In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 642058 8 pageshttpdxdoiorg1011552014642058

Research ArticleIn Vivo Lipid Regulation Mechanism of Polygoni MultifloriRadix in High-Fat Diet Fed Rats

Pei Lin Na Li Wan Gen Wang Wen Gu Jie Yu and Rong Hua Zhao

Yunnan University of Traditional Chinese Medicine Kunming Yunnan 650500 China

Correspondence should be addressed to Jie Yu yujieynzyxygmailcom and Rong Hua Zhao kmzhaoronghuahotmailcom

Received 6 January 2014 Revised 12 February 2014 Accepted 12 February 2014

Academic Editor Kashmira Nanji

Copyright copy 2014 Pei Lin 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

Mechanisms of the water extracts of PolygoniMultiflori Radix (PMR) and its processed products (PMRP) on liver lipidmetabolismwere observed in this paper Aqueous extract of PMR and PMRPwas given to nonalcoholic fatty liver model rats respectively PMRwas better in reducing the contents of very low density lipoprotein (VLDL) than PMRP and the positive control groups In theaspect of regulating TG medium dose PMR reduced the activity of diacylglycerol acyltransferase (DGAT) to 1536 plusmn 4769 pgmL(119875 lt 0001) and promoted the expression of hepatic lipase (HL) to 2359 plusmn 02758UmL (119875 lt 005) HL promotion ability ofmedium dose PMR was similar with the Ssimvastatin positive control Both medium and high dose of PMR showed significantalterations in TC which were related to the downregulation effects on hydroxyl methyl-glutaryl coenzyme A reductase (HMGCR)and upregulation effects on cholesterol 7-alpha-hydroxylase or cytochrome P450 7A (CYP7A) Quantitative relationships researchindicated that the prominent effect on inhibiting the content of HMGCR (119903 = 0756 119875 lt 005) was strongly positive correlatedwith to the TC regulation effects Effects of PMR on enhancing decomposition rate or reducing de novo synthesis rate of TG andTC were better than PMRP

1 Introduction

Fatty liver disease (FLD) a kind of lipid metabolic disorderof liver is a reversible condition in which large vacuolesof triglyceride fat accumulate in liver cells via the processof steatosis (abnormal retention of lipids within a cell)According to the different inducements of fatty liver FLD isdivided into alcoholic fatty liver disease (AFLD) and nonal-coholic fatty liver disease (NAFLD) NAFLD is increasinglyrecognized as the hepatic manifestation of insulin resistanceand the systemic complex known as metabolic syndrome [12] NAFLD is the most common form of chronic liver diseasein adults in the United States Australia Asia and Europe[3ndash5] NAFLD is also gaining recognition as a significantearly sign of liver cirrhosis and liver cancer [6] prevalenceestimates of NAFLD have used a variety of laboratory andimaging assessments1

In NAFLD pathogenic process the accumulation oflipid within the liver especially total cholesterol (TC) andtriglycerides (TG) accumulation has been confirmed bydynamics research These researches point out that TC TG

and esterification of free fatty acid (FFA) accumulation in theliver for the treatment of NAFLDmight have direct influence[7 8]

Polygoni Multiflori Radix (PMR Heshouwu in Chi-nese) and Polygoni Multiflori Radix Praeparata (PMRPZhiheshouwu in Chinese) are originated from the root ofPolygonum multiflorum Thunb (Polygonaceae) (Figure 1)They mainly contain anthraquinone stilbene glycosidesphospholipids and other ingredients and are used in theprevention and treatment of NAFLD hyperlipidemia orrelated diseases in oriental counties for centuries [9]

Previously a sensitive accurate and rapid in vitromodel(the steatosis L02 cells obtained after being cultured with1 fat emulsion 10 fetal bovine serum (FBS) and RPMI1640 medium for 48 h) was used in our research groupto investigate the lipid regulation effects of 2 3 5 41015840-tetrahydroxystilbene-2-O-120573-D-glucoside (TSG) emdin andphyscion [10] Hereafter in vivomodel (high-fat diet fed rats) 2was applied to explore the lipid regulation effects of extractsof PMR and PMRP [11] The results show that both the raw

2 Evidence-Based Complementary and Alternative Medicine

Hand drawing

(a)

PMR

(b)

PMRP

(c)

Figure 1 Photographs of Polygoni Multiflori Radix and its processed products

TG

FFA

VLDL

TG

VLDL

HDL

Acetyl-CoA

TC

HDLTC

CACDCA

TC

CYP7A

LDL

LDL

TC

FFA

HL

HMGCR

DGAT

Figure 2 Lipid synthesis and lipolysis procedure of TG and TC

Group A

(a)

