research article moxibustion activates macrophage...

Research ArticleMoxibustion Activates Macrophage Autophagy and ProtectsExperimental Mice against Bacterial Infection

Xiaojuan Li12 Guanhua Guo13 Feng Shen1 Lihong Kong1

Fengxia Liang12 and Guojie Sun12

1 Acupuncture and Moxibustion College Hubei University of Chinese Medicine Wuhan Hubei 430061 China2Hubei Provincial Collaborative Innovation Center of Preventive Treatment by Acupuncture and MoxibustionWuhan Hubei 430061 China

3 Shanxi University of Traditional Chinese Medicine Taiyuan Shanxi 030024 China

Correspondence should be addressed to Fengxia Liang 315938821qqcom and Guojie Sun sunguojiesinacom

Received 11 May 2014 Accepted 8 July 2014 Published 23 July 2014

Academic Editor Gerhard Litscher

Copyright copy 2014 Xiaojuan Li 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

Moxibustion is one of main therapies in traditional Chinese medicine and uses heat stimulation on the body surface from theburning of moxa to release pain or treat diseases Emerging studies have shown that moxibustion can generate therapeutic effectsby activating a series of signaling pathways and neuroendocrine-immune activities Here we showmoxibustion promoted profoundmacrophage autophagy in experimental Kunming mice with reduced Akt phosphorylation and activated eIF2120572 phosphorylationConsequently moxibustion promoted bacterial clearance by macrophages and protected mice from mortality due to bacterialinfection These results indicate that moxibustion generates a protective response by activating autophagy against bacterialinfections

1 Introduction

Autophagy is an evolutionary conserved physiological pro-cess by which protein aggregates and damaged organellesinside cells are selectively engulfed and delivered to anddegraded in lysosomes [1 2] This ldquoself-eatingrdquo processis tightly associated with diverse physiological conditionsincluding development and innate defense [3 4] and a num-ber of pathological conditions and diseases including cancersneurodegenerative disease and infectious diseases [5ndash7]Emerging studies have shown that modulation of autophagyacts as a potential therapeutic strategy for treatment of disease[8 9] even though autophagy is a double-edged sword in theprogression of cancer and certain metabolic diseases [10ndash12]Autophagymay restrict bacterial replication in the cytoplasmby clearance or be hijacked to support bacterial replicationand survival [13 14] a unique evasion technique of bacteriawhich uses a variety of machinery to evade host defense sys-tems to facilitate survival inside cells [15] Autophagy primar-ily acts as an innate defense against bacterial infection [16]and enhanced autophagy provides a protective response

against bacterial and virus infection [17 18]However it is dif-ficult to modulate autophagy in unique lesions in vivo whichlimits the application of autophagy as a therapeutic strategy

Moxibustion is a physical therapy in traditional Chinesemedicine that uses heat from the burning of mincedherb moxa to stimulate acupuncture points for pain relief ordisease treatment [19ndash21]Moxibustion can affect neuroendo-crineimmune functions and activate self-healing [22 23]Although the substitution of moxibustion by thermalphysical therapy remains equivocal [23 24] they both sharethe similarmechanism in self-healing and pain reduction [2526] Emerging studies have shown thatmoxibustion alters theprofile of gene expression including cytokines [27ndash30] andsignaling pathways [31ndash33] indicating it might be beneficialto patients with autoimmune and inflammatory diseases

Previously a protective response of moxibustion againstHSV-1 infection was demonstrated in experimental mice[34] However the mechanisms of how moxibustion mightbe useful against infectious diseases remain to be fullydefined The present study shows that moxibustion activates

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 450623 6 pageshttpdxdoiorg1011552014450623

2 Evidence-Based Complementary and Alternative Medicine

autophagy of intraperitoneal macrophages in experimentalKunming mice following the inactivation of Akt and theinduction of eIF2120572 phosphorylation Consequently it con-tributes to a protective response against bacterial infectionindicating moxibustion activates innate defenses to preventinfectious disease

2 Methods and Materials

21 Mice and Ethics Statement Specific-pathogen-free maleKunming mice (6ndash8 weeks of age 18ndash22 g weight) weremaintained and bred according to the guidelines approved bythe Laboratory Animal Center Sun Yat-sen University

