wentong huoxue cream can inhibit the reduction of the pain

Research ArticleWenTong HuoXue Cream Can Inhibit the Reduction ofthe Pain-Related Molecule PLC-1205733 in the Dorsal Root Ganglionof a Rat Model of Diabetic Peripheral Neuropathy

Chengcheng Feng1 Lijuan Xu2 Shiyun Guo2 Qian Chen1 Yuguo Shen1

Deng Zang2 and Li Ma 1

1Traditional Chinese Medicine Hospital Xinjiang Medical University Urumqi China2Xinjiang Medical University Urumqi China

Correspondence should be addressed to Li Ma lvgangxj163com

Received 11 September 2017 Revised 4 January 2018 Accepted 16 January 2018 Published 11 February 2018

Academic Editor I-Min Liu

Copyright copy 2018 Chengcheng Feng et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

WenTong HuoXue Cream (WTHX-Cream) has been shown to effectively alleviate clinical symptoms of diabetic peripheralneuropathy (DPN)This study investigated the gene and protein expression of the pain-related molecule PLC-1205733 in the dorsal rootganglion (DRG) of DPN rats 88 specific pathogen-free male Wistar rats were randomly divided into placebo (10 rats) and DPNmodel (78 rats) groups and the 78 model rats were used to establish the DPN model by intraperitoneal injection of streptozotocinand were then fed a high-fat diet for 8 weeksThese rats were randomly divided into themodel group the high- medium- and low-dose WTHX-Cream + metformin groups the metformin group the capsaicin cream group and the capsaicin cream + metformingroup After 4 weeks of continuous drug administration the blood glucose body weight behavioral indexes and sciatic nerveconduction velocity were measured The pathological structure of the DRG and the sciatic nerve were observed PLC-1205733 mRNAand protein levels in the DRG of rats weremeasured Compared with themodel group the high-doseWTHX-Cream group showedincreased sciatic nerve conduction velocity improved sciatic nerve morphological changes and increased expression of PLC-1205733mRNA and protein in the DRGThis study showed that WTHX-Cream improves hyperalgesia symptoms of DPN by inhibiting thereduction of PLC-1205733 mRNA and protein expression in the diabetic DRG of DPN rats

1 Introduction

Diabetic peripheral neuropathy (DPN) is a common chroniccomplication of diabetes and involves distal symmetricalneuropathy (major manifestation) numbness pain hyper-algesia and myasthenia The neuropathic pain generatedby DPN severely impacts the quality of life of diabeticpatients [1] Currently DPN treatment includes improvingmicrocirculation promoting neurotrophy and relieving pain[2] However due to the multifactor interactive pathogenesisof DPN clinical drugs targeting a single pathway have beenshown to have poor curative effects [3] Traditional Chinesemedicine has the advantages of multiple components andmultiple targets and thus may be valuable in treating DPNWTHX-Cream (a local heating cream for improving blood

perfusion) is produced based on the theory of traditionalChinese medicine and consists of seven traditional Chi-nese herbs Chuanxiong safflower Ramulus CinnamomiAsarum Angelica Allolobophora caliginosa trapezoids andpepper this treatment is believed to promote blood circu-lation and eliminate stasis External use of this cream onthe affected region is believed to activate the blood resolvestasis and dredge collaterals to relieve pain Our previousstudy showed that WTHX-Cream can effectively relieve thesymptoms of DPN and reduce allergic pain [4]

Pain can be divided into nociceptive pain and neu-ropathic pain (or mixed pain of the two types of pain)DPN is classified as neuropathic pain and its pathologicalmechanism is complex phospholipase C-1205733 (PLC-1205733) playsan important role in pain occurrence PLC-1205733 belongs to the

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 3416936 10 pageshttpsdoiorg10115520183416936

2 Evidence-Based Complementary and Alternative Medicine

phospholipase molecule C family which contains a variety ofmembers including PLC-120573 -120574 -120575 and -120576 PLC-120573 includesPLC-1205731 PLC-1205732 PLC-1205733 and PLC-1205734 PLC-1205731 PLC-1205733and PLC-1205734 are distributed in the dorsal root ganglion(DRG) and PLC-1205733 has the highest transcription levels[5] Many studies have confirmed that PLC-1205733 is associatedwith pain Related studies have shown that in spared nerveinjury the PLC-1205733 content in the DRG of the same side wassignificantly decreased but did not change on the noninjuryside indicating that the PLC-1205733 level of the DRG afterperipheral nerve injury is reduced [6]

In combinationwith analysis of the pain perception trans-mission mechanism histopathology real-time quantitativePCR and immunoblotting were used to explore the effects ofWTHX-Cream on DPN based on histopathological changesand pain-related molecule PLC-1205733 mRNA and protein levelchanges This study aimed to provide a theoretical basis andguidance for the clinical treatment of DPN with WTHX-Cream and to develop new ideas for the external use ofChinese traditional medicine in the treatment of DPN

2 Materials and Methods

21 Animals 88 specific pathogen-free male Wistar ratsweighing 80ndash130 g were purchased from Xinjiang Experi-mental Animal Research Center (production license num-ber SCXK (new) 2011-0001 animal certificate number65000200000408) Rats were fed separately with 5 rats ineach cage Humidity was controlled within the range of30ndash50 and temperature was controlled within 20ndash26∘CRats were fed for 1 week under natural light with freelyavailable food and drinking water and they were randomlydivided into the placebo (10 rats) and DPN model (78 rats)groups according to the random number table methodPlacebo cream was applied to rats in the placebo group andthey were given distilled water via gavageThe remaining ratsin the DPN model group were injected with streptozotocin(STZ) and were fed a high-fat diet for 8 weeks to developthe DPN model The DPN model groups were subdividedinto the model group (placebo cream + lavage with distilledwater 11 rats) high-dose WTHX-Cream + metformin group(high-dose WTHX-Cream + lavage with metformin 11 rats)medium-dose WTHX-Cream +metformin group (medium-dose WTHX-Cream + lavage with metformin 11 rats) low-dose WTHX-Cream + metformin group (low-dose WTHX-Cream + lavage with metformin 11 rats) metformin group(placebo cream + lavage with metformin 10 rats) capsaicingroup (capsaicin cream + lavage with distilled water 10 rats)and capsaicin cream + metformin group (capsaicin cream +lavage with metformin 11 rats)

