ldquoRegulation of pancreatic120573-cell growth and survival by the serinethreonine proteinkinase Akt1PKB120572rdquoNatureMedicine vol 7 no 10 pp 1133ndash11372001

[32] M Hofer M Pospısil S Viklicka et al ldquoEffects of postirra-diation carboxymethylglucan administration in micerdquo Interna-tional Journal of Immunopharmacology vol 17 no 3 pp 167ndash174 1995

[33] G Kogan A Stasko K Bauerova et al ldquoAntioxidant propertiesof yeast (1rarr 3)-120573-d-glucan studied by electron paramagneticresonance spectroscopy and its activity in the adjuvant arthri-tisrdquo Carbohydrate Polymers vol 61 no 1 pp 18ndash28 2005

[34] M Mirjana A Jelena U Aleksandra et al ldquo120573-Glucan admin-istration to diabetic rats reestablishes redox balance and stim-ulates cellular pro-survival mechanismsrdquo Journal of FunctionalFoods vol 5 no 1 pp 267ndash278 2013

[35] A Uskokovic M Mihailovic S Dinic et al ldquoAdministration ofa 120573-glucan-enriched extract activates beneficial hepatic antiox-idant and anti-inflammatory mechanisms in streptozotocin-induced diabetic ratsrdquo Journal of Functional Foods vol 5 no4 pp 1966ndash1974 2013

[36] S Rahbar and J L Figarola ldquoInhibitors and breakers ofadvanced glycation endproducts (AGEs) a reviewrdquo Cur-rent Medicinal Chemistry-Immunology Endocrine amp MetabolicAgents vol 2 no 2 pp 135ndash161 2002

[37] N Selvaraj Z Bobby and V Sathiyapriya ldquoEffect of lipidperoxides and antioxidants on glycation of hemoglobin an invitro study on human erythrocytesrdquo Clinica Chimica Acta vol366 no 1-2 pp 190ndash195 2006

[38] R M P Gutierrez E G Baez M del Socorro Lopez Cortezand S A Cardenas ldquoExtracts of bixa inhibit glycation andAGEsformation in vitrordquo Journal of Medicinal Plants Research vol 5no 6 pp 942ndash948 2011

[39] S Rahbar ldquoNovel inhibitors of glycation and AGE formationrdquoCell Biochemistry and Biophysics vol 48 no 2-3 pp 147ndash1572007

[40] E D Schleicher E Wagner and A G Nerlich ldquoIncreasedaccumulation of the glycoxidation product N(120576)- (carboxym-ethyl)lysine in human tissues in diabetes and agingrdquo Journal ofClinical Investigation vol 99 no 3 pp 457ndash468 1997

[41] S T Chang and S P Wasser ldquoThe role of culinary-medicinalmushrooms on human welfare with a pyramid model forhuman healthrdquo International Journal of Medicinal Mushroomsvol 14 no 2 pp 95ndash134 2012

[42] S P Wasser ldquoCurrent findings future trends and unsolvedproblems in studies of medicinal mushroomsrdquo Applied Micro-biology and Biotechnology vol 89 no 5 pp 1323ndash1332 2011

[43] A G Kayali K van Gunst I L Campbell et al ldquoThe stromalcell-derived factor-1120572CXCR4 ligand-receptor axis is critical forprogenitor survival and migration in the pancreasrdquoThe Journalof Cell Biology vol 163 no 4 pp 859ndash869 2003

[44] E Aboumrad A M Madec and C Thivolet ldquoThe CXCR4CXCL12 (SDF-1) signalling pathway protects non-obese dia-betic mouse from autoimmune diabetesrdquo Clinical and Experi-mental Immunology vol 148 no 3 pp 432ndash439 2007

[45] Z Liu V Stanojevic S Avadhani T Yano and J FHabener ldquoStromal cellderived factor-1 (SDF-1)chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhancesbeta cell survivalrdquo Diabetologia vol 54 no 8 pp 2067ndash20762011

[46] J A Engelman J Luo and L C Cantley ldquoThe evolutionof phosphatidylinositol 3-kinases as regulators of growth andmetabolismrdquoNature Reviews Genetics vol 7 no 8 pp 606ndash6192006

[47] BM BurgeringTh and P J Coffer ldquoProtein kinase B (c-Akt) inphosphatidylinositol-3-OHkinase signal transductionrdquoNaturevol 376 no 6541 pp 599ndash602 1995

[48] I Galetic M Andjelkovic R Meier D Brodbeck J Park andB A Hemmings ldquoMechanism of protein Kinase B activationby insulinInsulin-like growth Factor-1 revealed by specificinhibitors of phosphoinositide 3-kinase-significance for dia-betes and cancerrdquo PharmacologyampTherapeutics vol 82 no 2-3pp 409ndash425 1999

[49] T Yano Z Liu J Donovan M K Thomas and J F HabenerldquoStromal cell derived factor-1 (SDF-1)CXCL12 attenuates dia-betes in mice and promotes pancreatic beta-cell survival byactivation of the prosurvival kinase Aktrdquo Diabetes vol 56 no12 pp 2946ndash2957 2007

