Mulberry Bark Alleviates Effect of STZ Inducing Diabetic Mice ...downloads.hindawi.com/journals/ecam/2019/2182865.pdfEvidence-BasedComplementaryandAlternativeMedicine Inourlaboratory,westudiedtheextractionofactivecom-ponentssuchaspolysaccharides[,],-deo

Research ArticleMulberry Bark Alleviates Effect of STZ Inducing DiabeticMice through Negatively Regulating FoxO1

Fan Qiu JiangWang Hua-Yu Liu and Yu-Qing Zhang

School of Biology and Basic Medical Sciences Soochow University RM702-2303 No 199 Renai RoadDushuhu Higher Edu Town Suzhou China

Correspondence should be addressed to Yu-Qing Zhang sericultsudaeducn

Received 2 October 2018 Revised 22 November 2018 Accepted 4 December 2018 Published 21 January 2019

Academic Editor Kuttulebbai N S Sirajudeen

Copyright copy 2019 Fan Qiu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dysfunction of insulin secretion and hyperglycaemia were commonly found due to damaged 120573 cells of pancreas In our previousresearch it was found that mulberry branch bark powder (MBBP) was effective in treating diabetes in mice which were inducedby STZ and high fat diet The present study was designed to evaluate the protective effect of MBBP on STZ-induced 120573 cells injuryand investigate underlying mechanisms By preventive administration of branch bark powder the damage caused by STZ injectionwas found to be alleviated In MBBP feed groups pathological weight loss was inhibited fasting blood glucose was controlledthe incidence of diabetes decreased and blood lipid level and antioxidant capacities were restored The PI3KAKTFoxO1 signalpathway was found to be activated by key proteins expression and gene testing In liver the increased PI3K and phosphorylatedAKT the phosphorylated and inactivated FoxO1 which regulates the expression of gluconeogenic gene and explains the effect ofrelieving insulin resistance of MBBP Therefore the MBBP improves the tolerance of pancreas to the toxicity of STZ involving thePI3KAKTFoxO1 signalling pathway

1 Introduction

Streptozotocin or streptozocin (STZ) is a naturally occurringalkylating antineoplastic agent that is particularly toxic to theinsulin-producing 120573 cells of the pancreas in mammals It isused in medicine for treating certain cancers of the isletsof Langerhans and used in medical research to produce ananimal model for hyperglycemia as well as type 2 diabetes ortype 1 diabetes with multiple low doses [1]

As a glucosamine-nitrosourea compound STZ is similarenough to glucose to be transported into the cells by theglucose transport protein GLUT2 which is expressed rela-tively high in 120573 cells and causing alkylation of DNA [2] Theother toxic effect of STZ involves activation of inducible NO-synthase increase in NO concentration [3] and increasedH2O2generation [4] A single dose of 50 mgkg body weight

in a rat will cause necrosis of 120573 cells followed by 120573 cells lossand atrophy of the islets [5]

Mulberries (Morus alba L) are a deciduous tree in thefamily ofMoraceae and are widely cultivated in China KoreaIndia Brazil and othersThe leaves of mulberry are valued as

the primary food for silkworms supporting the silk industryfor centuries The branch of cultivated mulberry is a one ortwo year branch and used as fodder in agriculture

More importantly mulberries have medicinal propertiesand have been used in China for a long history Accordingto the Compendium of Materia Medica records mulberryhad been used in ldquoXiao kerdquo (diabetes) in ancient ChinaModern research has proven that the leaves and fruits ofthe mulberry tree have antidiabetic bioactivity [6ndash8] Thechemical components of Morus alba have been clearly listedin several reviews [9ndash11]

The mulberry has rich bioactive compounds in its sec-ondary metabolites such as alkaloids phenols polysaccha-rides and flavonoids With flavonoids as major constituentsmulberry leaves possess antioxidant glucosidase inhibi-tion antihyperlipidemic antiatherosclerotic and antiobesityactivities [12] Besides mulberry leaves more studies havesuggested that branch bark also has various biological func-tions such as hypolipidemic [13] hypoglycemic [14 15]antioxidant [16] anti-inflammatory [9 17] and antitumor[18 19] functions

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2019 Article ID 2182865 9 pageshttpsdoiorg10115520192182865

2 Evidence-Based Complementary and Alternative Medicine

In our laboratory we studied the extraction of active com-ponents such as polysaccharides [20 21] 1-deoxynojirimycin[22] mulberroside A [19] and morusin [23] from mulberrybranch bark The results showed that the extract of mul-berry branch bark has bioactivities of antioxidation [19]hypoglycaemia [24] hypolipidemia and anticancer [25] Inaddition our group investigated the interference effect of oraladministration of mulberry branch bark powder (MBBP) onthe incidence of type 2 diabetes induced by STZ in mice [26]

The aim of this study was to evaluate the protectiveproperties after the preventive administration of branchbark powder in mice which were against 120573 cells injuryinduced by STZ and examined whether the activation ofPI3KAKTFoxO1 signalling is involved in the protectiveeffect of MBBP treat

2 Materials and Methods

21 MBBP Preparation We followed the methods of Liu etal (2016) [26] and the branches of the mulberry (HuSang32 a cultivar from M multicaulis L) were obtained fromthe mulberry garden of Soochow University Suzhou Chinain November 2016 The bark which was peeled from themulberry branches was dried at 100∘C for 2 h pulverizedinto powder twice and passed through a 100-mesh sieveThepowder was weighted and mixed with standard diet to get25 5 and 10 MBBP diets for mice