22 Drug WTHX-Cream is a Chinese drug preparationcontaining Chuanxiong safflower Ramulus CinnamomiAsarum Angelica Allolobophora caliginosa trapezoids andpepper The preparation was generated as follows after theabove seven Chinese drugs were washed they were crushedinto coarse particles then 70 ethanol was added 8 times thedrugs were extracted twice (2 h each time) and theywere con-centrated into a liquidwith a relative density of approximately

105 by exposure to low temperature and decompression thenthey were added to the selected cream substrate [7] Thecream substrate included stearic acid glyceryl monostearateAzone and hydroxyphenethyl ester and was provided by theTraditional Chinese Medicine Hospital affiliated with Xin-jiang Medical University (batch number 20151115) the cap-saicin cream contained capsaicin (075mg per gram) whichwas purchased from Guizhou Green Sun PharmaceuticalCo Ltd (batch number 20150601)Metformin hydrochloridetablets were purchased from Beijing Zhonghui Pharmaceu-tical Co Ltd (batch number CO120150407) The placebocream was the basic substance used for the preparation ofWTHX-Cream and was provided by the Traditional ChineseMedicine Hospital affiliated to Xinjiang Medical University

23 Modeling Grouping and Administration According toprevious research methods [8 9] Wistar male rats wereadaptively fed for one week and were then randomly dividedinto two groups according to body weight Ten rats fed withbasic feedstuff were used as the placebo group the remainingrats were used as DPNmodel groups and these rats were feda high-sugar and high-fat diet (70 conventional feed 20sucrose and 10 lard) for 6 weeks fasted for 12 h and given35mgkg of low-dose STZ (dissolved into 01molL citratebuffer pH 42)The control group was given an equal volumeof 1 sodium citrate buffer After 7 days blood glucose wasmeasured via tail bleeds Fasting blood glucose ge 111mmolLwas used as the standard for diabetic rats The modeleddiabetic rats were fed a high-glucose high-fat diet for 8weeks to induce DPN The DPN rat model was establishedas follows the sensory and motor conduction velocity of thesciatic nerve of the lower limb of diabetic rats was measuredby electromyography The DPN rat model was successfullyestablished when the sensory or motor conduction velocitywas slowed by more than 11 Drug administration wasperformed following the method used by Leng et al [10] theplacebo group was given placebo cream (08 g per rat) andrats were lavaged with distilled water at 10mlkg the modelgroupwas given placebo cream (08 g per rat) andwas lavagedwith distilled water at 10mlkg the high- medium- andlow-dose WTHX-Cream + metformin groups were coatedwith WTHX-Cream (08 g per rat 04 g per rat and 02 g perrat resp) and were lavaged with metformin at 018 gkg themetformin group was coated with placebo cream (08 g perrat) and lavaged with metformin at 018 gkg the capsaicincream group was coated with capsaicin cream 02 g perrat and was lavaged with distilled water at 10mlkg andrats in the capsaicin cream + metformin group were coatedwith capsaicin cream 02 g per rat and were lavaged withmetformin at 018 gkgThe hind limbs of all rats were shavedand were coated with cream after 24 h Then the heads ofthe rats were fixed with a connector After 4 h the test drugwas washed off with warm water Drugs were continuouslyadministered for 4 weeks once per day

24 Determination of Nerve Conduction Velocity

241 Motor Nerve Conduction Velocity (MNCV) Determi-nation Rats were anesthetized and were fixed in a prone

Evidence-Based Complementary and Alternative Medicine 3

Table 1 PLC-1205733 primer sequence

Gene ID Gene name Primer sequence (51015840 to 31015840) Product size(bp)

29322 Rat PLC-1205733 F CAAGCCGTACCTCACTCTGG 169Rat PLC-1205733 R CATAGACATCTGGTCGCGCT

position A stimulating needle electrode was placed at theefferent location of the sciatic nerve on the incisura ischiad-ica and the needle electrode was used to record the passingsite of the sciatic nerve on the ankle joint The referenceelectrode was located at a 1 cm distance between the stimuluselectrode and recording electrode With single pulse squarewave stimulation 5 volts of constant voltage was used thecurrent size was 20ndash40mA the wave width was 01ms thestimulus intensity was 15 times the threshold and timefrom the stimulation of the nerve to the occurrence of thedistal muscle action potential was the latency The distancebetween the stimulus electrode and recording electrode wasaccurately measured on the animal surfaceTheMNCV fromthe ischiadic tuberosity to the ankle joint was calculated withthe following formula the distance between the stimuluselectrode and the recording electrodelatency

242 Sensory Nerve Conduction Velocity (SNCV) Determi-nation The sensory nerve stimulation point was located atthe distal end of the tail the recording electrode was locatedat the proximal end of the tail and distance between therecording electrode and stimulation point was approximately2-3 cm The amount of stimulation was gradually increasedThe sensory nerve conduction velocity (ms) = the distanceconducted by the composite action potentialthe latency ofthe compound action potential

25 Behavioral Observation

251Thermal Hyperalgesia Test In a quiet environment ratswere placed in the observation box for 15min to adapt tothe environment before determination The light illuminatesin the lateral metapedes footplate skin The time fromirradiation to the foot reaction which was defined as thelatent period was measured and the radiation treatment wasrepeated 3 times at 5-minute intervalsThe longest irradiationtime was 30 s to avoid damage to rats by long-term exposureto irradiation Meanwhile the average value was recorded

252 Cold Allodynia Test After rat fixation 01ml of acetonewas gently dropped on the lateral planta of the hind limbsand the heat dissipated by acetone was used to inducecold allodynia in rats The test was repeated 3 times withan interval of 5min Meanwhile the average time for footwithdrawal was recorded and the shortest time was 05 s

253 Mechanical Hyperalgesia Test After rat fixation asafety pin was used to appropriately puncture the lateralplanta The test was repeated 3 times with an interval of5min Meanwhile the average time for foot withdrawal wasrecorded and the shortest time was 05 s

254 Mechanical Pain Threshold Measurement Rats wereplaced in the observation box for 15min to adapt to theenvironment before determination Then 04 g of von Freyfiber filament was used to vertically stimulate the middleplanta to observe the foot withdrawal response The occur-rence of lifting the foot or licking foot behavior was definedas a positive reaction and the absence of these behaviors wasdefined as a negative reaction Each animal was stimulated 10times and the tests were conducted with an interval of 30 sThe percentage of the foot withdrawal response = the numberof foot withdrawals10 times 100

