Total Flavonoids Extracted from Oxytropis falcata...

Research ArticleTotal Flavonoids Extracted from Oxytropis falcataBunge Improve Insulin Resistance through Regulation on theIKK120573NF-120581B Inflammatory Pathway

Lixia Yang1 ZhichengWang2 Liangen Jiang3 Wen Sun4 Qiang Fan3 and Tonghua Liu4

1Central Laboratory Gansu Province Academy of Chinese Medicine Lanzhou Gansu 730050 China2Third Hospital Affiliated to Beijing University of Traditional Chinese Medicine Beijing 100029 China3School of Clinical Medicine Gansu University of Traditional Chinese Medicine Lanzhou Gansu 730000 China4Key Laboratory of Traditional Chinese Medicine of the Ministry of Education Beijing University of Traditional Chinese MedicineBeijing 100029 China

Correspondence should be addressed to Zhicheng Wang wangzhicheng116sinacom

Received 18 June 2016 Accepted 28 November 2016 Published 26 March 2017

Academic Editor Dolores Garcıa Gimenez

Copyright copy 2017 Lixia Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Insulin resistance (IR) is the main etiology of type 2 diabetes mellitus (T2DM) It has been known that total flavonoidextracts canmarkedly improve the hypoglycemic symptoms caused by IR Nevertheless the relevantmolecularmechanism remainsunclarified Aim This study aimed to investigate the antihyperglycemic effects and mechanism of the total flavonoid extract fromOxytropis falcata Bunge Methods STZ-induced T2DM rats (119899 = 35) were divided into 5 groups model low- medium- andhigh-dose total flavonoids and pioglitazone groups Ten healthy rats were used as controls The serum insulin and inflammatorycytokines (MCP-1 TNF-120572 and IL-6) level was measured by ELISAThe concentration of IRS-1 p-IRS-1 PKB p-PKB PI3Kp85 andp-PI3K in skeletal muscles was determined by Western blot The mRNA level of GLUT4 I120581B and NF-120581B in skeletal muscle wasdetected by qRT-PCR Results The treatment of medium- and high-dose total flavonoids significantly reduced the FPG and P2hPGand enhanced insulin level in T2DM rats (119875 lt 005) When compared with controls the serum level of MCP-1 TNF-120572 IL-6 IRS-1and p-IRS-1 was significantly increased in T2DM rats but the level of PKB p-PKB PI3Kp85 and p-PI3K expression was reduced(119875 lt 005)The GLUT4 and I120581BmRNA expression were significantly decreased and NF-120581BmRNA level was increased (119875 lt 005)The treatment of low- medium- or high-dose total flavonoids markedly reversed the changes above (119875 lt 005) Conclusion Ourstudy has confirmed the therapeutic effects of total flavonoids fromOxytropis falcata Bunge on IRThe flavonoids might reduce theproduction of inflammatory cytokines through downregulation of NF-120581B expression in inflammatory pathway and regulate theIRS-1-PI3-K-PKBAkt insulin pathway and thereby increased the GLUT4 expression

1 Introduction

With the rapid development of global economy the inci-dence of type 2 diabetes mellitus (T2DM) has substantiallyincreased The prevalence of T2DM in China and USA hasrecently reached as high as 116 and 113 respectively[1 2] Insulin resistance (IR) the main etiology of T2DMrefers to the physiological condition in which cells in thebody become resistant to the normal functions of insulinPatients with IR may also develop a variety of diseases such

as obesity hyperlipidemia cardiovascular diseases essentialhypotension and nonalcoholic fatty liver disease [3 4]Studies have suggested an important role of inflammation inIR [5ndash7] NF-kB (nuclear factor kappa-light-chain-enhancerof activated B cells) one of the important inflammatorypathways can decrease the expression of insulin receptorsubstrate 1 (IRS1) in patients with IR [8] A number of otherinflammatory factors are also involved in IR including TNF-120572 IL-1 IL-6 monocyte chemoattractant protein 1 (MCP-1) plasminogen activator inhibitor-1 (PAI-1) intercellular

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2405124 6 pageshttpsdoiorg10115520172405124

2 Evidence-Based Complementary and Alternative Medicine

adhesion molecule 1 (ICAM-1) and vascular cell adhesionmolecule 1 (VCAM-1) [9]

