| ObjectivesMacrophage (Macrophage, Mφ) are differentiated cells derived fromhematopoietic stem cells, and have high plasticity and multiple functions inkilling, clearance and wound repair. They are distributed in almost all organsand tissues including the blood, brain, lung, liver, spleen, kidney, bones, jointsand connective tissues. A range of stimuli, including the alteration of themicroenvironment and endocrine, exogenous materials and aging conditions,trigger the differentiation of macrophages quickly, efficiently and reversibly togenerate two types of phenotype: anti-inflammatory killing (M1) andanti-inflammatory repair (M2), with the key functions in host defense, tissueremodeling, immune modulation. Uncontrolled severe inflammatory responseis the major factor of tissue injury and death during acute and sub-acuteinfections, while excessive anti-inflammatory response is the key step in theprogress of chronic infection, metabolic disorders, cancer and aging-relateddiseases. Acupuncture and moxibustion have significant clinic effects and arewidely used in the prevention and treatment of diseases, the care of senilityand dementia, etc. Therefore, the study of the regulation on macrophage byacupuncture and moxibustion will greatly help to understand the mechanismand improve the clinical efficacy of acupuncture and moxibustion, as well asto definite the modern spectrum of disease for two therapies, even though themolecular and cell biology of acupuncture and moxibustion remains to befurther interpreted. Furthermore, a large number of ancient literatures and alarge panel of the therapeutic data of acupuncture and moxibustion show they activate the inflammatory response and release the pain, the effects arecontributed to the systemic, multi-central, multi-level comprehensiveanti-inflammatory and analgesic activities. However, the mild moxibustionmay have distinct anti-inflammatory effects. The topics of the present studiesare to reveal whether the mild moxibustion provides a protective effect againstacute infection of Staphylococcus aureus, promotes the phagocytosis,autophagy and bactericidal function of macrophage, and the profile of avariety of pro-inflammatory and anti-inflammatory cytokines, to provide theexperimental evidences that mild moxibustion prevent the bacterial infectionand inflammatory diseases by activating macrophage autophagy and theautophagy-related signal pathway, aimed to develop the clinical application ofacupuncture and moxibustion.Methods1. The protective effect of mild moxibustion against lethalStaphylococcus aureus infections:40SPF level male Kunming mice (18-22g in weight), were randomly divided into four groups: A, control group; B,model group; C,15min moxibustion group; D,30min moxibustion groups,10mice/group. Compared to the control group, the remaining30mice wereprimed by intraperitoneal injection of6%starch broth2days, followingintraperitoneal injection of Staphylococcus aureus as an acute infection model.After injection with bacteria for1hour, the mice of groups C and D wereadministered with moxibustion at Guanyuan for15min and30min. The deathof mice was recorded continuously in48h post-injection to draw survivalcurve. Peritoneal fluids were immediately collected once the mice died, andsubjected to bacterial counting assay in LB agar plates, the numbers ofcolonies were observed to evaluate the bacterial infection. All mice and theirorgans were weighted to detect anatomical reflect of inflammation by thethymus index, spleen index. 2. Mild moxibustion influences macrophage bactericidal activity andinflammatory cytokines:(1) The effect of instant moxibustion on macrophagebactericidal activity.24SPF level male Kunming mice were randomly dividedinto three groups: the model group, control group, and instant moxibustiongroup. All mice with intraperitoneal injection of6%starch broth for2dayswere administered with intraperitoneal injection of Escherichia coli (MOI=20), the model group were sacrificed at0h after bacterial infection, the controlgroup sacrificed at2h.The moxibustion group were performed with0.5hmoxibustion at Guanyuan immediately after infection and then sacrificed at2h later. The peritoneal fluids were collected and a series of dilution wereplated on LB agar plate to count the bacterial colonies.(2) Effect ofmacrophage phagocytosis by moxibustion.32SPF level male Kunming micewere randomly divided into four groups: A, model group; B, the control group;C,0.5h moxibustion group; D,1h moxibustion group. All mice were operatedwith intraperitoneal injection of6%starch broth for2days and intraperitonealinjection of Escherichia coli (MOI=20) as described above. The model andcontrol group were sacrificed at0h and4h after infection, respectively. GroupsC and D were administered with moxibustion at Guanyuan for0.5h and1hrespectively, then were sacrificed at4h after moxibustion. The peritonealfluids were collected and the bacterial colonies were tested as described above.(3) The effect of moxibustion on the expression of inflammatory factors.16SPF level male Kunming mice were divided and operated as (2) above, andperitoneal macrophages were collected at6h after infection, total RNA wasextracted to detect the expression levels of15key inflammatory factors andinflammation-related protein caspase-1by qRT-PCR.3. The effect of mild moxibustion on macrophage autophagy andautophagy related signal pathway:40SPF level male Kunming mice wererandomly divided into four groups: A, control group; B, model group; C,0.5h moxibustion group; D,1h moxibustion group. In B-D group, intraperitonealinjection of6%starch broth as macrophage activator was given to the micebefore moxibustion experiment. Two days later, the mice in C-D groups wereoperated with moxibustion at Guanyuan point and were sacrificed in1h aftermoxibustion. Peritoneal fluid cells in each group were collected and subjectedto autophagy assays. The LC3staining of fixed