26 Histopathology of DRG Tissue and an UltrastructuralObservation of the Ischiadic Nerve After drug administra-tion 05ml100 g (body weight) of 20 urethane was usedto anesthetize rats through intraperitoneal injection bloodwas drawn from the abdominal aorta and then L4L5 DRGtissue and part of the sciatic nerve were collected Thetissues were then fixed with 10 formaldehyde stained withHE and toluidine blue and histopathologically examinedvia light microscopy At the same time part of the sciaticnerve was fixed with glutaraldehyde and thin sections weregenerated for an electron microscopic evaluation of theultramicrostructure

27 PLC-1205733 Determination of the DRG After drug admin-istration anesthesia with 10 chloral hydrate (350mgkg)was performed and L4L5 dorsal root nerve tissue wasquickly removed PLC-1205733 mRNA expression was detected byfluorescence quantitative qPCR amplification (SYBR Greendye assay 120573-actin as an internal reference) after extrac-tion of total RNA and determination of RNA purity byelectrophoresis Fluorescent quantitative PCR primers weredesigned against the PLC-1205733 sequence in the NCBI databaseby Primer-BLAST software and were synthesized by UrumqiKuntairui Biotechnology Co Ltd The primer sequencesare shown in Table 1 Total protein was rapidly extractedfrom the remaining tissue with liquid nitrogen for Westernblot analysis Semidry electrophoresis gels were transferredto PVDF membranes and then placed in confining liquidcontaining 5 skim milk powder for 1 h Thereafter a rabbitanti-PLC-1205733 antibody (dilution ratio 1 1000 purchased fromAbnova batch number PAB17281) was added gently stirredand incubated at 4∘C overnight Then the correspondingsecondary antibody (G-anti-r-HRP) was applied to the blotand the blot was incubated at room temperature for 2 hfollowed by chemiluminescence development

28 StatisticalMethods SPSS 190 statistical softwarewas usedMultiple-group comparisons were performed by one-wayANOVA The LSD method and Dunnettrsquos T3 method were


used for pairwise multiple comparisons Data are expressedas the mean plusmn SD 119875 lt 005 was considered significantGraphPad Prism 50 was used to assist with mapping

3 Results

31 Establishment of the DPN Model During the modelestablishment period rats in the placebo group were in goodmental condition and their weight continued to increaseRats in the DPN model group lost weight (119875 lt 001) whilerats in the DPN model group displayed polyuria a yellowcoat color and slowmovement During the modeling periodsome rats had fundus lesions which showed white turbidityElectromyography was used to measure the motor neuronand sensory nerve conduction latencies and the MNCV andSNCV were calculated before and after modeling The motornerve and sensory nerve conduction latencieswere prolongedin the DPN model groups and MNCV and SNCV wereslower in the DPN model group than in the placebo group(119875 lt 001) The results indicated successful establishment ofthe DPN rat model (Figures 1(a) and 1(b)) Measurement ofthe rat behavioral index before drug administration showedthat the latency of foot withdrawal to heat pain durationof foot withdrawal to cold allodynia and duration of footwithdrawal to mechanical pain were shortened and that thepercentages of foot withdrawal response were increased (119875 lt001) indicating that the pain thresholds for cold allodyniaheat pain and mechanical pain were reduced (Figures 1(c)and 1(d)) After the data were measured three rats died inDPN model group before the group was given the drugDuring drug administration some rats died because of highblood sugar including one that died in the placebo groupthree that died in the model group one that died in the high-dose WTHX-Cream and metformin group two that died inthe middle-dose WTHX-Cream and metformin group andtwo that died in the capsaicin cream + metformin groupHence after the end of the experiment a total of 76 rats werestatistically analyzed

32 Effect of WTHX-Cream on the Body Weight and BloodGlucose of DPN Rats After 4 weeks of drug administrationthe differences in body weight and blood glucose weresignificantly different between the DPN model group andplacebo groupThere were no significant differences betweeneach administration group and the model group or amongthe administration groups (Figures 2(a) and 2(b))

33 WTHX-Cream Can Improve the Pain Threshold of DPNRats Compared with the model group the drug adminis-tration groups showed improved indexes Duration of footwithdrawal to cold allodynia and duration of foot withdrawalto mechanical pain were higher in the high-dose WTHX-Cream + metformin group than in the metformin groupand capsaicin cream + metformin group (119875 lt 001) Thepercentages of the foot withdrawal response were lowerthan those of the metformin group and capsaicin cream +metformin group (119875 lt 001) (Figures 3(a) and 3(b))

34 WTHX-Cream Can Improve Sciatic Nerve Conductionin Rats In the drug administration groups the MNCV andlatency were not significantly improved but the sensorynerve conduction effect was more pronounced Comparedwith the placebo group themodel group showed a prolongedlatent period of sensory nerve conduction (119875 lt 001)Compared with the model group the drug administrationgroups showed a shortened sensory nerve latency and accel-erated conduction velocity (119875 lt 001) which were morepronounced in the high-dose WTHX-Cream + metformingroup and the capsaicin cream+metformin group (119875 lt 001)(Figures 4(a) and 4(b))

35 Effects of WTHX-Cream on the Pathological Structureof the DRG and Ultrastructural Changes of the Sciatic Nervein Rats In the placebo group light microscopy indicatedthat the neuronal cell structures of the DRG were intactcytoplasm was rich nuclei were round or oval and werelocated in the middle of the cell body and Nissl bodieswere clearly observed in the cytoplasm Electron microscopyshowed dense and uniform myelinated nerve fibers con-centrically arranged myelinated lamellae axons withoutswelling and atrophy neatly arranged axonal nerve fibersand microtubules and normal Schwann cells (Figures 5(a)and 5(d)) In the model group light microscopy indicatedthat neuronal cell structures of the DRG were degeneratedmost of the cell bodies were atrophied the neuron poolwas broadened Nissl bodies within the cytoplasm weredissolved and had an unclear structure and some nucleihad dissolved and disappeared Electron microscopy showedwidened myelin in myelinated nerve fibers a loose structuredisorderly arrangement severe lamellar structure damageand lamellar isolation severe nerve demyelination decreasedaxonal density and obvious vacuolar degeneration (Figures5(b) and 5(e)) Both the metformin group and capsaicingroup showed the same morphological and ultrastructuralchanges as the model group In the high-dose WTHX-Cream + metformin group light microscopy indicated thatthe neuronal cell structures of the DRG were partiallydegenerated and atrophied the neuron pool was slightlywidened there was partial dissolution or disappearancewithin the cytoplasm and individual cells were fragmentedor disappeared Electron microscopy showed that the myeli-nated myelin sheath layer structure was basically normalthe overall vacuolar degeneration was significantly improvedcompared with that of the capsaicin cream + metformingroup only mild myelofibrosis was observed and the axonallesions were not obvious (Figures 5(c) and 5(f)) Underlight microscopy and electron microscopy the pathologicalchanges of the medium- and low-dose WTHX-Cream +metformin groups and capsaicin cream + metformin groupwere between those of the model group and the high-doseWTHX-Cream +metformin groupThe neurons of the DRGwere partially degenerated and atrophied the neuronal poolwas slightly widened Nissl bodies were dissolved in thecytoplasm or had disappeared and part of the nucleus wasfragmented or had disappeared Electronmicroscopy showedswelled myelination demyelination in some nerves varying