Oxytropis falcata Bunge a species belonging to the genusOxytropis is mainly distributed in Western China includingQinghai Gansu Tibet and SichuanThe plant has been usedin folk medicine for the treatment of inflammation Studieshave found that the chloroform extract of Oxytropis falcataBunge primarily flavonoid compounds has strong anti-inflammatory and antioxidant effect [10ndash12] Several studieshave also demonstrated that the total flavonoid extract canmarkedly improve the hypoglycemic symptoms caused by IR[13ndash15] Nevertheless the molecular mechanism behind thetherapeutic effect of the total flavonoid extract on IR remainsunclarified In the current study the antihyperglycemiceffects of the total flavonoid extract from Oxytropis falcataBunge were verified in streptozotocin- (STZ-) induced type 2diabetic ratsThe changes in inflammatory factors andNF-kBpathway as well as insulin signaling pathway were examinedto explore the possible underlying mechanisms

2 Material and Methods

21 Animals Specific pathogen-free (SPF) male Wistar ratsweighing (200plusmn20) g were purchased fromGansu Universityof Traditional Chinese Medicine (license number SCXK(Gan) 2004-0006) The rats were raised with free access towater and food in a barrier-level animal laboratory at theUniversity for a week before the experimentsThe experimentprotocol was approved by the Research Ethics Committee atthe University

22 Main Reagents Oxytropis falcata Bunge was purchasedfrom Youruntang Co Ltd (Qinghai China) Total flavonoid(3658 purity) was extracted by Usea Biotech (ShanghaiChina) Pioglitazone hydrochloride tablets were manufac-tured by Taiyang Pharmaceutical Co (Beijing China lotnumber 130605) STZ was purchased from Sigma (St LouisMO USA) The insulin ELISA detection kit and MCP-1ELISA kit were purchased from Cusabio (Wuhan China)ELISA kits for TNF-120572 and IL-6 were purchased fromDakeweBiotech (Shenzhen China) Roche Accu-Chek active bloodglucose test strips were purchased from Roche Diagnos-tics GmbH (Mannheim Germany) Protein extraction kitand BCA kit were purchased from Beyotime Institute ofBiotechnology (Shanghai China) Rabbit anti-mouse IRS1(ab52167 1 500 dilution) PKB (ab25893 1 1000 dilution)p-PKB (ab81283 1 5000 dilution) and p-PI3K (ab1826511 1000 dilution) antibodies were purchased from Abcam(Cambridge MA USA) Rabbit anti-mouse p-IRS1 (catalognumber 2381 1 1000 dilution) and PI3K (catalog number4292 1 1000 dilution) were purchased from Cell Signaling(Beverly MA USA) TRIzol extraction kit was purchasedfrom Invitrogen (Carlsbad CA USA) M-MLV reversetranscription kit and DreamTaq Green PCR Master Mix(2x) were purchased from Takara (Tokyo Japan) Real-timePCR amplification kit was purchased from Zhongyuan Ltd(Beijing China) The primers were synthesized by SangonBiotech (Shanghai China)

23 Construction of Animal Models and Medication Tenrats in the normal control group were given normal dietthroughout the experiment The remaining rats were givenhigh-sugar and high-fat diet for 8 weeks followed by aninjection of STZ (25mgsdotkgminus1sdotdminus1) for 1 week Blood wascollected via the tail vein and fasting plasma glucose (FPG)and postprandial 2-hour plasma glucose (P2hPG) were mea-sured with an Accu-Chek Active blood glucose meter (RocheDiagnostics) Rats with stable blood sugar above 139mmolLwere diagnosed as T2DM A total of 35 T2DM rats wererandomly divided into 5 groups model low- medium- andhigh-dose total flavonoids and pioglitazone groups (119899 = 7)Low- medium- and high-dose total flavonoids groups weregiven intragastric administration of 10mlsdotkgminus1 of 100 200and 400mgsdotkgminus1sdotdminus1 respectively for 4 weeks Pioglitazonewas given 10mlsdotkgminus1 of 10mgsdotkgminus1sdotdminus1 Normal control andmodel groups were given equal volume of saline After 4weeks all rats were fastened for 8 h and anesthetized Bloodsample was taken from the hearts Rats were sacrificed andskeletal muscles were collected for subsequent analysis

24 ELISA The level of serum insulin and inflammatorycytokines (MCP-1 TNF-120572 and IL-6) was measured usingthe ELISA kit according to manufacturerrsquos instructions TheOD value of each sample was detected at the wavelengthof 450 nm with a Bio-Rad 680 microplate reader (Bio-RadHercules CA USA)