22 Animals Male ICR mice (SPF) (4 weeks old 18ndash22 g)were obtained from the Experimental Animal Centre of Soo-chow University All animal experimental protocols used inthis study were approved by the Animal Ethics Committee atSoochowUniversity (number of animal license 201603A255)The mice were raised in standard experimental conditionsof humidity at 50ndash80 temperature of 18∘Cndash25∘C and12-hour lightdark cycle During the experiment the micecould freely obtain standard laboratory chow and standardlaboratory water

23 Drug Administration Drug administration was alsofollowed the methods of Liu et al (2016) [26] the mice wereallowed to adapt to the laboratory environment for one weekand then a total of 50 mice were randomly divided into 5groups normal group with standard feed model group withstandard feed 25MBBP feed group 5MBBP feed groupand 10MBBP feed groupThemicewere fed their individualdiets for 2 weeks On the 14st day the mice were given singletail-vein injection of freshly prepared citrate buffer (pH 45)solution of STZ (100 mgkg) [27] except for the normalgroup (STZ [S0130] was obtained from Sigma-Aldrich FineChemicalsUnited States) After injection allmice continuedto be fed as above

24 Fasting Blood Glucose Serum Insulin Levels and OrganCoefficient Fasting blood glucose (FBG) was measured bytail-vein sampling using a glucose meter from Johnson ampJohnson Medical (Shanghai) Ltd (China) on day 7 afterSTZ injection Then the mice were killed under anaesthe-sia The blood was collected and serum was prepared by

centrifuge (3000 rpm 15 min) The serum insulin levelswere determined by Insulin Assay Kit (Nanjing JianchengBioengineering Institute Nanjing China) The mice weredissected and then liver was weighed The ratio of tissueweight to body weight was calculated

25 Histological Examination and ImmunohistochemistryAssay Fresh mice pancreas was quickly removed rinsedwith cold phosphate buffered saline immersed in 4paraformaldehyde solution at pH 70 and embedded in paraf-fin Slices of 5 120583m thickness were cut After removing paraffinwith xylol the sections were stained with hematoxylin andeosin (HampE) and examined by optical microscope (400times)

To observe the immunohistological staining of insulinthe pancreas samples were soaked in 4 paraformaldehydeand then these tissues were sealed in paraffin and cut to 5120583m thicknessThe primary antibody anti-insulin (purchasedfrom Wuhan Boster Bio-engineering Limited Company[1200]) was incubated with tissues and then probed witha secondary antibody After rinsing the Elivison two-stepmethod was performed for immunohistochemical stainingAn optical microscope was used to observe the slices (400times)

26 Measurement of Antioxidant Status Glutathione peroxi-dase (GSH-PX) superoxide dismutase (SOD) and methanedicarboxylic aldehyde (MDA) in liver homogenates of micewere measured by using assay kits The GSH-PX assay kit(A005) SOD assay kit (A001-1) and MDA assay kit (A003-1)were purchased from the Nanjing Jiancheng BioengineeringInstitute Nanjing China In accordance with the assay kitguide book the absorbance of samples was determined at 412nm for GSH-PX 550 nm for SOD and 532 nm for MDA atthe end of the reaction

27 Quantitative Real Time-PCR Analysis The extraction ofpancreas and livers in total RNA determination of RNAand synthetization of the primer gene sequences of PI3KPDK-1 AKT FoxO1 and 120573-actin were carried out as reportedpreviously [26] Primer sequences are listed in Table 1 andthey were synthesized by Sangon Biotech (Shanghai) CoLtd China

The reverse transcription reaction system was assembledon ice according to the kit There are 250ndash300 ng mRNAfor reverse transcription reactions The reaction system wasset to 37∘C for 15 min The reverse transcriptase was theninactivated by heating to 85∘C for 5 sec and the cDNA wasobtainedThere is 2 120583L cDNA for PCR reaction PCR reactionconditions were as follows 94∘C for 4 min 94∘C for 20sec 60∘C for 30 sec and 72∘C for 30 sec Thirty-five cycleswere conducted from the second stage Using the differentconcentrations of cDNA as template the standard curvesof the experimental genes and the internal reference genewere generated The data were calculated according to thefollowing formula (T) = a times lg(copy) + b values of a varyamong the different genes and PCR fragments

28 Protein Expression by Western Blotting Western blottingwas done according to the methods described by Afrin etal [28] In the liver samples the all-protein concentration

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primers of reverse transcription-PCR analysis for genes

Gene Primer sequences (51015840 to 31015840) Product length (bp)AKT-F TGTCTGCCCTGGACTACTTGC 166AKT-R GGCGTTCCGCAGAATGTCPI3K-F AAGCCATTGAGAAGAAAGGACTG 176PI3K-R ATTTGGTAAGTCGGCGAGATAGPDK-1-F CTGGGCGAGGAGGATCTG 132PDK-1-R CACAGCACGGGACGTTTCFoxo1-F AGTGGATGGTGAAGAGCGTG 137Foxo1-R CTTTCCAGTTCCTTCATTCTGC120573-Actin-F GAGACCTTCAACACCCCAGC 263120573-Actin-R ATGTCACGCACGATTTCCC

was measured by the bicinchoninic acid method To analyzethe protein levels protein extracts of the same qualitywere separated by using 75ndash15 SDS polyacrylamide gelelectrophoresis Then the protein was transferred from elec-trophoresis membranes to nitrocellulose membranes Nitro-cellulose membranes were blocked by 5 skimmed dry milkPrimary antibodies against PI3K (BM5187) AKT (BM4808)pAKT (BM4744) and GAPDH (BM1623) were purchasedfrom Wuhan Boster Bio-Engineering Limited Companyprimary antibody pFoxO1 (9461T) FoxO1 (2880T) andLamin A (2032S) were from Cell Signaling TechnologyAmong primary antibodies GAPDH was used at a dilutionof 11300 and incubated overnight at 4∘C The following daythe membranes were washed with TBST three times eachfor 5 minutes The secondary antibodies which were boundto chemiluminescence developing agents were used at adilution of 15000 and incubated for 15 h After that themembranes werewashed againwith TBST four times each for5 minutes Films were scanned and the results were analyzedwith Quantity One