0

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duct

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velo

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(ms

)

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(a)


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Late

ncy

(ms)

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(b)


0

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Late

ncy

of fo

otw

ithdr

awal

tohe

at p

ain

(s)

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atio

n of

foot

with

draw

al to

cold

allo

dyni

a (s)

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atio

n of

foot

with

draw

al to

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cal p

ain

(s)

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(c)


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ntag

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spon

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DPN modelgroup

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(d)

Figure 1 Analysis of (a) nerve conduction velocity (b) nerve conduction latency and ((c) and (d)) behavioral indicators in rats All valuesgiven are the mean plusmn SD lowastlowast119875 lt 001 and lowast119875 lt 005 versus the placebo group

degrees of axonal degeneration normal nerve fiber filamentstructure and partial occurrence of vacuolar degeneration

36 WTHX-Cream Can Increase PLC-120573 Expression in theDRG of Rats The relative expression of PLC-1205733 mRNAin the model group was decreased compared with thatof the placebo group (119875 lt 005) the relative PLC-1205733mRNA levels in the high-dose WTHX-Cream + metformingroup medium-dose WTHX-Cream + metformin group

and capsaicin + metformin group were increased comparedwith that of the placebo group (119875 lt 005) and there was nosignificant difference among the three groups (Figure 6(a))The Western blot results showed that the expression of thePLC-1205733 protein in the DRG in the model group was lowerthan that in the placebo group (119875 lt 005) PLC-1205733 proteinexpression in the DRG in the drug administration groupwas increased compared with that in the model group (119875 lt005) The PLC-1205733 protein level was higher in the high-dose


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(a)

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cose

(mm

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(b)

Figure 2 Effect ofWTHX-Cream on the body weight and blood glucose of DPN rats (a) Body weight (b) blood glucose All values given arethe mean plusmn SD lowastlowast119875 lt 001 versus the placebo group A placebo group B model group C high-doseWTHX-Cream +metformin group Dmedium-dose WTHX-Cream + metformin group E low-dose WTHX-Cream +metformin group F metformin group G capsaicin groupH capsaicin cream + metformin group

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(s)

Latency of foot withdrawal to heat pain (s)Duration of foot withdrawal to cold allodynia (s)Duration of foot withdrawal to mechanical pain (s)

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(a)

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ntag

es o

f foo

t with

draw

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spon

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)

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(b)

Figure 3 Effect of WTHX-Cream on the behavioral indicators in rats (a) Latency of foot withdrawal to heat pain and duration of footwithdrawal to cold allodynia andmechanical pain (b) the percentages of foot withdrawal response all values given are the mean plusmn SD lowastlowast119875 lt001 versus the placebo group 119875 lt 005 and

119875 lt 001 versus the model group 119875 lt 005 and 119875 lt 001 versus the metformin group998771119875 lt 005 and 998771998771119875 lt 001 versus the capsaicin cream + metformin group A placebo group B model group C high-dose WTHX-Cream

+ metformin group D medium-dose WTHX-Cream + metformin group E low-dose WTHX-Cream + metformin group F metformingroup G capsaicin group H capsaicin cream + metformin group

WTHX-Cream + metformin group than in the middle-doseWTHX-Cream + metformin group or the low-dose WTHX-Cream + metformin group (119875 lt 005) but there was nosignificant difference between the high-dose WTHX-Cream+ metformin group and the capsaicin + metformin group(Figures 6(b) and 6(c))

4 Discussion

DPN is one of the most common chronic complicationsof diabetes mellitus and is a major risk factor for footulcers infections and gangreneThemajor clinical symptomsare hyperalgesia and sensory disturbances Because of itscomplicated pathogenesis clinical treatment with drugs islimited In traditional Chinese medicine DPN belongs to theldquoarthralgiardquo category which includes clinical symptoms such

as numbness pain and burning and formication sensationsTraditional Chinesemedicine defines themechanism ofDPNas follows for patients suffering from long-term diabetesblood and qi within the viscera and bowel are disharmoniousand pathological products such as phlegm toxin and staticblood are blocked in blood vessels and meridians whichwill not nourish limbs and eventually produce pain andeven numbness [11 12] WTHX-Cream can activate bloodresolve stasis and dredge collaterals to relieve pain whichis consistent with the treatment goal of DPN Previouslywe showed that WTHX-Cream can significantly improveclinical symptoms and signs relieve pain improve nerveconduction velocity and delay DPN progression [4 13] Thecurrent study showed that WTHX-Cream can improve DPNand hyperalgesia symptoms the mechanism of which mayinclude regulation of PLC-1205733 expression in the DRG


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(b)

Figure 4 Effect ofWTHX-Creamon the nerve conduction velocity inDPN rats (a) Nerve conduction latency (b) nerve conduction velocityall values given are the mean plusmn SD lowastlowast119875 lt 001 versus the placebo group 119875 lt 005 and

119875 lt 001 versus the model group 119875 lt 005and 119875 lt 001 versus the metformin group A placebo group B model group C high-dose WTHX-Cream + metformin group Dmedium-dose WTHX-Cream + metformin group E low-dose WTHX-Cream +metformin group F metformin group G capsaicin groupH capsaicin cream + metformin group

(a)

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(b)

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MSAxon

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(e)

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(f)