25 Western Blot Analysis The concentration of IRS-1 p-IRS-1 PKB p-PKB PI3Kp85 and p-PI3K in skeletal mus-cles was determined by Western blot Briefly tissues werehomogenized in liquid nitrogen Total protein was extractedusing protein extraction kits and quantified using the BCAkit following the manufacturerrsquos instructions Equal amountsof total protein (20120583g) were separated by SDS-PAGE elec-trophoresis and transferred to polyvinylidene difluoridemembranes The membrane was blocked in TBS buffercontaining 5 skim milk and 01 Tween 20 at room tem-perature for 2 h and incubated with the appropriate primaryantibody overnight at 4∘C with gentle shaking The HRP-labeled secondary antibodies were added and themembraneswere incubated at 37∘C for 1 h The membranes were washed3 times with TBST for 5min each and subjected to ECLdetectionThe intensity of bands was detected by aMolecularImager ChemiDoc XRS System (Bio-Rad Laboratories)The gray value of bands was analyzed by Image-Pro Plussoftware (Bio-Rad Laboratories) The relative expression oftarget protein was calculated as the ratio of its gray value tothat of the internal control GAPDH

26 Quantitative Reverse Transcription PCR (qRT-PCR)Total RNA was extracted using RNA extraction kit andquantified using a spectrometer under a wavelength of260 nm Total RNA was reverse-transcribed into cDNAusing reverse transcription kit following the manufacturerrsquosinstructions The mRNA level of GLUT4 I120581B and NF-120581Bin skeletal muscle was detected by qRT-PCR using cDNAas template and DreamTaq Green PCR Master Mix The

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Comparison of serum level of FPG P2hPG and insulin in difference groups (119899 = 7 119909 plusmn 119904)

Group Dosage (mgsdotkgminus1) FPG (mmoll) P2hPG (mmoll) Insulin (nIUml)Normal control mdash 430 plusmn 020 606 plusmn 044 9004 plusmn 1873

Model mdash 2534 plusmn 388(1) 2890 plusmn 338(1) 1942 plusmn 1936(1)

Pioglitazone 10 1180 plusmn 104(2) 1474 plusmn 226(2) 4344 plusmn 5710(2)

Low-dose total flavonoids 100 2286 plusmn 464 2569 plusmn 434 3652 plusmn 3385

Medium-dose total flavonoids 200 1609 plusmn 225(2) 1989 plusmn 191(2) 4740 plusmn 3477(2)

High-dose total flavonoids 400 1313 plusmn 080(2) 1434 plusmn 212(2) 4960 plusmn 4760(2)

(1)119875 lt 005 compared with normal control group and (2)119875 lt 005 compared with model group

Table 2 Comparison of serum level of MCP-1 TNF-120572 and IL-6 in different groups (119899 = 7 119909 plusmn 119904)

Group Dosage (mgsdotkgminus1) MCP-1 (pgml) TNF-120572 (pgml) IL-6 (pgml)Normal control mdash 8883 plusmn 2369 10444 plusmn 1084 6544 plusmn 561



Low-dose total flavonoids 100 15619 plusmn 9201 12053 plusmn 2742 9293 plusmn 2560(2)

Medium-dose total flavonoids 200 10184 plusmn 3171(2) 11662 plusmn 1556 6884 plusmn 2076(2)

High-dose total flavonoids 400 13354 plusmn 5155 11467 plusmn 2128 4868 plusmn 3257(2)


Table 3 Comparison of the expression of insulin signaling molecules in skeletal muscles in different groups (119899 = 3 119909 plusmn 119904)

Group Dosage (mgsdotkgminus1) IRS-1 p-IRS-1 PKB p-PKB PI3Kp85 p- PI3KNormal control mdash 012 plusmn 006 022 plusmn 008 190 plusmn 080 152 plusmn 039 156 plusmn 079 138 plusmn 024

Model mdash 099 plusmn 044(1) 075 plusmn 009(1) 076 plusmn 033(1) 040 plusmn 004(1) 059 plusmn 034(1) 052 plusmn 011(1)

Pioglitazone 10 087 plusmn 036 043 plusmn 028(2) 136 plusmn 050(2) 093 plusmn 024(2) 090 plusmn 055(2) 101 plusmn 010(2)

Low-dose total flavonoids 100 036 plusmn 012(2) 055 plusmn 035 101 plusmn 048(2) 101 plusmn 040(2) 078 plusmn 041 150 plusmn 016(2)

Medium-dose total flavonoids 200 036 plusmn 010(2) 056 plusmn 018 166 plusmn 057(2) 114 plusmn 050(2) 093 plusmn 023(2) 110 plusmn 038(2)

High-dose total flavonoids 400 044 plusmn 032(2) 042 plusmn 021(2) 082 plusmn 011 142 plusmn 009(2) 107 plusmn 024(2) 088 plusmn 021(1)119875 lt 005 compared with normal control group and (2)119875 lt 005 compared with model group

reaction condition was as follows 94∘C 10min 45 cyclesof 94∘C 15 s 60∘C 60 s and 72∘C 10min Primer sequenceswere I120581B forward 51015840-TGACCATGGAAGTGATTGGT-31015840reverse 51015840- AGCCAAGTGGAGTGGAGTCT-31015840 NF-120581Bforward 51015840-CGTGAGGCTGTTTGGTTTGA-31015840 reverse 51015840-CTTATGGCTGAGGTCTGGTC-31015840 GLUT4 forward 51015840-GTTGGTCTCGGTGCTCTTAG-31015840 reverse 51015840-GGCCAC-GATGGACACATAAC-31015840 and 120573-actin forward 51015840-GCA-GTTGGTTGGAGCAA-31015840 reverse 51015840-ATGCCGTGGATA-CTTGGA-31015840 The experiment was repeated three times Datawas analyzed using the 2-ΔCt method