29 Cell Fractionation The cytoplasmic and nuclear pro-tein was collected using nuclear and cytoplasmic proteinextraction kit (Boster AR0106) The liver was cut into smallpieces and made to be tissue homogenate with ice-coldbuffer A Buffer B was added to the samples followed by 1min centrifugation The cytoplasmic supernatant was thencollected 100 mL ice-cold buffer C was added to centrifugalprecipitate which containing 1 mL DTT and 5 mL 100 mMPMSF per mL Buffer C 5 120583L protease inhibitor Then thesupernatant was centrifuged at 4∘C for 16 000 g 10 minutesand transferred to ice-cold clean micro-centrifugal tube assoon as possible to obtain nucleoprotein Protein levels in thecytoplasmic and nuclear extracts were detected following thewestern blot procedure

210 Statistical Analysis The experimental data wereexpressed as the mean plusmn SD and were analysed with Origin85 software The one-way ANOVA was used to evaluate thedifference betweenmultiple groups with P lt 005 consideredas significant and P lt 001 as very significant

3 Results

31 Effect of MBBP on the BodyWeight of Mice and the Coeffi-cients of Organs to Body Weight During the experiment theweight of the normal mice increased steadily and the speedof weight gain slowed down a little within third weeks Thebody weight of the all five groups increased in the same trendduring the first two weeks On the 14th day the mice in themodel group and the mulberry powder feeding group wereinjected with STZ After that the weight of the mice in themodel group has a sharp drop The weight of mice in the25 and 5 doses of mulberry powder feeding group alsodecreased indicating that STZ could significantly affect thebody weight growth and development of mice After STZinjection there was a significant difference in body weightbetween the three mulberry powder feeding groups Therewas no significant difference between the 10 dose group andthe normal group As mice consume less MBBP they loseweight faster It indicated that the mulberry powder feedingcould inhibit the weight loss of mice caused by STZ in a dose-dependent way

The coefficients of the liver to body weight are expressedas milligrams (wet weight of tissues)grams (fasted bodyweight) As shown in Figure 1(a) the coefficients of liver in themodel group were obviously higher than that of the normalgroup (p lt 001 or p lt 005) those of themodel group reached511 mgg indicating that liver appeared to have pathologicalchanges The reduction of these coefficients was observedin MBBP-treated groups compared with the model groupparticularly the coefficient of the liver was the dose-effectmanner in MBBP-treated groups These data illustrated thatoral administration of MBBP could reduce liver pathologychanges induced STZ

32 Effect of MBBP on the Fast Blood Glucose After STZinjection the FBG level was measured on 21th days afterfasting 12 h As shown in Figure 1(b) the mean FBG level ofthe model group continually rose and was obviously higherthan that of the normal group (p lt 001 or p lt 001) The 25MBBP-treated group was also higher than that of the normalgroup (p lt 005 or p lt 001) Compared with themodel groupthe FBG level in the 10MBBP-treated group was lowest (p lt


Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

60

50

40

30

Live

rBW

(mg

g)

lowastlowast

(a)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

lowastlowastlowastlowast

0

5

10

15

20

25

Fast

Bloo

d G

luco

se (m

mol

L)

(b)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

lowast

0

2

4

6

7

10

Insu

lin (m

UL

)

(c)

Normal Model 25MBBP 5MBBP 10MBBP

(d)

Figure 1 (a)The effect ofMBBP on the coefficients of Liver to body weight (b)The effect ofMBBP on FBG after STZ injection (c) the insulinlevels of five groups (all data were presented as mean plusmn sd the animal number normal n = 10 model n = 7 each MBBP treat group n = 8 p lt005 p lt001 versus normal group lowast plt005 and lowastlowast plt001 versus model group) (d) the effect of mulberry branch bark powder onpathologic tissue in the pancreas and insulin immunohistochemistry of pancreas in mice Scale bar 50 120583m

001) followed by the 5MBBP-treated group (p lt 005) Onthe 21th day the dose-effect manner was more obvious to beobserved in the MBBP-treated groups These results showedthat MBBP had an effect on controlling the exaltation of theFBG level induced by STZ

Generally diabetes was defined as FBG level ge 111mmolL According to this standard the incidence of diabeteswas also counted There were no diabetic mice in the normalgroup the incidence of diabetes reached 75 in the modelgroup and there was a downward trend accompanied by thedose-effect manner in MBBP-treated groups 667 for 25MBBP group 125 for 5 MBBP group and 0 for 10MBBP group The incidence in the 10 MBBP group waslowest in theMBBP-treated groups Consequently the resultsshowed thatMBBP could efficiently decrease the incidence oftype 1 diabetes induced by STZ

33 Effect of MBBP on Serum Insulin and Insulin SecretionThe mean value calculated at the end of the experiment

is shown in Figure 1(c) The mean value of insulin levelwas obviously lower in the model group than that of thenormal group (p lt 005) The insulin level is 169 and 226mUL higher in the 5 and 10 MBBP-treated groupscompared with the model group respectively (p lt 005)The results represent the decreased secretion of insulin dueto the damage of pancreatic 120573-cells induced by STZ Dueto the intake of MBBP the occurrence of this process wasmitigated