Group B

(b)

Group D

(c)

Group I

(d)

Figure 3 Liver tissue samples Group A normal control group Group B model group Group D water extraction group of PMR (mediumdose) Group I positive control group (simvastatin)

Evidence-Based Complementary and Alternative Medicine 3

0

10

20

30

40

50

60

70

80

Groups

A

B

C

D

E

F

G

H

I

J

lowastlowastlowast

VL

DL

(120583

gm

L)

Figure 4 The content of VLDL of liver homogenate in all groupsThe lowast indicates a significant difference compared with controlgroup lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 The indicatesa significant difference compared with model group

119875 lt 005119875 lt 001 and

119875 lt 0001

crude drug and its processed products have significant lipid-lowering activities however obvious target organ selectivitywas found PMR was considered to possess better effectsin lipid regulation in liver sample and was recommended3for the treatment of early stage NAFLD On the other

4 hand PMRP displayed better effects in lipid regulation inblood circulation system for the treatment of hyperlipidemiaHowever the lipid regulation mechanism of P multiflorumis still not clearly elucidated So we chose the following fourkey enzymes as the investigation objectives in this paper(Figure 2)

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR)5most abundantly expressed in the liver plays a central role in

6 the regulation of TC concentration HMGCR is a key enzymecatalyzing cholesterol in de novo synthesis pathway in vivoHMGCR activity directly affects the speed of cholesterol syn-thesis and the level of cholesterol [12] Clinical results confirmthat HMGCR inhibitor reduced plasma concentrations of TCand TG low density lipoprotein (LDL) and very low densitylipoprotein (VLDL) and increased plasma concentrations ofhigh density lipoprotein (HDL) Therefore the inhibition ofthis enzyme could contribute to reduction of synthesis ofcholesterol

Cholesterol 7a-hydroxylase (CYP7A) is the first andrate-limiting enzyme in bile acid synthesis pathway and isexpressed only in the liver Lack of CYP7A results in highlevels of plasma cholesterol whereas induction of CYP7Aprevents elevation of blood cholesterol in rodents fed by acholesterol-rich diet indicating its importance inmaintainingplasma cholesterol homeostasis CYP7A is tightly regulated

by feedforward of cholesterol and negative feedback of bileacids [13]

The diacylglycerol acyltransferase (DGAT) is rate-limiting enzyme for triglyceride synthesis DGAT catalyzesthe final step in TG biosynthesis by converting diacylglycerol(DAG) and fatty acyl-coenzyme A into TG [14]

Hepatic lipase (HL) is a lipolytic enzyme that contributesto the regulation of TG levels Hepatic lipase facilitates theclearance of TG from VLDL pool and this function is gov-erned by the composition and quality of HDL particles HDLregulates the release of HL from the liver and HDL structurecontrols HL transport and activation in the circulation [15]HL could catalyze the chylomicrons (CM) and promote thehydrolysis of triglycerides in VLDL

Based on the key role of above enzyme in the lipidmetabolism we used the in vivo model (high-fat diet fed 7rats) to investigate lipid regulation mechanisms and possibleregulatory targets of TC and TG by RPM and RPMP furtherand systematically

2 Materials and Methods

21 Samples [11] PMR was collected in Luquan Yunnanby the authors The plants were identified as the root ofPolygonum multiflorum Thunb by Prof Rong-hua ZhaoYunnan University of Traditional Chinese Medicine PMRPwas made by PMR with black bean decoction accordingto the method recorded in the Pharmacopoeia of PeoplersquosRepublic of China [8] Processed products were detectedby high liquid chromatography (HPLC) the content ofstilbene glycoside was greater than 070 in accordance withthe Pharmacopoeia standards while the content of stilbeneglycoside of raw products was greater than 10

22 Preparation of Extraction of RPM and RPMP [11] 300 gpowder of PMR and 472 g powder of PMRP were extractedfor 1 hr with 10 times boiling water Then the residue wasextracted for 40min with 10 8 and 6 times volume boilingwater respectively Extracts were combined condensed andlyophilized The concentrations of PMR and PMRP extractswere 06980 gmL and 08580 gmL respectively