22 Mild Moxibustion on Lethal Infections of Staphylococcusaureus Forty mice were randomly divided into four groupsA control group B model group C 15 min moxibustiongroup and D 30 min moxibustion group with 10 miceper group Except for the control group the remaining 30mice were primed with intraperitoneal injection of 6 starchbroth for 2 days followed by intraperitoneal injection of 10LD50Staphylococcus aureus as an acute infection model One

hour after injection with bacteria mice in groups C and Dwere restrained and administered 1206014mm stick moxibustionfor 15min and 30min at Guanyuan acupuncture point (CV4) at the front of the small intestine In the control groupand model group 1206014mm sticks of unlighted moxa herbswere used as the control for moxibustion The death ofmice was monitored continuously for 48 h after injectionto prepare a survival curve Peritoneal fluids were collectedimmediately upon death or sacrifice at 48 h aftermoxibustionand subjected to bacterial colony formation inLuria-Bertani(LB) agar plates The numbers of colonies were used toevaluate bacterial clearance

23MildMoxibustion onMacrophage Autophagy Fortymicewere randomly divided into four groups A control group Bmodel group C 15 min moxibustion group and D 30 minmoxibustion group In B C and D groups mice receivedintraperitoneal injection of 6 starch broth as a macrophageinducer and two days later mice in C and D groups wereadministered moxibustion at CV4 as described above andsacrificed at 1 h after moxibustion Peritoneal fluid cellswere collected and subjected to autophagy assays To detectthe dynamic Akt and eIF2120572 phosphorylation under longermoxibustion an additional group with 60 min moxibustionwas performed following the procedure above

24 Assays of Autophagy and Autophagic Pathways A smallnumber of fresh macrophages were fixed with 3 formalde-hyde in phosphate buffer (PBS) for 10min and permeabilizedwith 05 Triton X-100 in PBS for 10min After washingwith PBS twice the cells were blocked with 1 bovine serumalbumin in PBS with 01 Triton X-100 for 30min andthen incubated with rabbit polyclonal anti-LC3 antibody(1 200 MBL International MA USA) for 1 h After threewashes with PBS with 01 Triton X-100 the cells wereincubated with FITC-conjugated anti-rabbit IgG secondary

antibody (1 500 Life Technologies) for 05 h counterstainedwithDAPI (410158406-diamidino-2-phenylindole Sigma St LouisMO) and mounted in antifade agent (Invitrogen Carls-bad CA) Finally LC3 staining was visualized by confocalfluorescence microscopy The remaining macrophages werelysed and total protein extracts were subjected to westernblots with anti-LC3 antibody phospho-eIF2120572 (Ser51) rabbitmonoclonal antibody (mAb) phospho-Akt (Ser473) rabbitmAb and anti-Akt and eIF2120572 rabbit antibodies (1 1000 CellSignaling Technology MA USA) Images were visualized byLicor Odyssey or Pierce ECL system

25 Statistical Analysis The survival rates were analyzed by alog-rank test and the statistical analysis was performed withone-way analyses of variance (ANOVA)

3 Results

31 Moxibustion Promotes Autophagy in Macrophages Toassess whether moxibustion affected autophagy moxibustionwith a small size moxa stick was applied to the mouseabdomen Guanyuan point (CV4) for 15 and 30 minutesMacrophages were then isolated from the peritoneal fluidafter mice were sacrificed and subjected to autophagy assayWhen macrophages were primed by starch and albumenautophagic LC3-postive vacuoles were not observed inmacrophages similar to that seen in control mice whereasLC3-positve puncta were greatly increased after 15 min or30 min moxibustion (Figures 1(a) and 1(b)) Furthermorewhen LC3 was detected in cell extracts the ratio of LC3-II was elevated following moxibustion compared with mockand primed conditions (Figure 1(c)) The levels of LC3-IIprotein and LC3 punctawere similar under bothmoxibustionconditions These results suggest that moxibustion inducesthe macrophage autophagy in mice