The pancreas is an important organ that regulates bloodglucose and insulin secretion This experiment used HampEstaining to evaluate the protective effect of MBBP on thepancreas as shown in Figure 1(d)The pancreas of the normalgroup showed normal structure The pancreas of the modelgroup showed minute inflammatory cells infiltration slightinterstitial proliferation angionecrosis and tiny congestionof vascellsum and prominent vasodilation The extent ofpancreas injury in MBBP-treated groups was better thanthat of the model group and was in a dose-effect manner


Table 2 The effect of MBBP on antioxidant capacity

Groups T-SOD (mmolL) GSH-PX (mmolL) MDA (mmolL)normal group 14135plusmn808 100644plusmn14591 0859plusmn0028Model group 12930plusmn315 90053plusmn7679 1459plusmn022425 MBBP 13621plusmn933 109203plusmn8538 1097plusmn02025 MBBP 14449plusmn1454lowast 109748plusmn12036lowast 0987plusmn0027lowast10 MBBP 16136plusmn1828lowastlowast 111412plusmn8516lowastlowast 0963plusmn0121lowastlowastplt005 and plt001 versus normal group lowastplt005 and lowastlowastplt001 versus model group

These pictures indicated that MBBP could effectively preventpancreas injury induced by STZ to a certain degree

To study insulin secretion in pancreas tissue theimmunohistological method was used to observe the stateof insulin secretion (Figure 1(d)) The insulin was dyed darkbrown and a large dark brown area was observed in thenormal group Insulin secretion was barely visible in themodel group Insulin secretions in MBBP-treated groupswere dramatically better than that of the model groupparticularly notable in the dark brown area of the 10MBBP-treated group These pictures show that MBBP hadan outstanding effect on preventing the decline of insulinsecretion

34 Measurement of Antioxidant Capacity To evaluate theinfluence of MBBP on antioxidant capacity the levels ofT-SOD GSH-PX and MDA were investigated The resultsin Table 2 show that the pancreas injury was correlatedwith the depletion of antioxidant status (SOD and GSH-PX)and excessive accumulation of one final oxidation product(MDA) Compared with the normal group the MDA levelin the liver was significantly increased (p lt 001) and theactivities of antioxidant enzymes (T-SOD GSH-PX) weresignificantly decreased (p lt 001) in the model group How-ever the situation changed with MBBP treatment before STZinjection MDA level was significantly decreased (p lt 005 orp lt 001) and the activities of antioxidant enzyme (T-SODGSH-PX) were significantly increased (p lt 001) in MBBP-treated groups compared with the model group

35 Effect of Expression of MBBP on PI3KATKFoxO1 Sig-nal Pathway Key Protein The present experiment furtherinvestigated PI3KAKTFoxO1 signal pathway The expres-sion levels of PI3K AKT phosphorylated AKT FoxO1 andphosphorylated FoxO1 protein in liver tissuewere detected byWestern blotting (Figure 2) in all mice The data in Figure 2showed that the expression of those proteins had an upwardtrend in MBBP-treated groups compared with the modelgroup the expression levels of PI3K pAKT and pFoxO1 in10MBBP-treated group showed a significant rise Althoughthe level of FoxO1 increased the ratio of pFoxO1 to FoxO1wasalso high inMBBP treatment groupwhen comparedwith thatin model group The FoxO1 in cytoplasm and nucleus alsohave been tested and presented in Figure 3 The expressionlevels of FoxO1 in cytoplasm increased significantly in 10MBBP treated group comparing with that in model groupCorrespondingly the FoxO1 level in nucleus was observeddecreased with MBBP treatment in dose dependent manner

36 Effect of MBBP on the Gene Expression of Signal PathwayPI3KAKTFoxO1 Gene expression of PI3K PDK1 AKTand FoxO1 in the pancreas and liver was analysed in the studyAs shown in Figure 4 the expression of each gene in thenormal group was obviously higher than that of the modelgroup (plt 005 or plt 001) Gene expression inMBBP-treatedgroups obviously increased (p lt 005 or p lt 001) comparedto the model group and the phenomenon presented a dose-effectmanner the expression in the 10MBBP-treated groupwas obviously higher than that of the model group (p lt 005or plt 001)These data indicate thatMBBP could increase theexpression of PI3K PDK1 AKT and FoxO1 which were lowerin pancreas and liver of mice of model group

4 Discussion

In this study the pancreas injury ofmice was induced by STZand the dysfunction of insulin secretion and hyperglycaemiawere observed due to damaged 120573 cells of pancreas Withpreventive administration of branch bark powder in micethe extent of pancreatic injury and hyperglycaemia wereobserved relieved

Under normal circumstances the pancreas releasesinsulin into the bloodstream when the level of blood glu-cose is high With damaged 120573 cells of pancreas due toSTZ injection in mice hyperglycaemia and low insulinlevel namely insulin resistance were observed in non-MBBP feed group (model group) Several markers of lipidmetabolism were also observed to be abnormal during theprocess The total triglycerides (TG) level was significantlyincreased and the high-density lipoprotein cholesterol (HDL-C) level was significantly decreased in model group Onthe other hand hyperglycaemia can lead to glycosylation ofantioxidant enzymes [29 30] and the decreased activity ofantioxidant enzymes such as Superoxide Dismutase (SOD)and Glutathione peroxidase (GSH-Px) were also observedMalondialdehyde (MDA) is one of the final products ofpolyunsaturated fatty acids peroxidation in the cells Anincrease in free radicals causes overproduction of MDA [31]Thus the increasedMDA level of themice inmodel group alsoindicates the imbalance of antioxidant capacity Comparedwith these results of model group the MBBP feed groupsshowed opposite tendency in a dose-effect mannerThe levelsof blood glucose insulin TG HDL-C MDA SOD GSH-Pxand body weight were all observed to significantly change inthe opposite direction compared with the model group Afteronly two weeks of MBBP feeding the phenotypic detection


Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH

Figure 2 Protein levels of PI3K p-AKT AKT pFoxO1 and FoxO1 in the liver assayed by Western blot Density values were normalized toGAPDH levels (ALL data were presented as mean plusmn sd n = 4 p lt 005 and p lt 001 versus normal group lowast p lt 005 and lowastlowast p lt 001versus model group)

results of these mice in MBBP feed groups presentedimproved ability to resist STZ damage

In vivo insulin resistance in the liver appears to resultfrom the failure of the insulin to inhibit hepatic glucoseproduction The PI3KAKT signalling pathway plays a role

in glucose storage and uptake in the liver [32] FoxO1 alsoknown as FKHR is a transcription factor in the downstreamof this insulin signalling pathway [33 34] Insulin causes theactivation of PI3K which subsequently phosphorylates AKTThe p-AKT then phosphorylates FoxO1 causing nuclear


105ModelNormal 105ModelNormal

FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A

Figure 3 Protein levels of FoxO1 in the cytoplasm and nucleus assayed by Western blot Density values were normalized to GAPDH andLamin A levels (all data were presented as mean plusmn sd n = 3 p lt 005 and p lt 001 versus normal group lowast p lt 005 and lowastlowast p lt 001 versusmodel group)

10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K

Normal Model5MBBP 10MBBP

FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

Figure 4 Preventive effect of MBBP to PI3K PDK-1 AKT and FoxO1 mRNA expression in the liver and pancreas (all data were presentedas mean plusmn sd n = 3 p lt 005 and p lt 001 versus normal group lowast p lt 005 and lowastlowast p lt 001 versus model group)

exclusionThe translocation of FoxO1 from the nucleus to thecytoplasm suppresses the expression of FoxO1 gluconeogenictargets phosphoenolpyruvate carboxylase (PEPCK) andglucose-6-phosphatase (G6Pase) in nuclear [35ndash37] Proteinsof PEPCK and G6Pase are rate-limiting enzymes for gluco-neogenesis In light of these reports our studies show thatMBBP feed resulted in enhancement of the PI3K phospho-rylation of both AKT and FoxO1 This will prolong insulinrsquosinhibitory effect on hepatic glucose production accompanied

by suppression of the expression of PEPCK and G6P in theliverThese results are in accord with the reduced blood sugarin MBBP treat groups

On the other hand the activation of the PI3KAKTFoxO1 pathway was also found in pancreas of MBBP groupmice According to the existing paper FoxO1 also playan important role in apoptosis and cells cycle regulationIncreasingly evidence showed that disruption of AKTFoxO1signalling might cause the apoptosis of 120573 cells Inhibition of


FoxO1 (phosphorylates FoxO1 and translocate to cytoplasm)could promote 120573 cells survival [38 39] This is in accord withour results of histopathological observation But the exactmolecular mechanism of apoptosis regulation has not beenclarifiedTherefore it would be interesting to conduct furtherstudies to detect downstream gene target of FoxO1

In conclusion our results indicate thatMBBP feed attenu-ates STZ induced dysfunction and injury in pancreatic 120573 cellsfor its beneficial metabolic effects The PI3KAKTFoxO1signalling is involved in the protective effect of MBBP byactivation of AKT inhibition of FoxO1ThusMBBP improvethe tolerance of pancreas to the injury of STZ and may bepromising for the prevention of pancreas injury

Data Availability

All data used to support the findings of this study are includedwithin the article All data created during this research areavailable from the corresponding author upon request

Conflicts of Interest

The authors have no conflicts of interest to declare

Acknowledgments

The authors gratefully acknowledge the earmarked fund(CARS-18-ZJ0502) for China Agriculture Research System(CARS) a project funded by the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions PRChina

References

[1] J Sviglerova J Kuncova and M Stengl ldquoCardiovascular mod-els heart secondarily affected by diseaserdquo in Animal Models forthe Study of Human Disease P Michael Conn Ed Chapter 9pp 195ndash220 Academic Press USA 2013

[2] Z Wang and H Gleichmann ldquoGLUT2 in pancreatic isletscrucial target molecule in diabetes induced with multiple lowdoses of streptozotocin in micerdquo Diabetes vol 47 no 1 pp 50ndash56 1998

[3] N S Kwon S H Lee C S Choi T Kho and H S Lee ldquoNitricoxide generation from streptozotocinrdquoThe FASEB Journal vol8 no 8 pp 529ndash533 1994

[4] N Takasu I Komiya T Asawa Y Nagasawa and T YamadaldquoStreptozocin- and alloxan-inducedH

2O2generation andDNA

fragmentation in pancreatic islets H2O2as mediator for DNA

fragmentationrdquo Diabetes vol 40 no 9 pp 1141ndash1145 1991[5] J R Thomas A D Ronald W H Philip and S Catherine

ldquoEndocrine Systemrdquo in Haschek and Rousseauxrsquos Handbook ofToxicologic Pathology W M Haschek C G Rousseaux andM A Wallig Eds Chapter 58 pp 2391ndash2492 Academic PressBoston 3rd edition 2013

[6] B Andallu V Suryakantham B Lakshmi Srikanthi and GKesava Reddy ldquoEffect of mulberry (Morus indica L) therapy onplasma and erythrocyte membrane lipids in patients with type2 diabetesrdquo Clinica Chimica Acta vol 314 no 1 pp 47ndash53 2001