27 Statistical Analysis All data were expressed as mean plusmnstandard deviation and analyzed using SPSS 190 (SPSS IncChicago IL USA) Difference between groups was comparedby ANOVA 119875 values smaller than 005 are consideredstatistically significant

3 Results

31 Comparison of Serum Level of Insulin FPG and P2hPGAs shown in Table 1 the level of FPG and P2hPG in modelgroup was significantly increased compared with normal

controls whereas serum insulin level wasmarkedly decreased(119875 lt 005) The treatment of medium- and high-dose totalflavonoids as well as pioglitazone had significantly reducedthe FPG and P2hPG and enhanced insulin level in T2DM rats(119875 lt 005) The low-dose total flavonoids treatment had littleeffect on serum level of insulin FPG and P2hPG

32 Comparison of Serum MCP-1 TNF-120572 and IL-6 Con-centration The serum level of MCP-1 TNF-120572 and IL-6 inT2DM rats was significantly increased (119875 lt 005) butwas substantially reduced after pioglitazone treatmentWhileboth medium- and high-dose total flavonoids significantlydecreased the level of IL-6 only medium-dose treatmentreduced the production of MCP-1 (119875 lt 005) None of thetotal flavonoids group affected the TNF-120572 level (Table 2)

33 Comparison of the Expression of Insulin Signaling Mol-ecules in Skeletal Muscles The relative expression of proteinsinvolved in the insulin signaling pathway was examined byWestern blot including IRS-1 PKB and PI3Kp85 (Table 3Figures 1(a)ndash1(c)) The expression of IRS-1 and p-IRS-1 inmodel group was significantly higher than that in normalcontrols (119875 lt 005) The IRS-1 level in low- medium- and


Table 4 Comparison of the mRNA expression of GLUT4 I120581B and NF-120581B in skeletal muscles in different groups (119899 = 3 119909 plusmn 119904)

Group Dosage (mgsdotkgminus1) GLUT4 I120581B NF-120581BNormal control mdash 1351 plusmn 118 1003 plusmn 009 1253 plusmn 079


Pioglitazone 10 0674 plusmn 014(2) 0842 plusmn 078 3609 plusmn 169(2)

Low-dose total flavonoids 100 0182 plusmn 006(2) 0526 plusmn 033 3968 plusmn 145(2)


High-dose total flavonoids 400 0234 plusmn 008(2) 0867 plusmn 062 2695 plusmn 070(2)


Nor

mal

cont

rol

Mod

el g

roup

Piog

litaz

one

Low

-dos

e tot

al fl

avon

oids

Med

ium

-dos

e tot

al fl

avon

oids

Hig

h-do

se to

tal

avon

oids

IRS-1

p-IRS-1

GAPDH

(a)

Nor

mal

cont

rol

Mod

el g

roup

Piog

litaz

one

Low

-dos

e tot

al fl

avon

oids

Med

ium

-dos

e tot

al fl

avon

oids

Hig

h-do

se to

tal

avon

oids

PKB

P-PKB

GAPDH

(b)

Nor

mal

cont

rol

Mod

el g

roup

Piog

litaz

one

Low

-dos

e tot

al fl

avon

oids

Med

ium

-dos

e tot

al fl

avon

oids

Hig

h-do

se to

tal

avon

oids

GAPDH

P-PI3K

PI3Kp85

(c)

Figure 1 Western blot analysis of the expression of insulin signaling molecules in skeletal muscles in different groups (a) IRS-1 and p-IRS-1(b) PKB and p-PKB and (c) PI3Kp85 and p-PI3Kp85 (1) Normal control (2) model group (3) pioglitazone (4) low-dose total flavonoids(5) medium-dose total flavonoids and (6) high-dose total flavonoids

high-dose total flavonoids groups was significantly reduced(119875 lt 005) The p-IRS-1 expression in pioglitazone groupwas also decreased (119875 lt 005) The expression of PKB p-PKB PI3Kp85 and p-PI3K in model group was significantlydecreased compared with normal control group (119875 lt 005)The p-PKB level in all treatment groups was markedly higherthan that in model group (119875 lt 005) The PKB and p-PI3Kexpression in pioglitazone and low- and medium-dose total

flavonoids groups were significantly increased (119875 lt 005)The expression of PI3Kp85 in pioglitazone and medium- andhigh-dose total flavonoids groups was also increased (119875 lt005)