32 Moxibustion Induces eIF2120572 Phosphorylation andSuppresses Akt Phosphorylation We attempted to addressupstream signaling pathways by whichmoxibustion activatesautophagy Akt inhibits endoplasmic reticulum (ER) stress byphosphorylating eIF2120572-kinase PERK [35] and the PI3KAktmTOR pathway acts as a well-characterized negative regu-lator of autophagy [36] Because moxibustion delivers heatstimulation to the operative site we hypothesized it induceda stress response and then Akt activity and ER stress wereexamined after moxibustion To diminish the individualdifferences in mice we investigated ER stress in mixedmacrophages (Figure 2(a)) and random macrophages(Figure 2(b)) As shown in Figures 2(a) and 2(b) in top panelphosphorylation of eIF2120572 in macrophages was significantlyelevated at 30min and then slightly fell back at 1 h duringmoxibustion indicating a rapid ER stress response wasactivated Furthermore the phosphorylation of Akt wasdecreased dramatically at both 30min and 1 h suggestingmoxibustion suppressed Akt activation (Figures 2(a) and2(b) in bottom panel) Thus we concluded that moxibustioninduced autophagy in macrophages by suppressing the Aktpathway and activating ER stress

Evidence-Based Complementary and Alternative Medicine 3

Mock Primed

Mox 15998400 Mox 30

998400

(a)

0

1

2

3

4

5

6

Mock Primed

LC3

punc

ta ra

tio

Mox 15998400 Mox 30

998400

lowastlowastlowastlowast

(b)

LC3

Actin

LC3-I

LC3-II

Moc

k

Prim

ed

Mox

15998400

Mox

30998400

(c)

Figure 1 Autophagy is accelerated by moxibustion Macrophages were collected frommice with or without moxibustion as indicated fixedand stained with anti-LC3 antibody and LC3 staining was visualized by confocal fluorescence microscopy Representative images (a) and themeans of LC3 punctacell counted from 20 individual cells (b) are shown lowastlowast119875 lt 001 Macrophages were lysed and equal mixtures of wholecell extracts were detected by western blots with anti-LC3 and anti-actin antibodies (c)

Akt

pAkt

Actin

Moc

k

Prim

ed

eIF2120572

peIF2120572

Mox

30m

in

Mox

60m

in

(a)

Prim

ed

Akt

pAkt

eIF2120572

peIF2120572

15m

in

30m

in

60m

in

(b)

Figure 2 Autophagic Akt and eIF2120572 pathways are activated by moxibustion (a) Four mice per group were untreated or treated as indicatedand equal whole cell extracts of macrophages from mice were mixed and signaling pathways detected as indicated (b) Two randommacrophage samples from six individual mice per group are shown


0

20

40

60

80

100

0

No infection primed With bacteria primed

12 24

(h)36 48

No infection mox 15998400


With bacteria mox 15998400


()

(a)

0

1

2

3

Primed Mox 15998400 Mox 30

998400

lowastlowast

lowastlowast

11 plusmn 07

017 plusmn 012

0042 plusmn 0023

Bact

eria

left

(cfu

times107m

L)(b)

Figure 3 Bacterial infection is alleviated by moxibustion (a) Mice were injected intraperitoneally with 10 LD50Staphylococcus aureus as an

acute bacterial model At 1 h later mice were untreated or administeredmoxibustion as indicatedThe parallel tests without bacterial infectionwere performed following the same procedures Survival was monitored for 48 h after moxibustion (119899 = 10micegroup) and the log-rank testwas performed for the survival curve119875 lt 005 primed group versus 30minmoxibustion group (b) Oncemice died or were sacrificed at 48 hafter moxibustion peritoneal fluids were collected immediately and plated on LB agar to measure the activity of bacterial colony formationlowastlowast119875 lt 001