[7] A Bondada V K Allagadda Venkata and V Nallan-chakravarthula ldquoInfluence ofmulberry (Morus indica L) leaves

on antioxidants and antioxidant enzymes in STZ-diabetic ratsrdquoInternational Journal of Diabetes in Developing Countries vol34 no 2 pp 69ndash76 2014

[8] J F Andrade V Silva andTMelnik ldquoMulberry therapy for type2 diabetes mellitusrdquo Cochrane Database of Systematic Reviewsvol 5 2015

[9] V Kuete D C Fozing and W F Kapche ldquoAntimicrobialactivity of the methanolic extract and compounds fromMorusmesozygia stem barkrdquo Journal of Ethnopharmacology vol 124no 3 pp 551ndash555 2009

[10] Y Ishrat F Anab M A Syed et al ldquoA review of ethnobotanyphytochemistry antiviral and cytotoxic anticancer potentialof morus alba linnrdquo International Journal of Advanced ResearchReview vol 1 pp 84ndash96 2016

[11] H Wei J Zhu X Liu et al ldquoReview of bioactive compoundsfrom root barks of Morus plants (Sang-Bai-Pi) and theirpharmacological effectsrdquo Cogent Chemistry vol 2 p 123202016

[12] E W-C Chan P-Y Lye and S-K Wong ldquoPhytochemistrypharmacology and clinical trials of Morus albardquo Chinese Jour-nal of Natural Medicines vol 14 no 1 pp 17ndash30 2016

[13] X M He S T Liao Y X Zou Y M Wu and J P Liu ldquoStudieson the hypolipidemic and antioxidative activity of Ramulusmori extracts for hypercholesterolaemic ratsrdquo Natural ProductResearch and Development vol 19 pp 462ndash464 2007

[14] F Ye Z F Shen F X Qiao and M Z Xie ldquoExperimentaltreatment of comp lications in alloxan diabetic rats with a-glucosidase inhibitor from the Chinese medicine herb RamulusMorirdquoActa Pharmaceutia Sinica vol 37 no 2 pp 108ndash112 2002

[15] L-Z Wan B Ma and Y-Q Zhang ldquoPreparation of morusinfrom Ramulus mori and its effects on mice with transplantedH22hepatocarcinomardquo BioFactors vol 40 no 6 pp 636ndash645

2014[16] O Mazimba R R T Majinda and D Motlhanka ldquoAntioxidant

and antibacterial constituents fromMorus nigrardquo African Jour-nal of Pharmacy and Pharmacology vol 5 no 5 pp 751ndash7542011

[17] Z Zhang J Jin and L Shi ldquoProtective function of cis-mulberroside A and oxyresveratrol from Ramulus mori againstethanol-induced hepatic damagerdquo Environmental Toxicologyand Pharmacology vol 26 no 3 pp 325ndash330 2008

[18] T Nomura T Fukai Y Hano S Yoshizawa M SuganumaandH Fujiki ldquoChemistry and anti-tumor promoting activity ofMorus flavonoidsrdquo Progress in Clinical and Biological Researchvol 280 pp 267ndash281 1988

[19] S Wang M Fang Y-L Ma and Y-Q Zhang ldquoPrepara-tion of the branch bark ethanol extract in mulberry morusalba its antioxidation and antihyperglycemic activity in vivordquoEvidence-Based Complementary and Alternative Medicine vol2014 Article ID 569652 7 pages 2014

[20] T Z He L Li XM Liu Y L Ma and Y Q Zhang ldquoExtractioncomposition and in vitro bioactivity of polysaccharide frommulberry branch barkrdquo Science of Sericulture vol 36 pp 1033ndash1036 2010

[21] F Qiu T-Z He and Y-Q Zhang ldquoThe isolation and thecharacterization of two polysaccharides from the branch barkof mulberry (Morus alba L)rdquo Archives of Pharmacal Researchvol 39 no 7 pp 887ndash896 2016

[22] M Fang A Q Huang and Y Q Zhang ldquoThe correla-tion between 1-deoxynojirimycin content and 120572-glucosidaseinhibitory activity in the bark ethanol extract from Ramulus


morirdquo in Proceedings of the International Conference on Biomed-ical Engineering and Biotechnology pp 1795ndash1798 2012

[23] B Ma F Y Cui and Y Q Zhang ldquoRapid analysis of morusinfrom ramulus mori by HPLC-DAD its distribution in vivo anddifferentiation in the cultivated mulberry speciesrdquo Journal ofFood and Nutrition Sciences vol 4 no 2 pp 189ndash194 2013

[24] H-Y Liu M Fang and Y-Q Zhang ldquoIn vivo hypoglycaemiceffect and inhibitory mechanism of the branch bark extractof the mulberry on STZ-induced diabetic micerdquo The ScientificWorld Journal vol 2014 Article ID 614265 11 pages 2014

[25] S Wang X M Liu J Zhang and Y Q Zhang ldquoAn EfficientPreparation of mulberroside A from the branch bark of mul-berry and its effect on the inhibition of tyrosinase activityrdquo PLoSONE vol 9 Article ID e109396 2014

[26] H-Y Liu J Wang J Ma and Y-Q Zhang ldquoInterference effectof oral administration of mulberry branch bark powder on theincidence of type II diabetic inmice induced by streptozotocinrdquoFood and Nutrition Research vol 60 pp 1ndash11 2016

[27] A A Hemmati M T Jalali I Rashidi and T K HormozildquoImpact of aqueous extract of black mulberry (Morus nigra) onliver and kidney function of diabetic micerdquo Jundishapur Journalof Natural Pharmaceutical Products vol 5 pp 18ndash25 2010