23 Animals Groups [11] SD rats were provided by theExperimental Animal Center of Yunnan University of Tra-ditional Chinese Medicine They were aged 8 weeks andweighed 245 plusmn 20 g and were acclimated in the controlledenvironment (temperature 22 plusmn 1∘C 60 plusmn 10 humidityand a 12 h12 h lightdark cycle) with free access to waterand a commercial laboratory complete food All animalexperiments were performed in compliance with the AnimalExperimental Ethics Committee of Yunnan University ofTraditional Chinese Medicine All reasonable efforts weremade to minimize the animalsrsquo suffering

120 SD male rats were randomly divided into 10 groups(Table 1) normal control group (A) model group (B) waterextraction group of PMR (low medium and high dosegroups C D and E) water extraction group of PMRP (lowmedium and high dose groups F G and H) and positive


A

B

C

D

E

F

G

H

I

J

1200

1300

1400

1500

1600

1700

1800

1900

2000

lowastlowast

lowastlowast

lowastlowast

lowast lowastlowastlowast lowastlowastlowast

lowast

DG

AT

(p

gm

L)

Groups

(a)

A

B

C

D

E

F

G

H

I

J

lowastlowast

lowast lowast

lowast lowast lowast

16

18

20

22

24

26

HL

(U

mL

)

Groups

(b)

Figure 5 The activity of key enzymes of TG metabolism The lowast indicates a significant difference compared with control group lowast119875 lt 005lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 The indicates a significant difference compared with model group

119875 lt 005 119875 lt 001 and

119875 lt 0001

HM

GC

R (

ng

mL

)

4000

3500

3000

2500

2000

1500

1000

500

lowastlowast

lowastlowast lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Groups

JIHGFEDCBA

(a)

Groups

JIHGFEDCBA

CY

P7

A (

pg

mL

)

140000

130000

120000

110000

100000

90000

(b)

Figure 6 The activity of key enzymes of TC metabolism The lowast indicates a significant difference compared with control group lowast119875 lt 005lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 The indicates a significant difference compared with model group

119875 lt 005 119875 lt 001 and

119875 lt 0001

control groups (fenofibrate and simvastatin control I and J)In addition to the normal control group other groups werefed with a high-fat diet (containing 1 cholesterol 10 lard02 methyl thiouracil and 888 usual feed) to the end ofthe experiment (42 days)

24 Drug Delivery Process after the Success of Modeling [11]After giving high-fat diet for 18 days group C to H receivedthe PMR and PMRP treatments till 42 days the end ofthe research In the meantime normal control group andhyperlipidemia model group were given 09 saline 1mLPositive groups I and J received 0033 gsdotkgminus1 fenofibrate

and 00012 gsdotkgminus1 simvastatin daily respectively The lowdose group of PMR and PMRP was given 0405 gsdotkgminus1 and0810 gsdotkgminus1 daily [15] the middle and high dosages of PMRandPMRPwere 2 and 4 times of the lowdosages respectivelyAll rats were fasted for 2 h every day before administration oftherapeutic agents (Table 1)

25 The Preparation and Detection of Animal LiverHomogenate Rats were sacrificed by cervical dislocationLiver samples were collected (Figure 3) and weighed afterwashing with 09 saline 100mg tissues were rinsed withPBS and homogenized in 1mL of PBS and then stored


Table 1 Animal grouping and treatments in this research

Groups DietsTreatment

(from the nineteenth day of theexperiment)

Dosage(gkg body weight)

A Normal diets Physiological saline 1mL per ratB High-fat diets Physiological saline 1mL per ratC High-fat diets Water extraction of RPM 04050D High-fat diets Water extraction of RPM 08100E High-fat diets Water extraction of RPM 1620F High-fat diets Water extraction of RPMP 08100G High-fat diets Water extraction of RPMP 1620H High-fat diets Water extraction of RPMP 3240I High-fat diets Simvastatin 0001200J High-fat diets Fenofibrate 003300

Table 2 Lipid indexes in the liver samples

Groups TC (mgdL) TG (mgdL) LDL-C (mgdL)A 6663 plusmn 4093 14722 plusmn 6180 1074 plusmn 2186

B 1002 plusmn 1922lowastlowastlowast 2000 plusmn 3256lowastlowastlowast 2836 plusmn 1257lowastlowast

C 1058 plusmn 1501lowastlowastlowast 1798 plusmn 1856lowastlowastlowast 2879 plusmn 7821lowastlowastlowast

D 8771 plusmn 1719lowastlowast 1808 plusmn 1594lowastlowastlowast 2449 plusmn 6547lowastlowastlowast