33 Moxibustion Protects Experimental Mice from BacterialInfection Because elevated autophagy promotes bacterialclearance of macrophages we hypothesized moxibustionwould protect mice against bacterial infection with Staphy-lococcus aureus Bacterial challenge caused 90 mortality inprimedmice at 48 h duringwhich the bacteria did not escapefrom killing by macrophages postinfection [37 38] Thegroup receiving 15 min moxibustion showed little differencein survival compared with the control group whereas the30 min moxibustion group was significantly protected fromdeath and acute infection (Figure 3(a))This suggested the 30minmoxibustion induced a protective effect against bacterialinfection Furthermore remaining bacteria were detectedafter moxibustion As shown in Figure 3(b) the bacterialloads in the peritoneal fluid were dramatically reducedwhen mice were administered moxibustion for 15min or30min although the latter was more effective In conclusionmoxibustion provides a protective response and enhancedclearance of bacterial infection in macrophages

4 Discussion

In the present study we revealed that macrophage autophagyin the peritoneal fluid was elevated when moxibustionwas applied to experimental mice Moxibustion provided aprotective response against bacterial infection This effect

probably contributes to the physiological stress response withAkt inhibition and activation of ER stress

Moxibustion is one physical therapy in traditionalmedicine that utilizes heat stimulation to cure disease orquell pain It might induce a series of signal pathwaysand stress through heat shock proteins [22 39] includingthe Akt pathway and ER stress as shown in the presentstudy Although the effect of moxibustion in the whole bodyrequires more evidence moxibustion is a suitable techniquefor local heat therapy to stimulate self-healing recoveryand innate defense mechanisms It is important to preventsevere heat stimuli bymoxibustion that can cause burn injuryfollowing inflammation However heat therapy is beneficialwhen the heat stimulation is artificially controlled to a uniquesite without injury [40 41]

Even though the effects of moxibustion have not yet beensystemically evaluated emerging studies show moxibustioncan alter gene expression and signal transduction The long-distance effect of moxibustion may be due to neural reflexes[25] whereas local effects are mediated through cytokinesand signal transductionThepresent study and previous stud-ies have revealed moxibustion activates a unique cytokineprofile and signal transduction pathways [22 27ndash34 4243] as well as elevating macrophage autophagy (Figure 1)and natural killer cell activity [34] These results suggestmoxibustion generates a protective response against viral


and bacterial infection although it is not clear whether itfunctions locally or systemically

Although the studies have shown moxibustion con-tributes to the systemic and comprehensive immune inflam-matory and analgesic activities our finding represents a novelprotective mechanism that the mild moxibustion promotesthe autophagy and bactericidal function of macrophageThiseffect is probably contributed to the inhibition of Akt phos-phorylation and the activation of eIF2120572 phosphorylationtwo key signal pathways in a variety of stresses Howeverthe upstream signaling of moxibustion remains elusive andthe further systematic analysis regarding metabolism andendocrine under moxibustion will greatly help to understandthe mechanism Alternatively it is urgent to improve theclinical efficacy of moxibustion as well as to definete theindications and usage

5 Conclusion

Our data provide evidence that moxibustion augmentsautophagy in macrophages and innate defense against bacte-rial infection Based on our present study mild moxibustionis effective at preventing and treating infectious diseasein mice acting as a simple and convenient activator ofautophagy in physical therapy

Conflict of Interests

The authors declare that there is no potential conflict ofinterests disclosed in this investigation

References

[1] D J Klionsky ldquoAutophagy from phenomenology to molecularunderstanding in less than a decaderdquoNature Reviews MolecularCell Biology vol 8 no 11 pp 931ndash937 2007

[2] Z Yang andD J Klionsky ldquoMammalian autophagy coremolec-ular machinery and signaling regulationrdquo Current Opinion inCell Biology vol 22 no 2 pp 124ndash131 2010

[3] B Levine N Mizushima and H W Virgin ldquoAutophagy inimmunity and inflammationrdquo Nature vol 469 no 7330 pp323ndash335 2011

[4] N Mizushima and B Levine ldquoAutophagy in mammaliandevelopment and differentiationrdquo Nature Cell Biology vol 12no 9 pp 823ndash830 2010

[5] A M Choi S W Ryter and B Levine ldquoAutophagy in humanhealth and diseaserdquo The New England Journal of Medicine vol368 no 7 pp 651ndash662 2013

[6] B Levine and G Kroemer ldquoAutophagy in the pathogenesis ofdiseaserdquo Cell vol 132 no 1 pp 27ndash42 2008