34 Comparison ofGLUT4 I120581B andNF-120581BmRNAExpressionin Skeletal Muscles As shown in Table 4 the GLUT4 and I120581BmRNA in model group were significantly reduced compared


with normal controls whereas the NF-120581B mRNA was sig-nificantly increased (119875 lt 005) When compared with themodel group GLUT4 mRNA in pioglitazone and the threetotal flavonoids groups was markedly enhanced and NF-120581BmRNA expression was reduced (119875 lt 005)

4 Discussion

Diabetes is one of the most common chronic diseases withincreasingly higher prevalence The pathogenesis of diabetesis a complex process involving many factors T2DM hasbeen known to be induced by IR Therefore investigationon improving IR is of significant clinical importance for theprevention and treatment of T2DM

In most T2DM patients insulin postreceptor signalingtransduction disorder in target cells is the main mecha-nism leading to IR [16] Numerous studies have suggestedthat IR is a chronic nonspecific inflammation involvinginflammatory cytokines immune system and adipose tissues[17ndash20] Inflammation has interfered the insulin postrecep-tor signaling pathway IRS-1-PI3-K-PKBAkt pathway andthereby induces the RI [21 22] Specifically inflammation caninduce the serinethreonine phosphorylation of IRS-1 whichaffects the interaction between IRS-1 and insulin receptorand inhibits the normal tyrosine phosphorylation of IRS-1 The reduced IRS-1 activity leads to reduced phosphoryla-tion of PI3-K and thereby decreased expression of GLUT4in skeletal muscle cells and adipocytes Consequently thebiological activity of insulin was reduced resulting in theoccurrence of IR IKK120573NF-120581B is one of the most frequentlystudied inflammatory signaling pathways associated withinflammation-induced IR Under normal conditions theI120581B protein (inhibitor of NF-120581B) binds to NF-120581B in thecytoplasm whereas IKK120573 the kinase of I120581B is activated bythe stimulation of inflammatory cytokines such as TNF-120572IL-1 and IL-6 during inflammation leading to the serinephosphorylation of I120581B The NF-120581B is translocated into thenucleus where it binds to the genomic DNA and regulatesthe expression of inflammatory cytokines initiating or aggre-gating the inflammatory response and ultimately leadingto the occurrence of IR Studies have suggested that theIR can be prevented by the inhibition of the IKK120573NF-120581Bpathway [23] In this study an IR rat model was successfullyconstructed by high-fat high-sugar diet and injection ofSTZ as indicated by increased blood FPG and P2hPG anddecreased serum insulin level We detected unusually highlevel of inflammatory cytokines (MCP-1 TNF-120572 and IL-6)in the serum of these IR rats Moreover these rats developedsignificantly enhanced IRS-1 p-IRS-1 and NF-120581B as wellas reduced PI3Kp85 p-PI3K PKB p-PKB GLUT4 andI120581B level in the skeletal muscles These results suggested aclose association between the inflammatory pathway and theIRS-1-PI3-K-PKBAkt insulin postreceptor pathway which isconsistent with previous studies

Currently the drawbacks of Western medicine in thetreatment of IR have been frequently reported includingadverse side effects and drug resistance Therefore explo-ration of herbal medicines for IR has recently become aresearch hotspot due to their high safety It has been found

that the flavonoids compounds in several plants such asbuckwheat leaf corn silk and kudzu root have hypoglycemiceffects [24ndash26] suggesting the potential of flavonoids com-pounds as effective and safe medicines for the treatment ofIR and diabetes Consistently in our study the treatmentwith medium- and high-dose total flavonoids fromOxytropisfalcata Bunge significantly reduced the blood FPG andP2hPG and stimulated the secretion of insulin in IR ratssuggesting the therapeutic effects of the total flavonoids

Nevertheless the molecular mechanism underlying thehypoglycemic effects of total flavonoids has not been wellclarified Studies have found that the total flavonoids fromOxytropis falcata Bunge are mainly composed of flavonoidglycoside flavanone flavonols chalcone dihydrochalconeflavanones and isoflavane andhave strong anti-inflammatoryand antioxidant effects [27] In this study we monitored thechanges in the IRS-1-PI3-K-PKBAkt insulin postreceptorpathway and inflammatory pathway after the treatment ofOxytropis falcata Bunge total flavonoids It was found thatOxytropis falcata Bunge total flavonoids significantly reducedthe level of MCP-1 and IL-6 when compared with modelgroup The flavonoids compounds also inhibited the expres-sion of IRS-1 p-IRS-1 PKB p-PKB PI3Kp85 and p-PI3KMoreover the flavonoids markedly stimulated the expressionof GLUT4 and reduced NF-120581B in the treatment groups