E 5718 plusmn 6754lowastlowast 1536 plusmn 2734 2486 plusmn 4385lowastlowastlowast

F 6629 plusmn 2808 1621 plusmn 3988 3839 plusmn 1853lowastlowastlowast

G 8948 plusmn 1875lowastlowast 2058 plusmn 2990lowastlowastlowast 4898 plusmn 1202lowastlowastlowast

H 6982 plusmn 2430 1650 plusmn 3211 4189 plusmn 1649lowastlowastlowast

I 4367 plusmn 2936lowastlowastlowast 1508 plusmn 1882 9990 plusmn 3548

J 4466 plusmn 5379lowastlowastlowast 1958 plusmn 2596lowastlowastlowast 1579 plusmn 9505

The (lowast) indicates a significant difference compared with control group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001The () indicates a significant difference compared with model group 119875 lt 005 119875 lt 001 and

119875 lt 0001

overnight at minus20∘C After two freeze-thaw cycles wereperformed to break all cell membranes the homogenateswere centrifuged for 5 minutes at 5000 g 2ndash8∘C Thesupernatants were collected and analysed immediately

Contents of TC TG LDL and HDL in the supernatantwere measured by AB-1020 automatic biochemical analyzer(Sunostik Medical Technology Co Ltd) and assay kits pur-chased from Shanghai Rongsheng Biological PharmaceuticalCo Ltd and SichuanMaker Biotechnology Co Ltd China

The VLDL DGAT HMGCR HL and CYP7A contentswere tested by assay kits purchased fromCusabio BiotechCoLtd China

26 Statistical Analysis All data in this research wereexpressed in the form of mean plusmn SD All data were analyzedby single factor analysis of variance (ANOVA) statistics andthe test results of 119875 lt 005 119875 lt 001 and 119875 lt 0001 as astatistically significant difference criterion

Relationships between these enzymes and proteins andTG and TC were assessed with Pearsonrsquos correlation coeffi-cient Results were classified into two significance levels usingthe 119875 value of 005

3 Results

31 Effects of Lipid Regulation Using Raw and ProcessedRadix Polygoni Multiflori [11] Morphologic observationswere carried in every group Livers in high-diet fed groupwere obviously smaller and paler than normal livers Treat-ment of simvastatin PMR and PMRP relieved the steatosisprocedure however none of these treatments could reverse it(Figure 3)

As listed in Table 2 TC TG and LDL-C in liver tissuewere all significantly higher than in model rats Both PMRand PMRP revealed TC-lowering effects however dose-dependent TC- and TG-lowering effects were observed onlyin PMR groups

32 Effects of RPM and RPMP on VLDL in Liver Samples 42days of high-fat diet intake significantly increased the liverVLDL level from 3322plusmn6445 ugmL to 6436plusmn6455 ugmL(119875 lt 0001) in rats Fortunately the liver VLDL levels werereduced in RPM RPMP and positive control drugs Fromcomparison of PMR groups and PMRP groups PMR wasbetter in reducing VLDL than PMRP Low dosage of PMRcould reduce the content of VLDL to 3520 plusmn 1503 ugmLwhich was similar to the normal group VLDL-lowering


Table 3 Contents of TC HMGCR and CYP7A in every group

TC (mgdL) HMGCR (ngmL) CYP7A (pgmL)A 6663 plusmn 4093 2109 plusmn 1687 1178 plusmn 4900

B 1002 plusmn 1922lowastlowastlowast 3250 plusmn 2533lowastlowastlowast 1110 plusmn 5464

C 1058 plusmn 1501lowastlowastlowast 2773 plusmn 1994lowastlowast 1191 plusmn 3396

D 8771 plusmn 1719lowastlowast 1435 plusmn 2389lowastlowast 1247 plusmn 8746

E 5718 plusmn 6754lowastlowast 1401 plusmn 1655lowastlowastlowast 1203 plusmn 7391

F 6629 plusmn 2808 2284 plusmn 2464 1180 plusmn 6437

G 8948 plusmn 1875lowastlowast 2788 plusmn 0920lowastlowastlowast 1161 plusmn 7016

H 6982 plusmn 2430 2676 plusmn 3432lowast 1212 plusmn 10359

I 4367 plusmn 2936lowastlowastlowast 1277 plusmn 2050lowastlowastlowast 1222 plusmn 2774



119875 lt 0001

Table 4 Relationships between enzymes and proteins and TG and TC

Correlation coefficient and significance Pearsonrsquos correlation coefficient Significance (119875)