[7] M Kundu and C B Thompson ldquoAutophagy basic principlesand relevance to diseaserdquo Annual Review of Pathology Mecha-nisms of Disease vol 3 pp 427ndash455 2008

[8] D C Rubinsztein P Codogno and B Levine ldquoAutophagymod-ulation as a potential therapeutic target for diverse diseasesrdquoNature Reviews Drug Discovery vol 11 no 9 pp 709ndash730 2012

[9] Z J Yang C E Chee S Huang and F A Sinicrope ldquoThe roleof autophagy in cancer therapeutic implicationsrdquo MolecularCancer Therapeutics vol 10 no 9 pp 1533ndash1541 2011

[10] C M Kenific A Thorburn and J Debnath ldquoAutophagy andmetastasis another double-edged swordrdquo Current Opinion inCell Biology vol 22 no 2 pp 241ndash245 2010

[11] E White and R S DiPaola ldquoThe double-edged sword ofautophagy modulation in cancerrdquo Clinical Cancer Research vol15 no 17 pp 5308ndash5316 2009

[12] T Shintani andD J Klionsky ldquoAutophagy in health and diseasea double-edged swordrdquo Science vol 306 no 5698 pp 990ndash9952004

[13] J Huang and J H Brumell ldquoBacteria-autophagy interplay abattle for survivalrdquo Nature Reviews Microbiology vol 12 no 2pp 101ndash114 2014

[14] S Mostowy ldquoAutophagy and bacterial clearance a not so clearpicturerdquo Cellular Microbiology vol 15 no 3 pp 395ndash402 2013

[15] A Choy and C R Roy ldquoAutophagy and bacterial infection anevolving arms racerdquo Trends in Microbiology vol 21 no 9 pp451ndash456 2013

[16] J Huang and J H Brumell ldquoAutophagy in immunity againstintracellular bacteriardquo Current Topics in Microbiology andImmunology vol 335 no 1 pp 189ndash215 2009

[17] S Shoji-Kawata R Sumpter M Leveno et al ldquoIdentification ofa candidate therapeutic autophagy-inducing peptiderdquo Naturevol 494 no 7436 pp 201ndash206 2013

[18] E Kuang C Y M Okumura S Sheffy-Levin et al ldquoRegulationof ATG4B stability by RNF5 limits basal levels of autophagy andinfluences susceptibility to bacterial infectionrdquo PLoS Geneticsvol 8 no 10 Article ID e1003007 2012

[19] S-H Yi ldquoThermal properties of direct and indirect moxibus-tionrdquo Journal of Acupuncture andMeridian Studies vol 2 no 4pp 273ndash279 2009

[20] S Y Kim S H Yi J H Cho C S Yin H Lee and H JPark ldquoHeat stimulation on the skin for medical treatment canit be controlledrdquo Journal of Alternative and ComplementaryMedicine vol 17 no 6 pp 497ndash504 2011

[21] J H Chiu ldquoHow does moxibustion possibly workrdquo Evidence-Based Complementary and Alternative Medicine vol 2013Article ID 198584 8 pages 2013

[22] Y O Cakmak ldquoA review of the potential effect of elec-troacupuncture andmoxibustion on cell repair and survival therole of heat shock proteinsrdquoAcupuncture inMedicine vol 27 no4 pp 183ndash186 2009

[23] H Deng and X Shen ldquoThemechanism of moxibustion ancienttheory and modern researchrdquo Evidence-Based Complementaryand Alternative Medicine vol 2013 Article ID 379291 7 pages2013

[24] I Jang and J Park ldquoCan carbon dioxide laser substitutemoxibustionrdquo Lasers in Medical Science vol 24 no 2 p 2902009

[25] E Noguchi H Ohsawa and K Takagi ldquoNeural mechanismof localized changes in skeletal muscle blood flow causedby moxibustion-like thermal stimulation of anesthetized ratsrdquoJournal of Physiological Sciences vol 59 no 6 pp 421ndash427 2009

[26] X Shen L Zhao G Ding et al ldquoEffect of combined laseracupuncture on knee osteoarthritis a pilot studyrdquo Lasers inMedical Science vol 24 no 2 pp 129ndash136 2009