Figure 5 Effect of WTHX-Cream on the pathology of the DRG and ultrastructure of the sciatic nerve in DPN rats Light microscopy (a)placebo group (b) model group and (c) high-dose WTHX-Cream + metformin group Electron microscopy (d) placebo group (e) modelgroup and (f) high-dose WTHX-Cream + metformin group uArr Nissl body (light microscopy) NP neuronal pool nucleus MS myelinuarr vacuolization (electron microscopy) Schwann Schwann cells


002040608

11214

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PLC-

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A

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(a)

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(b)

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137

(＋＄

)

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(c)

Figure 6WTHX-Cream can increase PLC-120573 expression in theDRGof rats (a) PLC-1205733mRNA ((b) and (c)) PLC-1205733 protein All values givenare the mean plusmn SD lowast119875 lt 005 versus the placebo group 119875 lt 005 versus the model group 998787119875 lt 005 versus the high-dose WTHX-Cream +metformin group 998810119875 lt 005 versus the middle-dose WTHX-Cream + metformin group X119875 lt 005 versus the low-dose WTHX-Cream +metformin group I119875 lt 005 versus the capsaicin group A placebo group B model group C high-doseWTHX-Cream +metformin groupD medium-dose WTHX-Cream + metformin group E low-dose WTHX-Cream + metformin group F metformin group G capsaicingroup H capsaicin cream + metformin group

In this study we used a classic STZ injection method toestablish diabetic ratmodels and assessed theDPN ratmodelsfor sciatic nerve conduction velocity after 8 weeks of con-tinuous observation The established DPN models showedthe typical characteristics of DPN such as elevated bloodglucoseweight loss andpolyuria Adecreased pain thresholdand increased pain sensitization are typical manifestationsof DPN After 4 weeks of WTHX-Cream treatment thepain threshold of rats was increased suggesting thatWTHX-Cream can reduce pain by decreasing pain sensitization toimprove the pain symptoms of DPN However no significantimprovement in body weight or blood glucose was observedafter 4 weeks of administration and previous studies haveshown that WTHX-Cream has no direct effect on bloodglucose in DPN rats [14] The possible reasons for the lackof effects were as follows (1) the number of experimentalanimals was limited (2) drug administration time was shortand drug plasma concentration was not fully maintainedHowever WTHX-Cream had a definite effect on improv-ing the pathological structure of DPN rats for examplelight microscopy showed that neuronal degeneration andnuclear fragmentation in the WTHX-Cream groups weresignificantly lower than those in the model groups electronmicroscopy showed that the nerve myelin structure andvacuolar changes in WTHX-Cream groups were improvedand these improvements were more obvious with higherdosages of WTHX-Cream

PLC-1205733 is a subtype of the phospholipase C familyand can bind to specific receptors on the cell membraneStudies have shown that the PLC-1205733 molecule in the ratDRG is a protein kinase C upstream molecule that leadsto inflammatory pain However the mechanism underlyingthe pain caused by PLC-1205733 is still unclear The possiblemechanisms are as follows (1) PLC-1205733 activation causes

hydrolysis of phosphatidylinositol-45-diphosphate (PIP2)resulting in inositol triphosphate (IP3) and diacylglycerol(DAG) production IP3 binds to the IP3 receptor on the endo-plasmic reticulum and opens Ca2+ channels thus promotingthe release of intracellular stores of Ca2+ and increase of intra-cellular Ca2+ (2) PLC-1205733 activation may cause the releaseof some irritating neurotransmitters such as glutamate andirritant nerve polypeptide [15] These neurotransmitters cancause pain demonstrating that PLC-1205733 has a pain-promotingeffect Local injection of the PLC-1205733 inhibitor U73122 inmouse paws inhibited inflammatory hyperalgesia caused bya carrageenan injection at the same site [16] indicating thatPLC-1205733 can enhance pain sensitivity In this study the PLC-1205733 mRNA or protein levels were significantly higher in themedium- and high-doseWTHX-Cream +metformin groupsthan in the model group at 4 weeks after using WTHX-CreamThe improvement wasmore obvious when the dosagewas higher Therefore we hypothesized that WTHX-Creamcan inhibit the decrease of PLC-1205733 in DPN rats Moreoverafter drug application the pain threshold of cold allodyniaheat pain andmechanical pain was increased nerve conduc-tion velocity and sciatic nerve morphology were improvedcompared with those of the model group and efficacy wasbetter than that of the capsaicin cream + metformin groupindicating that WTHX-Cream may improve hyperalgesia ofDPN by inhibiting downregulation of PLC-1205733

In WTHX-Cream Chuanxiong not only promotes bloodcirculation to regulate the meridians but also promotesthe operation of ldquoqirdquo (the energy of life) to achieve ananalgesic effect Safflower is an important traditional Chinesemedicine that promotes blood circulation and smooths themeridians The combined use of the two herbs can increaseblood circulation and dredging of the channel Modernpharmacological studies have shown that Chuanxiong and its


extracts have a direct expansion effect on peripheral bloodvessels and its effective component ligustrazine can dilateblood vessels reduce platelet aggregation reduce blood vis-cosity reduce thrombosis improve blood flow and improvemicrocirculation effects [17] Safflower contains ingredi-ents such as carthamin and carthamin yellow which canreduce blood viscosity inhibit platelet aggregation improveplasma fibrinolytic activity prevent microthrombosis anddissolve thrombolysis eliminate the effect on blood vesselscaused by adrenaline andnorepinephrine and relieve smoothmuscle spasm thus leading to expansion of blood vesselsimprovement in microcirculation [18] and improvement inneuronal ischemia and hypoxia The herb ldquoGuizhirdquo primar-ily contains components that include cinnamaldehyde 120573-paclitaxel and cinnamic acid which result in pain reliefand dilation of blood vessels Asarum was first reportedin ldquoShennongrsquos Classic of Materia Medicardquo and was said todispel wind dissipate cold move water and relieve painModern pharmacological studies have shown that Asarumhas antipyretic analgesic and sedative effects [19] Angelicacan also improve blood circulation in the lesion and relievepain In this study the curative effect of WTHX-Cream wasbetter than that found in the capsaicin cream + metformingroup indicating thatWTHX-Creamdoes have a therapeuticeffect on DPN Furthermore WTHX-Cream is an externalmedicine that avoids the first-pass effect of oral drugs intothe body and reduces the damage to the gastrointestinal tractAdditionally it directly acts on the lesion is easy to use hasfast transdermal absorption and is widely used in clinicalpractice Modern research also shows that the external useof traditional Chinese medicine can hydrate the stratumcorneum which helps drug penetration and increases thedrug transdermal rate by 4-5 times Additionally it increasesskin temperature and hence accelerates blood circulationWTHX-Cream added to the transdermal enhancer Azonecan significantly promote transdermal absorption of mosthydrophilic and hydrophobic compounds