Overall our study has confirmed the therapeutic effectsof total flavonoids extracted from Oxytropis falcata Bungeon IR The flavonoids might reduce the production ofinflammatory cytokines through downregulation of NF-120581Bexpression in inflammatory pathway and regulate the IRS-1-PI3-K-PKBAkt insulin pathway and thereby increased theGLUT4 expression Consequently the glucose uptake andutilization in skeletal muscles are improved and glucosebalance is recovered Our study has provided insights intothe therapeutic mechanism of Oxytropis falcata Bunge totalflavonoids on IR which will be a basis for the developmentof effective and safe natural medicine for the treatment ofT2DM

Competing Interests

The authors declare no conflict of interests

Acknowledgments

This work is supported by National Natural Science Foun-dation of China (81202971) Independent Project of BeijingUniversity of Traditional Chinese Medicine and GansuYouth Science and Technology Foundation (1107RJYA081)The authors sincerely thank Dr Tonghua Liu at the BeijingUniversity of Traditional Chinese Medicine for his guidanceon the current study

References

[1] K M Flegal D Carroll B K Kit and C L Ogden ldquoPrevalenceof obesity and trends in the distribution of body mass indexamong US adults 1999ndash2010rdquo JAMAmdashJournal of the AmericanMedical Association vol 307 no 5 pp 491ndash497 2012


[2] Y Xu L Wang J He et al ldquoPrevalence and control ofdiabetes in Chinese adultsrdquoThe Journal of the AmericanMedicalAssociation vol 310 no 9 pp 948ndash959 2013

[3] G Reaven ldquoThe metabolic syndrome or the insulin resistancesyndrome different names different concepts and differentgoalsrdquo Endocrinology and Metabolism Clinics of North Americavol 33 no 2 pp 283ndash303 2004

[4] L Pedersen M Nybo M K J Poulsen J E R HenriksenJ Dahl and L M E Rasmussen ldquoPlasma calprotectin andits association with cardiovascular disease manifestations obe-sity and the metabolic syndrome in type 2 diabetes mellituspatientsrdquo BMC cardiovascular disorders vol 14 p 196 2014

[5] Q Huang J Xue R Zou et al ldquoNR4A1 is associated withchronic low-grade inflammation in patients with type 2 dia-betesrdquo Experimental andTherapeutic Medicine vol 8 no 5 pp1648ndash1654 2014

[6] B-C Lee and J Lee ldquoCellular and molecular players in adiposetissue inflammation in the development of obesity-inducedinsulin resistancerdquo Biochimica et Biophysica Acta (BBA)mdashMolecular Basis of Disease vol 1842 no 3 pp 446ndash462 2014

[7] H Xu G T Barnes Q Yang et al ldquoChronic inflammation in fatplays a crucial role in the development of obesity-related insulinresistancerdquoThe Journal of Clinical Investigation vol 112 no 12pp 1821ndash1830 2003

[8] G Boden P She M Mozzoli et al ldquoFree fatty acids produceinsulin resistance and activate the proinflammatory nuclearfactor-120581b pathway in rat liverrdquoDiabetes vol 54 no 12 pp 3458ndash3465 2005

[9] H Y Li G Z Cui and Y Zhang ldquoThe insulin signal trans-duction pathway in type 2 diabetesrdquo The Journal of ChangchunUniversity Chinese Medicine vol 24 pp 325ndash326 2008

[10] X J Zhang W X Liu P Wei et al ldquoChemical compositionand toxicity of Tibetan medicine Oxytropis falcata BungerdquoTraditional Chinese Medicine vol 39 no 7 pp 1157ndash1161 2014

[11] H Jiang W Q Zhan X Liu and S X Jiang ldquoAntioxidantactivities of extracts and flavonoid compounds from Oxytropisfalcate Bungerdquo Natural Product Research vol 22 no 18 pp1650ndash1656 2008

[12] H Jiang J R Hu W Q Zhan and X Liu ldquoScreening forfractions of Oxytropis falcata Bunge with antibacterial activityrdquoNatural Product Research vol 23 no 10 pp 953ndash959 2009

[13] S J Na and B L Liu ldquoProgress in improving insulin resistanceflavonoidsrdquo Pharmaceutical and Clinical Research vol 17 no 4pp 315ndash318 2009

[14] Y Wang and H Y Zhao ldquoAdvances in pharmacological mech-anisms of diabetes treatment plant flavonoidsrdquoMedical Reviewvol 16 no 4 pp 612ndash615 2010