[27] E Zhou H Liu H Wu et al ldquoDown-regulation of proteinand mRNA expression of IL-8 and ICAM-1 in colon tissueof ulcerative colitis patients by partition-herb moxibustionrdquoDigestive Diseases and Sciences vol 54 no 10 pp 2198ndash22062009


[28] C Bao LWuHWu et al ldquoMoxibustion inhibits apoptosis andtumor necrosis factor-alphatumor necrosis factor receptor 1 inthe colonic epithelium of crohnrsquos disease model ratsrdquo DigestiveDiseases and Sciences vol 57 no 9 pp 2286ndash2295 2012

[29] M Kogure N Mimura H Ikemoto et al ldquoMoxibustionat mingmen reduces inflammation and decreases IL-6 in acollagen-induced arthritis mouse modelrdquo Journal of Acupunc-ture and Meridian Studies vol 5 no 1 pp 29ndash33 2012

[30] X M Wang Y Lu L Y Wu et al ldquoMoxibustion inhibitsinterleukin-12 and tumor necrosis factor alpha and modulatesintestinal flora in rat with ulcerative colitisrdquo World Journal ofGastroenterology vol 18 no 46 pp 6819ndash6828 2012

[31] Y Shi Y Cui H Wu et al ldquoEffects of mild-warming moxibus-tion onBcl-2 andPKCexpressions of peripheral blood in elderlypeoplerdquo Journal of Traditional Chinese Medicine vol 32 no 1pp 45ndash51 2012

[32] Z H Yue X Q He X R Chang et al ldquoThe effect of herb-partition moxibustion on toll-like receptor 4 in rabbit aortaduring atherosclerosisrdquo Journal of Acupuncture and MeridianStudies vol 5 no 2 pp 72ndash79 2012

[33] XWang Y Lu L Wu et al ldquoMoxibustion inhibits the ERK sig-naling pathway and intestinal fibrosis in rats with Crohnrsquos dis-easerdquoEvidence-BasedComplementary andAlternativeMedicinevol 2013 Article ID 198282 12 pages 2013

[34] Y Takayama M Itoi T Hamahashi et al ldquoMoxibustionactivates host defense against herpes simplex virus type Ithrough augmentation of cytokine productionrdquo Microbiologyand Immunology vol 54 no 9 pp 551ndash557 2010

[35] ZMounir J L Krishnamoorthy SWang et al ldquoAkt determinescell fate through inhibition of the PERK-eIF2120572 phosphorylationpathwayrdquo Science Signaling vol 4 no 192 article ra62 2011

[36] G Kroemer G Marino and B Levine ldquoAutophagy and theintegrated stress responserdquo Molecular Cell vol 40 no 2 pp280ndash293 2010

[37] M Kubica K Guzik J Koziel et al ldquoA potential new path-way for Staphylococcus aureus dissemination the silent sur-vival of S aureus phagocytosed by human monocyte-derivedmacrophagesrdquo PLoS ONE vol 3 no 1 Article ID e1409 2008

[38] G J Jakab and G M Green ldquoDefect in intracellular killing ofStaphylococcus aureus within alveolar macrophages in Sendaivirus infected murine lungsrdquo Journal of Clinical Investigationvol 57 no 6 pp 1533ndash1539 1976

[39] J YuH Peng Y Lin and S Yi ldquoEffect ofmoxibustion treatmenton cell apoptosis and expressions of heat shock protein andsecond mitochondrial activator of caspase in acute gastricmucosal lesion of ratsrdquo Journal of Traditional Chinese Medicinevol 33 no 2 pp 258ndash261 2013

[40] S I Zeitlin ldquoHeat therapy in the treatment of prostatitisrdquoUrology vol 60 supplement 6 pp 38ndash41 2002

[41] A Chandler J Preece and S Lister ldquoUsing heat therapy for painmanagementrdquo Nursing Standard vol 17 no 9 pp 40ndash42 2002

[42] K Kimura H Takeuchi K Yuri and I Wakayama ldquoInhibitionof nitric oxide synthase attenuates cutaneous vasodilation dur-ing warm moxibustion-like thermal stimulation in humansrdquoJournal of Alternative and Complementary Medicine vol 18 no10 pp 965ndash970 2012