In summary the multitarget role of traditional Chinesemedicine in the treatment of DPN has been widely recog-nized and an in-depth study of its mechanism will helpelucidate the efficacy of Chinese medicine and thus providenew ideas for the treatment of diseases In this study weexamined the inhibitory effect of WTHX-Cream on thedownregulation of PLC-1205733 at the gene and protein levelsin the rat DRG and investigated the molecular mechanismof WTHX-Cream to reduce the sensory sensitivity of DPNwhich is important for the clinical treatment of DPN Ourfuture studies will focus on the role and mechanism of themajor components of WTHX-Cream

Conflicts of Interest

The authors declare that there are no conflicts of interest

Acknowledgments

This study is funded by the National Natural Science Foun-dation of China (no 81360529)

References

[1] J S Han ldquoHot Issues of Neuropathic Painrdquo Journal of MedicalResearch vol 40 no 7 pp 1-2 2011

[2] H L Zhou and C Q Yan ldquoResearch progress of diabeticperipheral neuropathyrdquo Journal ofMilitary Surgeon in SouthwestChina vol 19 no 1 pp 81ndash83 2017

[3] J J Duby CamPbell R K Setter S M et al ldquoDibaeticneuropaty an intensive reviewrdquo Am J Health-Syst Pharm vol61 no 2 pp 160ndash173 2004

[4] Z Jian-hong Z Xin-jun and L An-kun ldquoThe clinical researchinto painful diabetic neuropathy treated with wentong huoxuerugao in combination with pancreatic kininogenase enteric-coated tabletsrdquoHenan Traditional Chinese Medicine vol 37 no4 pp 663ndash665 2017

[5] S-K Han V Mancino and M I Simon ldquoPhospholipase C120573 3mediates the scratching response activated by the histamine H1receptor on C-fiber nociceptive neuronsrdquoNeuron vol 52 no 4pp 691ndash703 2006

[6] T-J S Shi S-X L Liu H Hammarberg M Watanabe Z-Q D Xu and T Hokfelt ldquoPhospholipase C1205733 in mouse andhuman dorsal root ganglia and spinal cord is a possible targetfor treatment of neuropathic painrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 105 no50 pp 20004ndash20008 2008

[7] F R Wang C L Xia and J Lu ldquoResearches about Wen TongHuoxue cream technologyrdquo Xinjiang Traditional Chinese Medvol 26 no 6 pp 48ndash50 2007

[8] T Klemm and R Paschke ldquoPossible genetic reasons for latecomplications in diabetes mellitusrdquoMedizinische Klinik - Inten-sivmedizin und Notfallmedizin vol 95 no 1 pp 31ndash39 2000

[9] G Conti E Scarpini P Baron et al ldquoMacrophage infiltrationand death in the nerve during the early phases of experimentaldiabetic neuropathy a process concomitant with endoneurialinduction of IL-1120573 and p75NTRrdquo Journal of the NeurologicalSciences vol 195 no 1 pp 35ndash40 2002

[10] X Y Leng X K Wang and Z H Li ldquoExperimental study ontreatment of Dan Gui detumescence cream for acute closed softtissue injuryrdquo Guide of China Medicine vol 9 no 36 pp 70-712011

[11] B X Zhao ldquoTreatment of diabetic peripheral neuropathy fromthe aspect of Qi and bloodrdquo Liaoning Journal of TraditionalChinese Medicine vol 31 no 12 pp 995-996 2004

[12] H u XL and W H Zhang ldquoDiscussion about diabetic periph-eral neuropathy from the spleen and stomach and phlegmpoisoningrdquo Xin Jiang Traditional Chinese Med vol 24 no 1 pp1-2 2006

[13] M A Li W Xianmin C Qian et al ldquoClinical observation onpainful diabetic neuropathy treated by wengtong huoxue creamplus limb air-pressure therapy a report of 30 casesrdquo Journal ofTraditional Chinese Med vol 52 no 19 pp 1665ndash1667 2011

[14] Z Hai-Ying and Z Yong-Bo ldquoAcute cutaneous toxicity skinirritation and allergy reaction irritation of wen-tong-huo-xuecreamrdquo in Chinese Journal of Spectroscopy Laboratory vol 30pp 2354ndash2356 5 edition 2013

[15] T-J S Shi M-D Zhang H Zeberg et al ldquoCoenzyme Q10prevents peripheral neuropathy and attenuates neuron loss inthe db-db- mouse a type 2 diabetes modelrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 110 no 2 pp 690ndash695 2013

[16] C Hou T Kirchner M Singer M Matheis D Argentieri andD Cavender ldquoIn Vivo Activity of a Phospholipase C Inhibitor


1-(6-((17120573 -3-Methoxyestra-135(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-25-dione (U73122) in Acute and Chronic Inflam-matory Reactionsrdquo The Journal of Pharmacology and Experi-mental Therapeutics vol 309 no 2 pp 697ndash704 2004

[17] J Zhongmei and C Zhuanxin ldquoTherapeutic effect of combinedtreatment of mecobalamin and salvia miltiorrhiza ligustrazineon diabetic peripheral neuropathyrdquo Chinese Journal of PrimaryMedicine and Pharmacy vol 22 pp 3467ndash3469 2015

[18] L Zhang ldquoEffect of safflower alprostadil and mecobalaminin treating diabetic peripheral neuropathyrdquo Chinese Journal ofTrauma Disability Medicine vol 23 pp 98-99 2015

[19] X Q Liang andDD Li ldquoProgress research on pharmacologicalactivities of asarumrdquo Journal of Henan University of Science ampTechnology (Medical Science) vol 29 no 4 pp 318ndash320 2011

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


phospholipase molecule C family which contains a variety ofmembers including PLC-120573 -120574 -120575 and -120576 PLC-120573 includesPLC-1205731 PLC-1205732 PLC-1205733 and PLC-1205734 PLC-1205731 PLC-1205733and PLC-1205734 are distributed in the dorsal root ganglion(DRG) and PLC-1205733 has the highest transcription levels[5] Many studies have confirmed that PLC-1205733 is associatedwith pain Related studies have shown that in spared nerveinjury the PLC-1205733 content in the DRG of the same side wassignificantly decreased but did not change on the noninjuryside indicating that the PLC-1205733 level of the DRG afterperipheral nerve injury is reduced [6]