[28] R Afrin S Arumugam M I I Wahed et al ldquoAttenuationof endoplasmic reticulum stress-mediated liver damage bymulberry leaf diet in streptozotocin-induced diabetic ratsrdquoAmerican Journal of Chinese Medicine vol 44 no 1 pp 87ndash1012016

[29] A C Maritim R A Sanders and J B Watkins III ldquoDiabetesoxidative stress and antioxidants a reviewrdquo Journal of Biochem-ical and Molecular Toxicology vol 17 no 1 pp 24ndash38 2003

[30] U Asmat K Abad and K Ismail ldquoDiabetes mellitus and oxida-tive stress-A concise reviewrdquo Saudi Pharmaceutical Journal vol24 pp 547ndash553 2016

[31] S Gaweł M Wardas E Niedworok and P Wardas ldquoMalondi-aldehyde (MDA) as a lipid peroxidation markerrdquo WiadomosciLekarskie vol 57 no 9 pp 453ndash455 2004

[32] S J Kim K Winter C Nian M Tsuneoka Y Koda and CH McIntosh ldquoGlucose-dependent insulinotropic polypeptide(GIP) stimulation of pancreatic beta-cell survival is dependentupon phosphatidylinositol 3-kinase (PI3K)protein kinase B(PKB) signaling inactivation of the forkhead transcriptionfactor Foxo1 and down-regulation of bax expressionrdquo TheJournal of Biological Chemistry vol 280 no 22 pp 297ndash3072005

[33] G J P L Kops and B M T Burgering ldquoForkhead transcriptionfactors New insights into protein kinase B (c-akt) signalingrdquoJournal of Molecular Medicine vol 77 no 9 pp 656ndash665 1999

[34] J Nakae V Barr and D Accili ldquoDifferential regulation ofgene expression by insulin and IGF-1 receptors correlates withphosphorylation of a single amino acid residue in the forkheadtranscription factor FKHRrdquo EMBO Journal vol 19 no 5 pp989ndash996 2000

[35] A W Kinyua C M Ko K V Doan et al ldquo4-hydroxy-3-methoxycinnamic acid regulates orexigenic peptides and hep-atic glucose homeostasis through phosphorylation of FoxO1rdquoExperimental amp Molecular Medicine vol 50 Article ID e4372018

[36] A Barthel D Schmoll and T G Unterman ldquoFoxO proteinsin insulin action and metabolismrdquo Trends in Endocrinology ampMetabolism vol 16 no 4 pp 183ndash189 2005

[37] A M Valverde D J Burks I Fabregat et al ldquoMolecular mech-anisms of insulin resistance in IRS-2-deficient hepatocytesrdquoDiabetes vol 52 no 9 pp 2239ndash2248 2003

[38] F Hao J Kang Y Cao et al ldquoCurcumin attenuates palmitate-induced apoptosis in MIN6 pancreatic 120573-cells throughPI3KAktFoxO1 and mitochondrial survival pathwaysrdquoApoptosis vol 20 no 11 pp 1420ndash1432 2015

[39] Y Furukawa-Hibi Y Kobayashi C Chen and N MotoyamaldquoFOXO transcription factors in cell-cycle regulation and theresponse to oxidative stressrdquo Antioxidants amp Redox Signalingvol 7 no 5 pp 752ndash760 2005

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


In our laboratory we studied the extraction of active com-ponents such as polysaccharides [20 21] 1-deoxynojirimycin[22] mulberroside A [19] and morusin [23] from mulberrybranch bark The results showed that the extract of mul-berry branch bark has bioactivities of antioxidation [19]hypoglycaemia [24] hypolipidemia and anticancer [25] Inaddition our group investigated the interference effect of oraladministration of mulberry branch bark powder (MBBP) onthe incidence of type 2 diabetes induced by STZ in mice [26]

The aim of this study was to evaluate the protectiveproperties after the preventive administration of branchbark powder in mice which were against 120573 cells injuryinduced by STZ and examined whether the activation ofPI3KAKTFoxO1 signalling is involved in the protectiveeffect of MBBP treat

2 Materials and Methods

21 MBBP Preparation We followed the methods of Liu etal (2016) [26] and the branches of the mulberry (HuSang32 a cultivar from M multicaulis L) were obtained fromthe mulberry garden of Soochow University Suzhou Chinain November 2016 The bark which was peeled from themulberry branches was dried at 100∘C for 2 h pulverizedinto powder twice and passed through a 100-mesh sieveThepowder was weighted and mixed with standard diet to get25 5 and 10 MBBP diets for mice

22 Animals Male ICR mice (SPF) (4 weeks old 18ndash22 g)were obtained from the Experimental Animal Centre of Soo-chow University All animal experimental protocols used inthis study were approved by the Animal Ethics Committee atSoochowUniversity (number of animal license 201603A255)The mice were raised in standard experimental conditionsof humidity at 50ndash80 temperature of 18∘Cndash25∘C and12-hour lightdark cycle During the experiment the micecould freely obtain standard laboratory chow and standardlaboratory water

23 Drug Administration Drug administration was alsofollowed the methods of Liu et al (2016) [26] the mice wereallowed to adapt to the laboratory environment for one weekand then a total of 50 mice were randomly divided into 5groups normal group with standard feed model group withstandard feed 25MBBP feed group 5MBBP feed groupand 10MBBP feed groupThemicewere fed their individualdiets for 2 weeks On the 14st day the mice were given singletail-vein injection of freshly prepared citrate buffer (pH 45)solution of STZ (100 mgkg) [27] except for the normalgroup (STZ [S0130] was obtained from Sigma-Aldrich FineChemicalsUnited States) After injection allmice continuedto be fed as above