macrophage was visualized byconfocal fluorescence microscopy and LC3processing was examined bywestern blots. Subsequently total protein extracts of macrophage fromperitoneal fluids were detected with the phosphorylated Akt and eIF2αchange.Results1. After a lethal Staphylococcus aureus infection at mortality of90%for48h, moxibustion for30min significantly increased the survival rate (P <0.05),moxibustion15min group slightly improved survival but the difference wasnot statistically significant (P>0.05). At the time points of death, the bacteriafrom the mice of15min moxibustion group and30min moxibustion groupwere significantly reduced compared to the model group (P <0.05, P <0.01),and the longer moxibustion time are more efficient (P <0.05). Comparison ofthe thymus and spleen index show30min moxibustion were significantlyreduced thymus and spleen index (P <0.01), as well as moxibustion15mingroup did so on thymus (P <0.05) and spleen index (P <0.01). The longermoxibustion has a smaller spleen index (P <0.05).2. Under MOI=20of E. coli infection, instant moxibustion groupshowed31.6%of bactericidal activity, decreased the activity compared to68.3%in the control group (P <0.05). While moxibustion at1h later, thebactericidal activity was significantly increased compared to the control group(P <0.01), and1h moxibustion raised a more efficient bactericidal activity than 0.5h moxibustion (P<0.05). Compared the expression of16genes in controlgroup with these in the model group, the expression of TGFb, Fizz1, IF1204genes were down-regulated, while the expression of other inflammatorycytokines were up-regulated in various degrees, in which IL-1β and iNOSwere up-regulated, respectively, up to100and500folds. Compared to themodel group, moxibustion from0.5h to1h, IL1α and IL1β were slightlyincreased (P<0.05) and later reduced (P<0.01), and TNFα gene expressiondeclined in continual manner (P <0.05), while the expression of iNOS andIL10were significantly down-regulated starting at a delayed time point (P<0.01). Compared the control group to the model group, the expression ofcaspase-1, an inflammatory proteins, was increased about six folds, andmoxibustion for both0.5h and1h restored caspase-1expression (P <0.05).The other10genes did not shown the significant differences before and aftermoxibustion.3. In mouse peritoneal macrophages, the control group and the modelgroup show a background level of LC3puncta, while after moxibustion for0.5h or1h, LC3staining shown a higher LC3puncta in the cytoplasm, indicatingthat moxibustion induced a significant autophagy (P <0.01). Confocalmicroscopy confirmed the elevated autophagic LC3puncta in the macrophageof moxibustion group, with the leaf and diffused nuclei which may be causedby excessive autophagic cell death. Compared the model group to the controlgroup, the combination of both Akt and eIF2α phosphorylation lead to theslightly elevated autophagy, by which the phosphorylation of Akt inhibitsautophagy and eIF2α phosphorylation induces autophagy. However,moxibustion for both0.5h and1h greatly reduced Akt phosphorylation andpromotes eIF2α phosphorylation, by which they played a cooperative role inthe activation of autophagy, indicating that moxibustion promote autophagythrough activating Akt and eIF2α related autophagic pathway. ConclusionThe results of this study showed that a lethal dose of S.aureus infectionthrough intraperitoneal injection induces the death in experimental mice withthe persist infection and excessive inflammatory damages, mild moxibustionprovide a significant protective effect by reducing the mortality rate of mice,bacterial infection, inflammation, and the organ damage. The protective effectwas relatively correlated with the operation time. This result indicates mildmoxibustion has anti-bacterial infection and anti-inflammatory effects.The protective effect of mild moxibustion against bacterial infectionoccurs after the bacteria are engulfed into macrophages, instant moxibustionimmediately after infection is not beneficial to bacterial clearance. Aftermacrophages capture and gulf bacteria, moxibustion can significantly improvethe bactericidal activity of macrophages with a positive correlation withmoxibustion time up to1h. Unlike direct moxibustion which activateinflammation, mild moxibustion raises the opposite effect which rapidlypromotes bacterial clearance and down-regulates the expression of IL1α, IL1β,IL10, TNFα, iNOS and other inflammatory genes except that IL1α and IL1βwere slight increased at the beginning time points. Continuingdown-regulation of the inflammatory genes caspase-1further confirm thatmild moxibustion mainly inhibits severe inflammation, which may be the keypoint distinct from the acupuncture and direct moxibustion.Autophagy is a cellular self-eating process by which the proteinaggregates, exogenous materials and damaged organelles inside cells wereengulfed and degraded through lysosme, and it can be triggered by stress,starvation, pathogens and other stimuli. Moxibustion can activate autophagy inmacrophage, marked by elevated LC3puncta and LC3-I/II shift. Themechanism is probably contributed to the fact that moxibustion resulted in theinhibition of Akt phosphorylation and the activation of eIF2α phosphorylation, two key upstream signal pathways of autophagy.In conclusion, the heat stimulation of mild moxibustion promotesautophagy through multiple pathways of metabolism and endocrine;subsequently accelerate the expression of inflammatory factors and bacterialclearance of macrophages, which represent an innate bactericidal defense andanti-inflammatory process to alleviate severe inflammatory injury and acuteinflammation. The present studies provide the experimental evidences thatmild moxibustion and its combination with acupuncture are effective therapyand intervention for infectious diseases and inflammatory diseases. |
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