In combinationwith analysis of the pain perception trans-mission mechanism histopathology real-time quantitativePCR and immunoblotting were used to explore the effects ofWTHX-Cream on DPN based on histopathological changesand pain-related molecule PLC-1205733 mRNA and protein levelchanges This study aimed to provide a theoretical basis andguidance for the clinical treatment of DPN with WTHX-Cream and to develop new ideas for the external use ofChinese traditional medicine in the treatment of DPN

2 Materials and Methods

21 Animals 88 specific pathogen-free male Wistar ratsweighing 80ndash130 g were purchased from Xinjiang Experi-mental Animal Research Center (production license num-ber SCXK (new) 2011-0001 animal certificate number65000200000408) Rats were fed separately with 5 rats ineach cage Humidity was controlled within the range of30ndash50 and temperature was controlled within 20ndash26∘CRats were fed for 1 week under natural light with freelyavailable food and drinking water and they were randomlydivided into the placebo (10 rats) and DPN model (78 rats)groups according to the random number table methodPlacebo cream was applied to rats in the placebo group andthey were given distilled water via gavageThe remaining ratsin the DPN model group were injected with streptozotocin(STZ) and were fed a high-fat diet for 8 weeks to developthe DPN model The DPN model groups were subdividedinto the model group (placebo cream + lavage with distilledwater 11 rats) high-dose WTHX-Cream + metformin group(high-dose WTHX-Cream + lavage with metformin 11 rats)medium-dose WTHX-Cream +metformin group (medium-dose WTHX-Cream + lavage with metformin 11 rats) low-dose WTHX-Cream + metformin group (low-dose WTHX-Cream + lavage with metformin 11 rats) metformin group(placebo cream + lavage with metformin 10 rats) capsaicingroup (capsaicin cream + lavage with distilled water 10 rats)and capsaicin cream + metformin group (capsaicin cream +lavage with metformin 11 rats)

22 Drug WTHX-Cream is a Chinese drug preparationcontaining Chuanxiong safflower Ramulus CinnamomiAsarum Angelica Allolobophora caliginosa trapezoids andpepper The preparation was generated as follows after theabove seven Chinese drugs were washed they were crushedinto coarse particles then 70 ethanol was added 8 times thedrugs were extracted twice (2 h each time) and theywere con-centrated into a liquidwith a relative density of approximately

105 by exposure to low temperature and decompression thenthey were added to the selected cream substrate [7] Thecream substrate included stearic acid glyceryl monostearateAzone and hydroxyphenethyl ester and was provided by theTraditional Chinese Medicine Hospital affiliated with Xin-jiang Medical University (batch number 20151115) the cap-saicin cream contained capsaicin (075mg per gram) whichwas purchased from Guizhou Green Sun PharmaceuticalCo Ltd (batch number 20150601)Metformin hydrochloridetablets were purchased from Beijing Zhonghui Pharmaceu-tical Co Ltd (batch number CO120150407) The placebocream was the basic substance used for the preparation ofWTHX-Cream and was provided by the Traditional ChineseMedicine Hospital affiliated to Xinjiang Medical University

23 Modeling Grouping and Administration According toprevious research methods [8 9] Wistar male rats wereadaptively fed for one week and were then randomly dividedinto two groups according to body weight Ten rats fed withbasic feedstuff were used as the placebo group the remainingrats were used as DPNmodel groups and these rats were feda high-sugar and high-fat diet (70 conventional feed 20sucrose and 10 lard) for 6 weeks fasted for 12 h and given35mgkg of low-dose STZ (dissolved into 01molL citratebuffer pH 42)The control group was given an equal volumeof 1 sodium citrate buffer After 7 days blood glucose wasmeasured via tail bleeds Fasting blood glucose ge 111mmolLwas used as the standard for diabetic rats The modeleddiabetic rats were fed a high-glucose high-fat diet for 8weeks to induce DPN The DPN rat model was establishedas follows the sensory and motor conduction velocity of thesciatic nerve of the lower limb of diabetic rats was measuredby electromyography The DPN rat model was successfullyestablished when the sensory or motor conduction velocitywas slowed by more than 11 Drug administration wasperformed following the method used by Leng et al [10] theplacebo group was given placebo cream (08 g per rat) andrats were lavaged with distilled water at 10mlkg the modelgroupwas given placebo cream (08 g per rat) andwas lavagedwith distilled water at 10mlkg the high- medium- andlow-dose WTHX-Cream + metformin groups were coatedwith WTHX-Cream (08 g per rat 04 g per rat and 02 g perrat resp) and were lavaged with metformin at 018 gkg themetformin group was coated with placebo cream (08 g perrat) and lavaged with metformin at 018 gkg the capsaicincream group was coated with capsaicin cream 02 g perrat and was lavaged with distilled water at 10mlkg andrats in the capsaicin cream + metformin group were coatedwith capsaicin cream 02 g per rat and were lavaged withmetformin at 018 gkgThe hind limbs of all rats were shavedand were coated with cream after 24 h Then the heads ofthe rats were fixed with a connector After 4 h the test drugwas washed off with warm water Drugs were continuouslyadministered for 4 weeks once per day

24 Determination of Nerve Conduction Velocity

241 Motor Nerve Conduction Velocity (MNCV) Determi-nation Rats were anesthetized and were fixed in a prone

















3 Results







0

10

20

30

40

50

60

70

80

90


Con

duct

ion

velo

city

(ms

)


lowastlowast

lowastlowast

(a)


0

05

1

15

2

25

3


Late

ncy

(ms)

lowastlowast

lowast

(b)


0

5

10

15

20

25

30

Late

ncy

of fo

otw

ithdr

awal

tohe

at p

ain

(s)

Dur

atio

n of

foot

with

draw

al to

cold

allo

dyni

a (s)

Dur

atio

n of

foot

with

draw

al to

mec

hani

cal p

ain

(s)

(s)

lowastlowast

lowastlowast

lowastlowast

(c)


The p

erce

ntag

es o

f foo

t with

draw

al re

spon

se (

)

0

20

40

60

80

100

120

Placebogroup

DPN modelgroup

lowastlowast

(d)