TCLDL 0487 0153

HMGCR 0756 0011CYP7A minus0453 0189

TGVLDL 0490 0150DGAT 0192 0596HL minus0019 0958


119875 lt 0001

effects were even better than the positive control groups(Figure 4)

33 Expression of Key Enzyme of Triglyceride (TG)Metabolism The activities of DGAT and HL wereinvestigated as the key enzymes of TG metabolism

Considering the two indexes in Figure 5 the effects ofaqueous extract of PMR on enzyme activity were better thanthose of PMRP Medium dose group of PMR showed moreprominent effect they reduced the activity of DGAT from1776 plusmn 5044 pgmL to 1536 plusmn 4769 pgmL and promotedthe expression of HL to 2359 plusmn 02758UmL HL expressionregulation ability of PMR was even similar to the positivecontrol group The way of PMR to regulate the TG contentin the liver was to reduce the activity of DGAT and promotethe expression of HLThe regulations of DGATwere not veryobvious in all PMRP groups while the promotion effects ofHL were much better

34 Expression of Key Enzyme of Total Cholesterol (TC)Metabolism TC level in high-fat diet group was signif-icantly higher than that in the control group (Table 3)no TC melioration effect was observed in the low dosagePMR group however both the middle (group D) and highdosage (group E) PMR groups showed significant changesin TC regulation These TC regulation effects were relatedto the downregulation on HMGCR and upregulation onCYP7A Comparing the expression of these two key enzymes

HMGCR played a leading role in TC metabolism the abilityof middle and high dosage PMR was similar to the positivecontrol group In the meantime all dosage groups of RPMPshowed the TC melioration effect however these are notbetter than PMR

35 Relationships between Enzymes and Proteins and TC andTG Pearsonrsquos correlation coefficients between these enzymesand proteins and TG and TC were displayed in Table 4HMGCR activities (119903 = 0756 119875 lt 005) were stronglypositive correlated with TC Moreover LDL was positivecorrelated with TC CYP7A was negative correlated with TCand VLDL was positive correlated with TG However we didnot find any significant relationship between DGAT and HLand TG regulation effects

4 Discussion and Conclusion

Liver was the main organ responsible for lipid metabolismLiver cells played important roles in lipid uptake transportmetabolism and excretion Generally the fatmetabolism dis-order leaded to liver steatosiswhichwas the initial step of fattyliver disease Fat metabolism related to fatty liver inducedtriglyceride metabolism cholesterol metabolism phospho-lipid metabolism and priority to triglycerides metabolicdisorder

However there was no specific drug for NAFLD treat-ment Considering the effectiveness and acceptable pricesof traditional Chinese medicine (TCM) the prevention and


treatment of NAFLD and hyperlipidemia by TCM were aresearch hotspot PMR and PMRP which displayed greatclinical effect in treating NAFLD seemed to be potentialchoices

Our research group had focused on the lipid regulationeffect of PMR and PMRP for decades we had confirmed thegreat lipid reducing effect of RPM and RPMP by both invitro [10] and in vivo [11] assays However the lipid regulationmechanism of P multiflorum was still not clearly elucidatedSo in this research we chose four key enzymes DGAT HLHMGCR and CYP7A as the major objectives to seek thepossible regulatory targets in the metabolisms of TC and TGby RPM and RPMP

After giving high-fat diet for 42 days we found that TCTG LDL and VLDL in liver tissue were all significantlyhigher than in model rats DGAT and HMGCR in modelgroup were upregulated and CYP7A and HL were all down-regulated These showed that the method of the rats inducedinto NAFLD was successful

Compared with model group PRM and PRMP hadgood lipid-lowering effects they could not only enhancedecomposition activity but also reduce synthase activity TCand TGmelioration effects of PRMwere better In particularmiddle dose group of RPM showedmore prominent effect oninhibiting the content of HMGCR and DGAT which couldblock the synthesis of TC and TG respectively Key enzymeregulation ability of PMR was similar to the positive controlgroup At the same time low dose group of RPM showed thebest VLDL-reducing effect and the VLDL-reducing effect ofRPMP was in a dose-dependent manner

According to the analysis of Pearsonrsquos correlation coef-ficients HMGCR activity (119903 = 0756 119875 lt 005) wasstrongly positive correlated with TC regulation effects Thiswas consistent with the above results In a conclusion TCregulation of PMR was mediated by HMGCR In someextension PMR showed similar activities and mechanismswith statins We also found the better TG regulation effectsin some dosage of PMR and RPMP however there was nosignificantly relationship between DGAT HL VLDL andTG Therefore we assumed that the regulation on TG mightbe related to some other mechanisms or targets