[43] H Y Yin Y Tang S F Lu et al ldquoGene expression profilesat moxibustioned site (ST36) a microarray analysisrdquo Evidence-Based Complementary and Alternative Medicine vol 2013Article ID 890579 7 pages 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


autophagy of intraperitoneal macrophages in experimentalKunming mice following the inactivation of Akt and theinduction of eIF2120572 phosphorylation Consequently it con-tributes to a protective response against bacterial infectionindicating moxibustion activates innate defenses to preventinfectious disease

2 Methods and Materials

21 Mice and Ethics Statement Specific-pathogen-free maleKunming mice (6ndash8 weeks of age 18ndash22 g weight) weremaintained and bred according to the guidelines approved bythe Laboratory Animal Center Sun Yat-sen University

22 Mild Moxibustion on Lethal Infections of Staphylococcusaureus Forty mice were randomly divided into four groupsA control group B model group C 15 min moxibustiongroup and D 30 min moxibustion group with 10 miceper group Except for the control group the remaining 30mice were primed with intraperitoneal injection of 6 starchbroth for 2 days followed by intraperitoneal injection of 10LD50Staphylococcus aureus as an acute infection model One

hour after injection with bacteria mice in groups C and Dwere restrained and administered 1206014mm stick moxibustionfor 15min and 30min at Guanyuan acupuncture point (CV4) at the front of the small intestine In the control groupand model group 1206014mm sticks of unlighted moxa herbswere used as the control for moxibustion The death ofmice was monitored continuously for 48 h after injectionto prepare a survival curve Peritoneal fluids were collectedimmediately upon death or sacrifice at 48 h aftermoxibustionand subjected to bacterial colony formation inLuria-Bertani(LB) agar plates The numbers of colonies were used toevaluate bacterial clearance

23MildMoxibustion onMacrophage Autophagy Fortymicewere randomly divided into four groups A control group Bmodel group C 15 min moxibustion group and D 30 minmoxibustion group In B C and D groups mice receivedintraperitoneal injection of 6 starch broth as a macrophageinducer and two days later mice in C and D groups wereadministered moxibustion at CV4 as described above andsacrificed at 1 h after moxibustion Peritoneal fluid cellswere collected and subjected to autophagy assays To detectthe dynamic Akt and eIF2120572 phosphorylation under longermoxibustion an additional group with 60 min moxibustionwas performed following the procedure above

24 Assays of Autophagy and Autophagic Pathways A smallnumber of fresh macrophages were fixed with 3 formalde-hyde in phosphate buffer (PBS) for 10min and permeabilizedwith 05 Triton X-100 in PBS for 10min After washingwith PBS twice the cells were blocked with 1 bovine serumalbumin in PBS with 01 Triton X-100 for 30min andthen incubated with rabbit polyclonal anti-LC3 antibody(1 200 MBL International MA USA) for 1 h After threewashes with PBS with 01 Triton X-100 the cells wereincubated with FITC-conjugated anti-rabbit IgG secondary

antibody (1 500 Life Technologies) for 05 h counterstainedwithDAPI (410158406-diamidino-2-phenylindole Sigma St LouisMO) and mounted in antifade agent (Invitrogen Carls-bad CA) Finally LC3 staining was visualized by confocalfluorescence microscopy The remaining macrophages werelysed and total protein extracts were subjected to westernblots with anti-LC3 antibody phospho-eIF2120572 (Ser51) rabbitmonoclonal antibody (mAb) phospho-Akt (Ser473) rabbitmAb and anti-Akt and eIF2120572 rabbit antibodies (1 1000 CellSignaling Technology MA USA) Images were visualized byLicor Odyssey or Pierce ECL system

25 Statistical Analysis The survival rates were analyzed by alog-rank test and the statistical analysis was performed withone-way analyses of variance (ANOVA)

