【作者】 李晓娟；

【导师】 孙国杰；

【作者基本信息】 湖北中医药大学 ， 针灸推拿学， 2014， 博士

【摘要】 目的巨噬细胞(Macrophage,Mφ)是来源于造血干细胞的具有杀伤、修复双重功能的高度可塑性分化细胞，驻留于几乎所有的器官组织中。在胞外微环境、内分泌、异源物质和衰老等条件刺激下，巨噬细胞能可逆性地分化为炎症杀伤（M1）和抗炎修复（M2）两种类型。正常的巨噬细胞功能是宿主防御、组织重建、免疫调节的中坚力量，失控的炎症反应是感染引起组织损伤和死亡的主要因素，而抗炎过度引起的免疫抑制则是各种慢性感染、代谢失调、癌症、自身免疫、衰老相关疾病的关键步骤。中医针灸具有双向良性调节作用而广泛应用于各种疾病的预防、治疗和防衰保健中，因此研究针灸对巨噬细胞的作用和机制对于了解针灸机理、提高针灸临床疗效和对现代疾病谱的适用具有重要意义。我们通过对比大量古代文献和现代研究发现，针灸很大一部分治疗作用与机体多系统、多中心、多层次的炎症与抗炎、疼痛与镇痛有关，但温和灸的抗炎作用明显不在此列。本课题以温和灸对金黄色葡萄球菌急性感染的治疗作用为切入点，围绕温和灸对巨噬细胞的吞噬、杀菌功能以及多种促炎、抗炎细胞因子表达的影响，探讨温和灸对于细菌感染和炎症的整体调节作用及机制，及更深层次的细胞自噬通路机制，为针灸的临床应用提供实验依据。方法1.温和灸对金黄色葡萄球菌致死性感染的保护作用：SPF级昆明小鼠雄性40只，体重18~22克，随机分为四组：A、空白对照组；B、模型组；C、艾灸15min组；D、艾灸30min组，每组10只。除空白对照组外，其余30只腹腔注射6%淀粉肉汤2天后再腹腔注射金黄色葡萄球菌作为感染模型。感染后模型组不做处理，C、D两组分别于感染后1小时艾灸小鼠关元穴15min和30min。随后持续48h观察和记录小鼠死亡情况，绘制生存力曲线。小鼠死亡后立即取腹腔液梯度稀释后用琼脂平板计数菌落数，定量观察细菌感染程度。解剖所有小鼠称重检测反映炎症程度的胸腺指数、脾指数。2.温和灸对巨噬细胞杀菌活性和炎症细胞因子表达的影响:○1感染后即刻艾灸对巨噬细胞杀菌活性的影响。SPF级昆明小鼠雄性24只随机分为三组：模型组；内对照组；即刻艾灸组。所有小鼠腹腔注射6%淀粉肉汤2天后再腹腔注射大肠杆菌（MOI=20）作为感染模型，模型组感染后0h处死，内对照组感染后2h处死，即刻艾灸组感染后立即艾灸关元穴0.5h并于2h处死。各组取腹腔液梯度稀释后用琼脂平板计数菌落数。○2感染吞噬后艾灸对巨噬细胞杀菌活性的影响。SPF级昆明小鼠雄性32只随机分为四组：A、模型组；B、内对照组；C、艾灸0.5h组；D、艾灸1h组。所有小鼠腹腔注射6%淀粉肉汤2天后再腹腔注射大肠杆菌（MOI=20）作为感染模型，模型组感染后0h处死，内对照组感染后4h处死，C、D两组感染1h后分别艾灸关元穴0.5h和1h并于4h处死。各组取腹腔液梯度稀释后用琼脂平板计数菌落数。○3感染吞噬后艾灸对炎症细胞因子表达的影响。SPF级昆明小鼠雄性16只，分组和处理同○2，各组于感染后6h处死取腹腔巨噬细胞，提取总RNA逆转录后用qRT-PCR定量检测15种炎症相关因子和炎症体蛋白caspase-1的表达水平。3.温和灸对巨噬细胞自噬和自噬信号通路的影响：SPF级昆明小鼠雄性40只随机分为四组：A、空白对照组；B、模型组；C、艾灸0.5h组；D、艾灸1h组。除空白对照组外，其余30只艾灸实验前2天腹腔注射6%淀粉肉汤作为巨噬细胞活化模型，2天后对C、D两组分别艾灸关元穴0.5h和1h后处死小鼠。每组取腹腔液细胞经固定免疫荧光染色和悬浮免疫荧光染色后分别用普通荧光显微镜和激光共聚焦荧光显微镜观察含LC3染色的自噬体光点的计数变化。