0

100

200

300

400

500

600

A B C D E F G H

Wei

ght (

g)

lowastlowast

(a)

0

5

10

15

20

25

30

A B C D E F G H

Glu

cose

(mm

oll)

lowastlowast

(b)


0

5

10

15

20

25

30

A B C D E F G H

(s)


lowastlowast


(a)

0

20

40

60

80

100

120

A B C D E F G H

The p

erce

ntag

es o

f foo

t with

draw

alre

spon

se (

)

lowastlowast

(b)





4 Discussion




0

05

1

15

2

25

3

A B C D E F G H

Late

ncy

(ms)


lowastlowast

(a)

0

10

20

30

40

50

60

70

80

90

A B C D E F G H

Con

duct

ion

velo

city

(ms

)


lowastlowast

(b)



(a)

NP

NP

NP NPNP

(b)

NP

NP

NP

NP

(c)

Schwann

MSAxon

(d)

Axon

(e)

Axon

SchwannMS

(f)



002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































3 Results







0

10

20

30

40

50

60

70

80

90


Con

duct

ion

velo

city

(ms

)


lowastlowast

lowastlowast

(a)


0

05

1

15

2

25

3


Late

ncy

(ms)

lowastlowast

lowast

(b)


0

5

10

15

20

25

30

Late

ncy

of fo

otw

ithdr

awal

tohe

at p

ain

(s)

Dur

atio

n of

foot

with

draw

al to

cold

allo

dyni

a (s)

Dur

atio

n of

foot

with

draw

al to

mec

hani

cal p

ain

(s)

(s)

lowastlowast

lowastlowast

lowastlowast

(c)


The p

erce

ntag

es o

f foo

t with

draw

al re

spon

se (

)

0

20

40

60

80

100

120

Placebogroup

DPN modelgroup

lowastlowast

(d)






0

100

200

300

400

500

600

A B C D E F G H

Wei

ght (

g)

lowastlowast

(a)

0

5

10

15

20

25

30

A B C D E F G H

Glu

cose

(mm

oll)

lowastlowast

(b)


0

5

10

15

20

25

30

A B C D E F G H

(s)


lowastlowast


(a)

0

20

40

60

80

100

120

A B C D E F G H

The p

erce

ntag

es o

f foo

t with

draw

alre

spon

se (

)

lowastlowast

(b)





4 Discussion




0

05

1

15

2

25

3

A B C D E F G H

Late

ncy

(ms)


lowastlowast

(a)

0

10

20

30

40

50

60

70

80

90

A B C D E F G H

Con

duct

ion

velo

city

(ms

)


lowastlowast

(b)



(a)

NP

NP

NP NPNP

(b)

NP

NP

NP

NP

(c)

Schwann

MSAxon

(d)

Axon

(e)

Axon

SchwannMS

(f)



002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

10

20

30

40

50

60

70

80

90


Con

duct

ion

velo

city

(ms

)


lowastlowast

lowastlowast

(a)


0

05

1

15

2

25

3


Late

ncy

(ms)

lowastlowast

lowast

(b)


0

5

10

15

20

25

30

Late

ncy

of fo

otw

ithdr

awal

tohe

at p

ain

(s)

Dur

atio

n of

foot

with

draw

al to

cold

allo

dyni

a (s)

Dur

atio

n of

foot

with

draw

al to

mec

hani

cal p

ain

(s)

(s)

lowastlowast

lowastlowast

lowastlowast

(c)


The p

erce

ntag

es o

f foo

t with

draw

al re

spon

se (

)

0

20

40

60

80

100

120

Placebogroup

DPN modelgroup

lowastlowast

(d)






0

100

200

300

400

500

600

A B C D E F G H

Wei

ght (

g)

lowastlowast

(a)

0

5

10

15

20

25

30

A B C D E F G H

Glu

cose

(mm

oll)

lowastlowast

(b)


0

5

10

15

20

25

30

A B C D E F G H

(s)


lowastlowast


(a)

0

20

40

60

80

100

120

A B C D E F G H

The p

erce

ntag

es o

f foo

t with

draw

alre

spon

se (

)

lowastlowast

(b)





4 Discussion




0

05

1

15

2

25

3

A B C D E F G H

Late

ncy

(ms)


lowastlowast

(a)

0

10

20

30

40

50

60

70

80

90

A B C D E F G H

Con

duct

ion

velo

city

(ms

)


lowastlowast

(b)



(a)

NP

NP

NP NPNP

(b)

NP

NP

NP

NP

(c)

Schwann

MSAxon

(d)

Axon

(e)

Axon

SchwannMS

(f)



002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

100

200

300

400

500

600

A B C D E F G H

Wei

ght (

g)

lowastlowast

(a)

0

5

10

15

20

25

30

A B C D E F G H

Glu

cose

(mm

oll)

lowastlowast

(b)


0

5

10

15

20

25

30

A B C D E F G H

(s)


lowastlowast


(a)

0

20

40

60

80

100

120

A B C D E F G H

The p

erce

ntag

es o

f foo

t with

draw

alre

spon

se (

)

lowastlowast

(b)





4 Discussion




0

05

1

15

2

25

3

A B C D E F G H

Late

ncy

(ms)


lowastlowast

(a)

0

10

20

30

40

50

60

70

80

90

A B C D E F G H

Con

duct

ion

velo

city

(ms

)


lowastlowast

(b)



(a)

NP

NP

NP NPNP

(b)

NP

NP

NP

NP

(c)

Schwann

MSAxon

(d)

Axon

(e)

Axon

SchwannMS

(f)



002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

05

1

15

2

25

3

A B C D E F G H

Late

ncy

(ms)


lowastlowast

(a)

0

10

20

30

40

50

60

70

80

90

A B C D E F G H

Con

duct

ion

velo

city

(ms

)


lowastlowast

(b)



(a)

NP

NP

NP NPNP

(b)

NP

NP

NP

NP

(c)

Schwann

MSAxon

(d)

Axon

(e)

Axon

SchwannMS

(f)



002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















002040608

11214

A B C D E F G H

PLC-

3

mRN

A


(a)

lowast

lowast lowastlowast

0010203040506

A B C D E F G H

PLC-

3

prot

ein

lowast

(b)

A B C D E F G H

PLC-3

-Actin

137

(＋＄

)

42

(c)











Acknowledgments


References


























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























Acknowledgments


References


























of




Disease Markers



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OncologyJournal of





PPAR Research



Volume 2018


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