3 Results

31 Moxibustion Promotes Autophagy in Macrophages Toassess whether moxibustion affected autophagy moxibustionwith a small size moxa stick was applied to the mouseabdomen Guanyuan point (CV4) for 15 and 30 minutesMacrophages were then isolated from the peritoneal fluidafter mice were sacrificed and subjected to autophagy assayWhen macrophages were primed by starch and albumenautophagic LC3-postive vacuoles were not observed inmacrophages similar to that seen in control mice whereasLC3-positve puncta were greatly increased after 15 min or30 min moxibustion (Figures 1(a) and 1(b)) Furthermorewhen LC3 was detected in cell extracts the ratio of LC3-II was elevated following moxibustion compared with mockand primed conditions (Figure 1(c)) The levels of LC3-IIprotein and LC3 punctawere similar under bothmoxibustionconditions These results suggest that moxibustion inducesthe macrophage autophagy in mice

32 Moxibustion Induces eIF2120572 Phosphorylation andSuppresses Akt Phosphorylation We attempted to addressupstream signaling pathways by whichmoxibustion activatesautophagy Akt inhibits endoplasmic reticulum (ER) stress byphosphorylating eIF2120572-kinase PERK [35] and the PI3KAktmTOR pathway acts as a well-characterized negative regu-lator of autophagy [36] Because moxibustion delivers heatstimulation to the operative site we hypothesized it induceda stress response and then Akt activity and ER stress wereexamined after moxibustion To diminish the individualdifferences in mice we investigated ER stress in mixedmacrophages (Figure 2(a)) and random macrophages(Figure 2(b)) As shown in Figures 2(a) and 2(b) in top panelphosphorylation of eIF2120572 in macrophages was significantlyelevated at 30min and then slightly fell back at 1 h duringmoxibustion indicating a rapid ER stress response wasactivated Furthermore the phosphorylation of Akt wasdecreased dramatically at both 30min and 1 h suggestingmoxibustion suppressed Akt activation (Figures 2(a) and2(b) in bottom panel) Thus we concluded that moxibustioninduced autophagy in macrophages by suppressing the Aktpathway and activating ER stress


Mock Primed

Mox 15998400 Mox 30

998400

(a)

0

1

2

3

4

5

6

Mock Primed

LC3

punc

ta ra

tio

Mox 15998400 Mox 30

998400


(b)

LC3

Actin

LC3-I

LC3-II

Moc

k

Prim

ed

Mox

15998400

Mox

30998400

(c)


Akt

pAkt

Actin

Moc

k

Prim

ed

eIF2120572

peIF2120572

Mox

30m

in

Mox

60m

in

(a)

Prim

ed

Akt

pAkt

eIF2120572

peIF2120572

15m

in

30m

in

60m

in

(b)



0

20

40

60

80

100

0


12 24

(h)36 48





()

(a)

0

1

2

3


998400

lowastlowast

lowastlowast

11 plusmn 07

017 plusmn 012

0042 plusmn 0023

Bact

eria

left

(cfu

times107m

L)(b)




4 Discussion








5 Conclusion




References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Mock Primed

Mox 15998400 Mox 30

998400

(a)

0

1

2

3

4

5

6

Mock Primed

LC3

punc

ta ra

tio

Mox 15998400 Mox 30

998400


(b)

LC3

Actin

LC3-I

LC3-II

Moc

k

Prim

ed

Mox

15998400

Mox

30998400

(c)


Akt

pAkt

Actin

Moc

k

Prim

ed

eIF2120572

peIF2120572

Mox

30m

in

Mox

60m

in

(a)

Prim

ed

Akt

pAkt

eIF2120572

peIF2120572

15m

in

30m

in

60m

in

(b)



0

20

40

60

80

100

0


12 24

(h)36 48





()

(a)

0

1

2

3


998400

lowastlowast

lowastlowast

11 plusmn 07

017 plusmn 012

0042 plusmn 0023

Bact

eria

left

(cfu

times107m

L)(b)




4 Discussion








5 Conclusion




References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

0


12 24

(h)36 48





()

(a)

0

1

2

3


998400

lowastlowast

lowastlowast

11 plusmn 07

017 plusmn 012

0042 plusmn 0023

Bact

eria

left

(cfu

times107m

L)(b)




4 Discussion








5 Conclusion




References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion




References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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