[15] C Cheng J Guo andC Y Zhang ldquoThe pharmacological effectsof flavonoidsrdquo North China University of Technology (NaturalScience) vol 12 no 2 pp 180ndash183 2011

[16] T D Filippatos V Tsimihodimos C S Derdemezis et alldquoIncreased plasma visfatin concentration is a marker of anatherogenic metabolic profilerdquo Nutrition Metabolism and Car-diovascular Diseases vol 23 no 4 pp 330ndash336 2013

[17] M Yu C L Piao Z Nan X L Tong and C Li ldquoAdvances intraditional Chinese and western medicine in the mechanismunderlying adipose tissue inflammation and insulin resistancein type 2 diabetesrdquo Chinese Journal of Hospital Infection vol 20no 1 pp 147ndash150 2010

[18] P A Tataranni and E Ortega ldquoA burning question does anadipokine-induced activation of the immune system mediate

the effect of overnutrition on type 2 diabetesrdquoDiabetes vol 54no 4 pp 917ndash927 2005

[19] JWang R X Zhou Y THan et al ldquoAdipocytokines and insulinresistancerdquoMedical Review vol 14 no 8 p 2411 2008

[20] X J Li Metabolic Syndrome Insulin Resistance SyndromePeopersquos Medical Publishing House Beijing China 2nd edition2007

[21] Y Zhou and L J Zhang ldquoInsulin resistance inflammatoryfactors and the relevant signaling pathwaysrdquo Chinese Journal ofCardiovascular Rehabilitation Medicine vol 19 no 1 pp 107ndash109 2010

[22] S Chang ldquoResearch progresses in PI3KAkt signaling pathwayand insulin resistancerdquo Guiding Journal of Traditional ChineseMedicine and Pharmacy vol 14 no 7 pp 113ndash115 2008

[23] Q Huang andH L Ding ldquoIKK120573NF-120581B signaling pathway andinsulin resistance in skeletal musclerdquo International Journal ofInternal Medicine vol 35 no 12 pp 705ndash708 2008

[24] B H Shang S Y Han L K Zhen et al ldquoEffects of totalflavonoids of buckwheat leaf on insulin resistance in ratwith type 2 diabetesrdquo Lishizhen Medicine and Materia MedicaResearch vol 20 no 10 pp 2468ndash2470 2009

[25] Y Jing R Q Jing and T H Hu ldquoEffect of corn silk totalflavonoids on blood lipid and glucose levels in diabetesrdquoPharmacology andClinics of ChineseMateriaMedica vol 27 no2 pp 85ndash86 2011

[26] Z C Zhang X Y Ye M H Xu and Y F Wang ldquoKudzu rootflavonoids in the prevention of complications associated withdiabetesrdquo Journal of East China Normal University (EducationalSciences) vol 2 pp 77ndash81 2010

[27] X Wei and L Jin ldquoAdvances in Oxytropis falcata Bunge aTibetan herbal medicinerdquo Chinese Pharmacological Bulletinvol 26 no 11 pp 1535ndash1538 2010

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


adhesion molecule 1 (ICAM-1) and vascular cell adhesionmolecule 1 (VCAM-1) [9]

Oxytropis falcata Bunge a species belonging to the genusOxytropis is mainly distributed in Western China includingQinghai Gansu Tibet and SichuanThe plant has been usedin folk medicine for the treatment of inflammation Studieshave found that the chloroform extract of Oxytropis falcataBunge primarily flavonoid compounds has strong anti-inflammatory and antioxidant effect [10ndash12] Several studieshave also demonstrated that the total flavonoid extract canmarkedly improve the hypoglycemic symptoms caused by IR[13ndash15] Nevertheless the molecular mechanism behind thetherapeutic effect of the total flavonoid extract on IR remainsunclarified In the current study the antihyperglycemiceffects of the total flavonoid extract from Oxytropis falcataBunge were verified in streptozotocin- (STZ-) induced type 2diabetic ratsThe changes in inflammatory factors andNF-kBpathway as well as insulin signaling pathway were examinedto explore the possible underlying mechanisms

2 Material and Methods

21 Animals Specific pathogen-free (SPF) male Wistar ratsweighing (200plusmn20) g were purchased fromGansu Universityof Traditional Chinese Medicine (license number SCXK(Gan) 2004-0006) The rats were raised with free access towater and food in a barrier-level animal laboratory at theUniversity for a week before the experimentsThe experimentprotocol was approved by the Research Ethics Committee atthe University