Previous researches [16 17] pointed out that water extractand total glycosides of PMR had shown a good lipid-lowering activity in animal experiments they could reducethe contents of the TG and TC in rats caused by high-fat dietMoreover the effect of total glycosides on reducing the TG inhyperlipidemia rat lack of Apo E gene was even better thanthe positive drugs These were in line with our results thathigh dosage of PMR had the similar TG-lowering effect withstatins

Other researches [18 19] consider that the lipid-loweringactivity of PMR was associated with the inhibition ofHMGCR the reduction of VLDL and LDL contents andthe decreasing of TG and TC absorption In this paperwe reported the effects on the activity of DGAT HL andCYP7A for the first time This research could contribute toour knowledge on the lipid regulation mechanism of PMR inhigh-fat diet fed rats

TG content in the liver was affected not only by theamount directly absorbed from the food but also by the denovo synthesis by FFA [8 20]Therefore whether FFA supplychainwas affected by PMRandPMRP will be in great worthy 8of study in the future On the other hand previous litera-tures [21 22] also showed that insulin resistance was oftenobserved in the NAFLD patients The increasing of insulinconcentration could reduce the oxidation and decompositionrate of TG so that TG content in the cells would increase [23]Therefore whether PMR and PMRP could control the insulinresistance status in NAFLD patients will be a subject also in 9our future research plan

Abbreviations

CM ChylomicronsCYP7A Cholesterol 7-alpha-hydroxylase or

cytochrome P450 7ADAG DiacylglycerolDGAT Diacylglycerol acyltransferaseFBS Fetal bovine serumFFA Free fatty acidFLD Fatty liver diseaseHMGCR 3-Hydroxy-3-methylglutaryl-CoA

reductaseHDL High density lipoproteinHL Hepatic lipaseHPLC High liquid chromatographyLDL Low density lipoproteinNAFLD Nonalcoholic fatty liver diseasePMR Polygoni Multiflori RadixPMRP Polygoni Multiflori Radix PraeparataRPMI 1640 Roswell Park Memorial Institute medium

1640TC Total cholesterolTCM Traditional Chinese medicineTG TriglycerideTSG 2 3 5 41015840-Tetrahydroxystilbene-2-O-120573-D-

glucosideVLDL Very low density lipoprotein

Conflict of Interests

The authors declare that there is no conflict of interests Theydeclare that they have no financial and personal relationshipswith other people or organizations that can inappropriatelyinfluence their work there are no professional or otherpersonal interests of any nature or kind in any productservice or company that could be construed as influencingthe position presented in this paper

Acknowledgments

This research was financially supported by the NationalNatural Science Foundation of China (Grant no 30760312and Grant no 81060337) the Key Public Project of Ministryof Science and Technology and Ministry of Finance China


(Grant no 201107007-08) and the Natural Science Founda-tion of Yunnan (Grant no 2010ZC105)10

References

[1] G Marchesini M Brizi G Blanchi et al ldquoNonalcoholic fattyliver disease a feature of themetabolic syndromerdquoDiabetes vol50 no 8 pp 1844ndash1850 2001

[2] G Marchesini E Bugianesi G Forlani et al ldquoNonalcoholicfatty liver steatohepatitis and the metabolic syndromerdquo Hep-atology vol 37 no 4 pp 917ndash923 2003

[3] McCullough and J M D Arthur ldquoUpdate on nonalcoholic fattyliver diseaserdquo Journal of Clinical Gastroenterology vol 34 no 3pp 255ndash262 2002

[4] G C Farrell ldquoNon-alcoholic steatohepatitis what is it andwhy is it important in the Asia-Pacific regionrdquo Journal ofGastroenterology and Hepatology vol 18 no 2 pp 124ndash1382003

[5] C E Ruhl and J E Everhart ldquoRelation of elevated serumalanineaminotransferase activity with iron and antioxidant levels in theUnited Statesrdquo Gastroenterology vol 124 no 7 pp 1821ndash18292003

[6] J B Schwimmer C Behling R Newberry et al ldquoThe his-tological features of pediatric nonalcoholic fatty liver dis-ease(NAFLD)rdquo Hepatology vol 36 p 412 2002

[7] G Musso R Gambino and M Cassader ldquoRecent insightsinto hepatic lipid metabolism in non-alcoholic fatty liver dis-ease(NAFLD)rdquoProgress in Lipid Research vol 48 no 1 pp 1ndash262009