[50] S Bonner-Weir ldquoLife and death of the pancreatic120573 cellsrdquoTrendsin Endocrinology and Metabolism vol 11 no 9 pp 375ndash3782000

[51] D O Adeyemi O A Komolafe O S Adewole E M ObuotorA A Abiodun and T K Adenowo ldquoHistomorphological andmorphometric studies of the pancreatic islet cells of diabetic ratstreated with extracts of Annona muricatardquo Folia Morphologicavol 69 no 2 pp 92ndash100 2010

[52] A Vetere and B KWagner ldquoChemical methods to induce beta-cell proliferationrdquo International Journal of Endocrinology vol2012 Article ID 925143 8 pages 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


essential contributor to the development and progression ofdiabetes and related complications the therapeutic approachto DM is based on improving hyperglycemia and the organ-ismrsquos endogenous antioxidant activities Recent investigationsthat have provided evidence for the antidiabetic effectivenessof mushrooms provide novel approaches for controlling DMand its complications [6] Edible mushrooms are rich invitamins (B D A C K) contain high protein contents andminerals and are low in saturated fats Many of the biologicalcharacteristics of mushrooms are mainly due to the presenceof dietary fiber in particular chitin and the 120572 and 120573 glucansthat have significant nonspecific immunostimulatory effects[7 8] Mushrooms have also proven to be effective ROSscavengers their antioxidant properties correlatingwith theirtotal content of phenolics [4 9 10]

Lactarius deterrimus also known as false saffronmilkcapis an edible mushroom from the family Russulaceae thatmainly grows in coniferous woods in northern northeast-ern and Central Europe L deterrimus and other Lactariusspecies such as Lactarius salmonicolor Lactarius deliciosusand Lactarius sanguifluus possess potentmedicinal activitiesThe antimicrobial activities against several Gram (+) andGram (minus) bacteria and the anticancer and antiviral activi-ties of their antioxidant constituents have been describedin addition it has been suggested that these mushroomsrepresent a potential source of natural immunostimulatorysubstances [11ndash13] Previously we described the in vitroantioxidant and scavenging properties of the L deterrimusextract especially NO-scavenging and metal-chelating activ-ities which correlatedwith the ability of the extract to preventlipid peroxidation and DNA damage during streptozotocin-induced oxidative stress in Rin-5F cells [14]

The aim of this study was to examine the potential invivo beneficial effect of the extract obtained from the ediblemushroom Lactarius deterrimus on the systemic antioxidantstatus and control of pancreatic damage in streptozotocin-(STZ-) induced diabetic rats

2 Materials and Methods

21 Mushroom Collection and Extract Preparation ThemushroomLactarius deterrimuswas collected near the villageMune from the Istra region in Croatia in the summer of 2008Fruiting bodies were gently cleansed of any residual compostFreshmushroomswere air-dried and stored in airtight plasticbags at room temperature The dried mushroom sampleswere broken up in a blender before extraction with 50ethanol at a sample solvent ratio of 1 10 (wv)The extractionprocess was carried out using an ultrasonic bath (B-220Branson and SmithKline Company) at 45∘C for 40min Afterfiltration the extraction solvent was removed by a rotaryevaporator (Devarot Elektromedicina) under vacuum Theobtained L deterrimus extracts (Ld) were dried at 60∘C to aconstant mass and stored in glass bottles at minus80∘C to preventoxidative damage

22 Phytochemical Analysis of the L deterrimus Extract Thetotal phenolic compounds and other oxidation substrates

Table 1 Phytochemical constituents of the L deterrimus extract

Phenolic content(mgGAEg extract)a

Flavonoid content(mgQEg extract)b

Lactarius deterrimus 148 plusmn 223 507 plusmn 197Total phenolic and flavonoid contents were determined by the Folin Cio-calteu and aluminium-chloride colorimetric methods respectively and areexpressed as milligrams of gallic acid (GA) per gram of dry mushroommateriala and as milligrams of quercetin (QE) per gram of dry mushroommaterialb Data are presented as means plusmn SD

contained in dry mushroom extracts were determined by theFolin-Ciocalteu colorimetric method based on absorbanceat 765 nm [15] and are shown in Table 1 The values areexpressed as g of gallic acid equivalents (GAE) per 100 gof the dry mushroom extract sample The total flavonoidcontent was analyzed by the aluminum-chloride colorimetricassay at 510 nm [16] and is expressed as g of quercetinequivalents (QE) per 100 g of the dry extract sample Previousdetailed qualitative and quantitative analyses of the extract byhigh-performance liquid chromatography with diode-arraydetection (HPLCDAD) revealed the presence of tryptophanp-hydroxybenzoic acid and unsaturated oxy(hydroxyl- orepoxy-) fatty acids [14]

23 Animals Experiments were performed on 25-month-old adult albino Wistar rats weighing 220ndash250 g All animalprocedures were in compliance with the EEC Directive(86609EEC) on the protection of animals used for experi-mental and other scientific purposes and were approved bythe Ethical Committee for the Use of Laboratory Animalsof the Institute for Biological Research ldquoSinisa StankovicrdquoUniversity of Belgrade