22 Main Reagents Oxytropis falcata Bunge was purchasedfrom Youruntang Co Ltd (Qinghai China) Total flavonoid(3658 purity) was extracted by Usea Biotech (ShanghaiChina) Pioglitazone hydrochloride tablets were manufac-tured by Taiyang Pharmaceutical Co (Beijing China lotnumber 130605) STZ was purchased from Sigma (St LouisMO USA) The insulin ELISA detection kit and MCP-1ELISA kit were purchased from Cusabio (Wuhan China)ELISA kits for TNF-120572 and IL-6 were purchased fromDakeweBiotech (Shenzhen China) Roche Accu-Chek active bloodglucose test strips were purchased from Roche Diagnos-tics GmbH (Mannheim Germany) Protein extraction kitand BCA kit were purchased from Beyotime Institute ofBiotechnology (Shanghai China) Rabbit anti-mouse IRS1(ab52167 1 500 dilution) PKB (ab25893 1 1000 dilution)p-PKB (ab81283 1 5000 dilution) and p-PI3K (ab1826511 1000 dilution) antibodies were purchased from Abcam(Cambridge MA USA) Rabbit anti-mouse p-IRS1 (catalognumber 2381 1 1000 dilution) and PI3K (catalog number4292 1 1000 dilution) were purchased from Cell Signaling(Beverly MA USA) TRIzol extraction kit was purchasedfrom Invitrogen (Carlsbad CA USA) M-MLV reversetranscription kit and DreamTaq Green PCR Master Mix(2x) were purchased from Takara (Tokyo Japan) Real-timePCR amplification kit was purchased from Zhongyuan Ltd(Beijing China) The primers were synthesized by SangonBiotech (Shanghai China)

23 Construction of Animal Models and Medication Tenrats in the normal control group were given normal dietthroughout the experiment The remaining rats were givenhigh-sugar and high-fat diet for 8 weeks followed by aninjection of STZ (25mgsdotkgminus1sdotdminus1) for 1 week Blood wascollected via the tail vein and fasting plasma glucose (FPG)and postprandial 2-hour plasma glucose (P2hPG) were mea-sured with an Accu-Chek Active blood glucose meter (RocheDiagnostics) Rats with stable blood sugar above 139mmolLwere diagnosed as T2DM A total of 35 T2DM rats wererandomly divided into 5 groups model low- medium- andhigh-dose total flavonoids and pioglitazone groups (119899 = 7)Low- medium- and high-dose total flavonoids groups weregiven intragastric administration of 10mlsdotkgminus1 of 100 200and 400mgsdotkgminus1sdotdminus1 respectively for 4 weeks Pioglitazonewas given 10mlsdotkgminus1 of 10mgsdotkgminus1sdotdminus1 Normal control andmodel groups were given equal volume of saline After 4weeks all rats were fastened for 8 h and anesthetized Bloodsample was taken from the hearts Rats were sacrificed andskeletal muscles were collected for subsequent analysis

24 ELISA The level of serum insulin and inflammatorycytokines (MCP-1 TNF-120572 and IL-6) was measured usingthe ELISA kit according to manufacturerrsquos instructions TheOD value of each sample was detected at the wavelengthof 450 nm with a Bio-Rad 680 microplate reader (Bio-RadHercules CA USA)

25 Western Blot Analysis The concentration of IRS-1 p-IRS-1 PKB p-PKB PI3Kp85 and p-PI3K in skeletal mus-cles was determined by Western blot Briefly tissues werehomogenized in liquid nitrogen Total protein was extractedusing protein extraction kits and quantified using the BCAkit following the manufacturerrsquos instructions Equal amountsof total protein (20120583g) were separated by SDS-PAGE elec-trophoresis and transferred to polyvinylidene difluoridemembranes The membrane was blocked in TBS buffercontaining 5 skim milk and 01 Tween 20 at room tem-perature for 2 h and incubated with the appropriate primaryantibody overnight at 4∘C with gentle shaking The HRP-labeled secondary antibodies were added and themembraneswere incubated at 37∘C for 1 h The membranes were washed3 times with TBST for 5min each and subjected to ECLdetectionThe intensity of bands was detected by aMolecularImager ChemiDoc XRS System (Bio-Rad Laboratories)The gray value of bands was analyzed by Image-Pro Plussoftware (Bio-Rad Laboratories) The relative expression oftarget protein was calculated as the ratio of its gray value tothat of the internal control GAPDH

26 Quantitative Reverse Transcription PCR (qRT-PCR)Total RNA was extracted using RNA extraction kit andquantified using a spectrometer under a wavelength of260 nm Total RNA was reverse-transcribed into cDNAusing reverse transcription kit following the manufacturerrsquosinstructions The mRNA level of GLUT4 I120581B and NF-120581Bin skeletal muscle was detected by qRT-PCR using cDNAas template and DreamTaq Green PCR Master Mix The



























3 Results














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OncologyJournal of





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Total Flavonoids Extracted from Oxytropis falcata...

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