[8] Y Li C H Gu and W Zhang ldquoThe research progress ofthe pathogenesis of non-alcoholic fatty liver diseaserdquo ChineseJournal of Coal Industry Medicine vol 10 pp 1648ndash1650 2009

[9] Commission of Chinese Pharmacopoeia Pharmacopoeia of thePeoplersquos Republic of China vol 1 ChinaMedico-PharmaceuticalScience amp Technology Publishing House Beijing China 2010

[10] M J Wang R H Zhao W G Wang et al ldquoLipid regulationeffects of Polygoni Multiflori Radix its processed products andits major substances on steatosis human liver cell line L02rdquoJournal of Ethnopharmacology vol 139 no 1 pp 287ndash2293 2012

[11] N Li Z Chen X J Mao et al ldquoEffects of lipid regulationusing raw and processed Polygoni Multiflori Radix in rats feda high-fat dietrdquo Evidence-Based Complementary and AlternativeMedicine vol 2012 Article ID 329171 10 pages 2012

[12] G Ji X Zhao L Leng P Liu and Z Jiang ldquoComparisonof dietary control and atorvastatin on high fat diet inducedhepatic steatosis and hyperlipidemia in ratsrdquo Lipids in Healthand Disease vol 10 article 23 2011

[13] C Juree N Kasem B William et al ldquoInduction of humancholesterol 7a-hydroxylase in HepG2 cells by 2 4 6 -trihydroxyacetophenonerdquo European Journal of Pharmacologyvol 515 no 1ndash3 pp 43ndash46 2005

[14] Y Wang J L Deng Z C Zuo et al ldquoEffect of INS and GLP onabundance of DGAT2mRNA in vitri culture bovine hepatocytecellsrdquo Chinese Journal of Veterinary Science vol 32 no 11 pp1716ndash1719 2012

[15] C Chatterjee and L Daniel ldquoHepatic lipase high densitylipoproteins and hypertriglyceridemiardquo The American Journalof Pathology vol 178 no 4 pp 1429ndash1433 2011

[16] X SWangG Y Xie X L Shi et al ldquoThe effect of different kindsof Chinese herb medicine such as Radix Polygoni Multiflori on

blood biochemical indexes in rats of fatty liverrdquo Journal of AnhuiTraditional Chinese Medical College vol 5 pp 39ndash40 2006

[17] W Fang Y W Qin L Y Wang et al ldquoThe protecting effect ofPMTGon the atherosclerotic lesion formationrdquoChinese Journalof Drug Application and Monitoring vol 1 pp 48ndash51 2005

[18] W X Gao Y J Hu and L C Fu ldquoLipid regulation effect of TSGof Polygonum Multiflorum Radixrdquo China Journal of ChineseMaterial Medica vol 4 pp 323ndash326 2007

[19] X Han C A Wu WWang et al ldquoLipid regulation mechanismof TSG of Polygonum Multiflorum Radixrdquo Chinese Archives ofTraditional Chinese Medicine vol 8 pp 1687ndash1689 2008

[20] J G Fan and Z J Xu ldquoDiscuss treatment measures ofnon-alcoholic steatohepatitis from the pathogenesisrdquo ModernMedicine amp Health vol 20 no 2 pp 75ndash76 2004

[21] J C Cohen J D Horton and H H Hobbs ldquoHuman fatty liverdisease old questions and new insightsrdquo Science vol 332 no6037 pp 1519ndash1523 2011

[22] A P Lin and J Yu ldquoResearch on diabetic patients with non-alcoholic fatty liverrdquo Progress inModern Biomedicine vol 10 no19 pp 3762ndash3764 2010

[23] H Li L Xue Y Song et al ldquoThe effect of insulin on triglyceridessynthesis and lipolysis of mice primary hepatocytesrdquo Progress inModern Biomedicine vol 3 pp 401ndash404 2013


Composition Comments

1 Please provide a short description (in paragraph style)

of the Supplementary Material This description along with

the Supplementary Material will be available online

2 We made the highlighted changes in the footer of

Table 2 as per journal style Please check similar cases

throughout

3 We made the highlighted change(s) for the sake of

clarity and correctness Please check

4 Please specify what the asterisk double asterisks and

triple asterisks in the footer of Table 4 refer to

5 There is a difference between the manuscript and the

new electronic version in Figure 2 and we followed the new

electronic version Please check

6 We redrew some parts in Figure 2 Please check

7 Please specify what the and in the footer

of Table 4 refer to





10 Please refer to Figure 6 in the text as per journal style

Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6


Hand drawing

(a)