24 Experimental Design The experimental model of multi-ple low dose (MLDS) STZ-induced diabetes was used Dia-betes was induced by injection (ip) of STZ (40mgkgday)to Wistar rats for five consecutive days STZ was dissolvedbefore use in sodium citrate buffer (01M pH 45) Bloodglucose was measured 24 h after the last STZ injectionBlood samples were obtained from the tail vein of rats thatfasted overnight and glucose was measured with a bloodglucose meter (Accu-Chech Active) Rats were considered tohave diabetes when the fasting blood glucose level exceeded20mmolL (the baseline glucose level measured before dia-betes induction by STZ was 62 plusmn 03) Male albino Wistarrats were randomly divided into four groups (i) NDMthe nondiabetic group (119899 = 7) also referred to as thenegative control received citrate buffer (ip) equivalent tothe STZ injection for 5 consecutive days (ii) NDM + Ldthe nondiabetic group (119899 = 7) also referred to as thepositive control was administered the mushroom extract(Ld 60mgkg ip) daily for four weeks (iii) DM the diabeticgroup (119899 = 8) was injected STZ and left untreated throughoutthe fourweek period (iv)DM+Ld the diabetic group (119899 = 8)was treated with the Ld daily for four weeks starting from thelast day of STZ administration







= [119860 sample14150












62 plusmn 02a

65 plusmn 03a

075 plusmn 003a

65 plusmn 03a

67 plusmn 03a

05 plusmn 002a

301 plusmn 11b

116 plusmn 05b

14 plusmn 006b

227 plusmn 09c

91 plusmn 04c

1 plusmn 003c

NDM + Ld DM + Ld

(a)


0010203040506070809

1

NDM DM

Abso

rban

ce a a

b

c

NDM + Ld DM + Ld

(b)



3 Results








SH groups

0105011

0115012

0125013

0135

NDM DM

(mol

SHg

pro

tein

s)a a

b

c

NDM + Ld DM + Ld

(a)

GSSP

0010203040506070809

1

a a

b

a

(120583m

olg

Hb)


(b)


0

5

10

15

20

25

30

35

(Ug

Hb)


a

a

a

a

a

a

b

b

b

c

c

a


(c)






4 Discussion



NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld






AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















= [119860 sample14150












62 plusmn 02a

65 plusmn 03a

075 plusmn 003a

65 plusmn 03a

67 plusmn 03a

05 plusmn 002a

301 plusmn 11b

116 plusmn 05b

14 plusmn 006b

227 plusmn 09c

91 plusmn 04c

1 plusmn 003c

NDM + Ld DM + Ld

(a)


0010203040506070809

1

NDM DM

Abso

rban

ce a a

b

c

NDM + Ld DM + Ld

(b)



3 Results








SH groups

0105011

0115012

0125013

0135

NDM DM

(mol

SHg

pro

tein

s)a a

b

c

NDM + Ld DM + Ld

(a)

GSSP

0010203040506070809

1

a a

b

a

(120583m

olg

Hb)


(b)


0

5

10

15

20

25

30

35

(Ug

Hb)


a

a

a

a

a

a

b

b

b

c

c

a


(c)






4 Discussion



NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld






AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















62 plusmn 02a

65 plusmn 03a

075 plusmn 003a

65 plusmn 03a

67 plusmn 03a

05 plusmn 002a

301 plusmn 11b

116 plusmn 05b

14 plusmn 006b

227 plusmn 09c

91 plusmn 04c

1 plusmn 003c

NDM + Ld DM + Ld

(a)


0010203040506070809

1

NDM DM

Abso

rban

ce a a

b

c

NDM + Ld DM + Ld

(b)



3 Results








SH groups

0105011

0115012

0125013

0135

NDM DM

(mol

SHg

pro

tein

s)a a

b

c

NDM + Ld DM + Ld

(a)

GSSP

0010203040506070809

1

a a

b

a

(120583m

olg

Hb)


(b)


0

5

10

15

20

25

30

35

(Ug

Hb)


a

a

a

a

a

a

b

b

b

c

c

a


(c)






4 Discussion



NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld






AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SH groups

0105011

0115012

0125013

0135

NDM DM

(mol

SHg

pro

tein

s)a a

b

c

NDM + Ld DM + Ld

(a)

GSSP

0010203040506070809

1

a a

b

a

(120583m

olg

Hb)


(b)


0

5

10

15

20

25

30

35

(Ug

Hb)


a

a

a

a

a

a

b

b

b

c

c

a


(c)






4 Discussion



NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld






AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NDM DM

HE

Insu

linCX

CL12

CXCR

4

NDM + Ld DM + Ld






AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AGE

0

100000

200000

300000

400000

500000

600000

700000

a a

b

a

Seru

m fl

uore

scen

ce in

tens

ityex350

em440

nm


(a)

CML

RAG

E


(b)


pAkt

PCN

A








5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusion





Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgment


References




























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Protective Effects of the Mushroom ...Protective Effects of the Mushroom Lactarius deterrimus Extract on Systemic Oxidative Stress and Pancreatic Islets in Streptozotocin-Induced