24 Fasting Blood Glucose Serum Insulin Levels and OrganCoefficient Fasting blood glucose (FBG) was measured bytail-vein sampling using a glucose meter from Johnson ampJohnson Medical (Shanghai) Ltd (China) on day 7 afterSTZ injection Then the mice were killed under anaesthe-sia The blood was collected and serum was prepared by

centrifuge (3000 rpm 15 min) The serum insulin levelswere determined by Insulin Assay Kit (Nanjing JianchengBioengineering Institute Nanjing China) The mice weredissected and then liver was weighed The ratio of tissueweight to body weight was calculated

25 Histological Examination and ImmunohistochemistryAssay Fresh mice pancreas was quickly removed rinsedwith cold phosphate buffered saline immersed in 4paraformaldehyde solution at pH 70 and embedded in paraf-fin Slices of 5 120583m thickness were cut After removing paraffinwith xylol the sections were stained with hematoxylin andeosin (HampE) and examined by optical microscope (400times)

To observe the immunohistological staining of insulinthe pancreas samples were soaked in 4 paraformaldehydeand then these tissues were sealed in paraffin and cut to 5120583m thicknessThe primary antibody anti-insulin (purchasedfrom Wuhan Boster Bio-engineering Limited Company[1200]) was incubated with tissues and then probed witha secondary antibody After rinsing the Elivison two-stepmethod was performed for immunohistochemical stainingAn optical microscope was used to observe the slices (400times)

26 Measurement of Antioxidant Status Glutathione peroxi-dase (GSH-PX) superoxide dismutase (SOD) and methanedicarboxylic aldehyde (MDA) in liver homogenates of micewere measured by using assay kits The GSH-PX assay kit(A005) SOD assay kit (A001-1) and MDA assay kit (A003-1)were purchased from the Nanjing Jiancheng BioengineeringInstitute Nanjing China In accordance with the assay kitguide book the absorbance of samples was determined at 412nm for GSH-PX 550 nm for SOD and 532 nm for MDA atthe end of the reaction

27 Quantitative Real Time-PCR Analysis The extraction ofpancreas and livers in total RNA determination of RNAand synthetization of the primer gene sequences of PI3KPDK-1 AKT FoxO1 and 120573-actin were carried out as reportedpreviously [26] Primer sequences are listed in Table 1 andthey were synthesized by Sangon Biotech (Shanghai) CoLtd China

The reverse transcription reaction system was assembledon ice according to the kit There are 250ndash300 ng mRNAfor reverse transcription reactions The reaction system wasset to 37∘C for 15 min The reverse transcriptase was theninactivated by heating to 85∘C for 5 sec and the cDNA wasobtainedThere is 2 120583L cDNA for PCR reaction PCR reactionconditions were as follows 94∘C for 4 min 94∘C for 20sec 60∘C for 30 sec and 72∘C for 30 sec Thirty-five cycleswere conducted from the second stage Using the differentconcentrations of cDNA as template the standard curvesof the experimental genes and the internal reference genewere generated The data were calculated according to thefollowing formula (T) = a times lg(copy) + b values of a varyamong the different genes and PCR fragments

28 Protein Expression by Western Blotting Western blottingwas done according to the methods described by Afrin etal [28] In the liver samples the all-protein concentration







3 Results





Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

60

50

40

30

Live

rBW

(mg

g)

lowastlowast

(a)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P


0

5

10

15

20

25

Fast

Bloo

d G

luco

se (m

mol

L)

(b)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

lowast

0

2

4

6

7

10

Insu

lin (m

UL

)

(c)


(d)















4 Discussion




Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH







FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

























3 Results





Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

60

50

40

30

Live

rBW

(mg

g)

lowastlowast

(a)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P


0

5

10

15

20

25

Fast

Bloo

d G

luco

se (m

mol

L)

(b)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

lowast

0

2

4

6

7

10

Insu

lin (m

UL

)

(c)


(d)















4 Discussion




Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH







FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

60

50

40

30

Live

rBW

(mg

g)

lowastlowast

(a)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P


0

5

10

15

20

25

Fast

Bloo

d G

luco

se (m

mol

L)

(b)

Nor

mal

Mod

el

25

M

BBP

5

MBB

P

10

MBB

P

lowast

0

2

4

6

7

10

Insu

lin (m

UL

)

(c)


(d)















4 Discussion




Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH







FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























4 Discussion




Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH







FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Normal 105Model

PI3K

pAKT

AKT

GAPDH

Normal 105Model

GAPDH

pFoxO1

FoxO1

lowastlowast

lowast

08

10

00

02

04

06

PI3

K Pr

otei

n le

velG

APD

H

lowast

lowast

lowastlowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

00

02

04

06

08

10

12A

KT p

rote

in le

velG

APD

H

00

02

04

06

08

10

pAKT

pro

tein

leve

lGA

PDH

00

01

02

03

04

05

FoxO

1G

APD

H

000

005

010

015

020

025

030

035

040

pFox

O1

GA

PDH







FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















FoxO1

GAPDH

Lamin A

Cytoplasm Nucleus

Cytoplasm Nucleus

lowast

lowastlowast

lowastlowast

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

Nor

mal

5

MBB

P

10

MBB

P

Mod

el

000

005

010

015

020

F0xO

1G

APD

H

000

002

004

006

008

010

012

014

016

FoxO

1L

amin

A


10

08

06

04

02

00

10

08

06

04

02

00

mRN

A in

live

r

-act

in

mRN

A in

pan

crea

s

-act

in

FoxO1AktPDK1PI3K


FoxO1AktPDK1PI3K



lowast lowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast








Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















Data Availability




Acknowledgments


References



















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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