PMR

(b)

PMRP

(c)

Figure 1 Photographs of Polygoni Multiflori Radix and its processed products

TG

FFA

VLDL

TG

VLDL

HDL

Acetyl-CoA

TC

HDLTC

CACDCA

TC

CYP7A

LDL

LDL

TC

FFA

HL

HMGCR

DGAT

Figure 2 Lipid synthesis and lipolysis procedure of TG and TC

Group A

(a)

Group B

(b)

Group D

(c)

Group I

(d)

Figure 3 Liver tissue samples Group A normal control group Group B model group Group D water extraction group of PMR (mediumdose) Group I positive control group (simvastatin)


0

10

20

30

40

50

60

70

80

Groups

A

B

C

D

E

F

G

H

I

J

lowastlowastlowast

VL

DL

(120583

gm

L)


119875 lt 005119875 lt 001 and

119875 lt 0001
















A

B

C

D

E

F

G

H

I

J

1200

1300

1400

1500

1600

1700

1800

1900

2000

lowastlowast

lowastlowast

lowastlowast


lowast

DG

AT

(p

gm

L)

Groups

(a)

A

B

C

D

E

F

G

H

I

J

lowastlowast

lowast lowast


16

18

20

22

24

26

HL

(U

mL

)

Groups

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001

HM

GC

R (

ng

mL

)

4000

3500

3000

2500

2000

1500

1000

500

lowastlowast

lowastlowast lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Groups

JIHGFEDCBA

(a)

Groups

JIHGFEDCBA

CY

P7

A (

pg

mL

)

140000

130000

120000

110000

100000

90000

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001























119875 lt 0001






3 Results

















119875 lt 0001



TCLDL 0487 0153




119875 lt 0001



















Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6


0

10

20

30

40

50

60

70

80

Groups

A

B

C

D

E

F

G

H

I

J

lowastlowastlowast

VL

DL

(120583

gm

L)


119875 lt 005119875 lt 001 and

119875 lt 0001
















A

B

C

D

E

F

G

H

I

J

1200

1300

1400

1500

1600

1700

1800

1900

2000

lowastlowast

lowastlowast

lowastlowast


lowast

DG

AT

(p

gm

L)

Groups

(a)

A

B

C

D

E

F

G

H

I

J

lowastlowast

lowast lowast


16

18

20

22

24

26

HL

(U

mL

)

Groups

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001

HM

GC

R (

ng

mL

)

4000

3500

3000

2500

2000

1500

1000

500

lowastlowast

lowastlowast lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Groups

JIHGFEDCBA

(a)

Groups

JIHGFEDCBA

CY

P7

A (

pg

mL

)

140000

130000

120000

110000

100000

90000

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001























119875 lt 0001






3 Results

















119875 lt 0001



TCLDL 0487 0153




119875 lt 0001



















Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6


A

B

C

D

E

F

G

H

I

J

1200

1300

1400

1500

1600

1700

1800

1900

2000

lowastlowast

lowastlowast

lowastlowast


lowast

DG

AT

(p

gm

L)

Groups

(a)

A

B

C

D

E

F

G

H

I

J

lowastlowast

lowast lowast


16

18

20

22

24

26

HL

(U

mL

)

Groups

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001

HM

GC

R (

ng

mL

)

4000

3500

3000

2500

2000

1500

1000

500

lowastlowast

lowastlowast lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Groups

JIHGFEDCBA

(a)

Groups

JIHGFEDCBA

CY

P7

A (

pg

mL

)

140000

130000

120000

110000

100000

90000

(b)


119875 lt 005 119875 lt 001 and

119875 lt 0001























119875 lt 0001






3 Results

















119875 lt 0001



TCLDL 0487 0153




119875 lt 0001



















Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6



















119875 lt 0001






3 Results

















119875 lt 0001



TCLDL 0487 0153




119875 lt 0001



















Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6














119875 lt 0001



TCLDL 0487 0153




119875 lt 0001



















Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6










Abbreviations








Acknowledgments




References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6



References
































throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6








throughout










of Table 4 refer to






Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6

Author(s) Name(s)

Author 1

Author 2

Author 3

Author 4

Author 5

Author 6

In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats

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Transcript of In Vivo Lipid Regulation Mechanism of Polygoni Multiflori Radix in High-Fat Diet Fed Rats