随后提取腹腔液细胞总蛋白用Western blot检测磷酸化Akt和eIF2α含量变化。结果1.金黄色葡萄球菌致死感染后，艾灸30min组显著提高48h内的生存率(P<0.05),艾灸15min组生存率有所提高但差异不显著（P>0.05）。感染致死后，艾灸15min组、艾灸30min组腹腔细菌感染程度与模型组比较明显减轻（P<0.05，P<0.01），艾灸时间长者感染较轻（P<0.05）。致死感染后模型组与空白组对比胸腺与脾脏指数变大（P<0.01）,艾灸30分钟组显著减小胸腺与脾脏指数（P<0.01），艾灸15min组也使胸腺（P<0.05）与脾脏指数减小（P<0.01）。艾灸时间长者脾脏指数更小（P<0.05）。2.在20=MOI大肠杆菌感染后即刻艾灸组杀菌活性为31.6%，与内对照组的68.3%相比反而有所下降（P<0.05）。感染后允许巨噬细胞吞噬1h后艾灸的杀菌活性与内对照组比较显著上升（P<0.01），艾灸1h组杀菌活性比艾灸0.5h组升高明显（P<0.05）。内对照组与模型组比较，所检测的16个基因中除TGFb,Fizz1,IF1204等基因被下调外，其余炎症因子基因表达均不同程度的上调，其中IL-1β和iNOS分别被上调100和500倍。与模型组比较，从艾灸0.5h至1h之间， IL1α和IL1β先轻微上调（P<0.05）然后下调（P<0.01），TNFα基因表达持续下降（P<0.05）。iNOS和IL10开始变化不大之后显著下调（P<0.01）。内对照组与模型组比较炎症体蛋白caspase-1表达量约提高6倍，艾灸0.5h和1h均使caspase-1基因表达下降（P<0.05）。艾灸后其余10个基因表达变化差异不显著。3.在空白对照组和模型组中，自噬标志物LC3光点数为较低的本底水平；普通荧光显微镜下，艾灸0.5小时或1小时后,小鼠腹腔巨噬细胞胞质中的LC3颗粒变多,在细胞质中形成弥散的颗粒状分布,数量与未艾灸对照活化组的差异具有统计学意义(P<0.01)，说明自噬水平显著增加。激光共聚焦结果与普通显微镜一致，并且显示艾灸1h情况下细胞核具有分叶、弥散状况，可能与自噬过度引起的凋亡有关。模型组与空白对照组比较，mTOR通路中的Akt磷酸化上调可以抑制自噬活化，mTOR非依赖通路的eIF2α磷酸化上调可以诱导自噬活化，二者结合的情况下自噬活化增加不多。与模型组比较，艾灸0.5h组和艾灸1h组使Akt磷酸化下调，eIF2α磷酸化上调，故起到了全面激活自噬的作用，艾灸促进自噬的作用与mTOR依赖的p-Akt的下调和mTOR非依赖的p-eIF2α的上调均有关。结论1.以致死剂量的金黄色葡萄球菌感染小鼠腹腔诱发的动物死亡与感染的持续存在、器官的炎症损伤过度均有关，艾条温和灸可以起到使小鼠死亡率显著下降、细菌感染减少、炎症损伤减轻的保护作用，效果与时间正相关，表明温和灸有明显的抗细菌感染、抗炎作用。2.温和灸的抗细菌感染作用发生在细菌被巨噬细胞吞噬步骤之后，过早艾灸不利于感染的清除。在巨噬细胞吞噬细菌之后进行艾灸能显著地提高巨噬细胞的杀菌活性，显著性与艾灸时间呈正相关，但是过长时间的艾灸有可能对巨噬细胞不利。温和灸的作用主要是抑制炎症，这可能是温和灸作用机制不同于针刺、直接灸之处。3.温和灸诱导巨噬细胞内自噬反应显著上升。温和灸对自噬的激活作用与Akt磷酸化下调和eIF2α磷酸化上升有关。温和灸的温热效应可能通过多个途径促进自噬，加快对内吞细菌的清除，是一种不依赖于免疫炎症活化的杀菌抗炎过程，对引起组织损伤的急性感染和过度炎症能够实施有效的干预，对于温和灸单独和与其它方法协同应用于多种感染、炎症、免疫异常相关疾病提供了理论根据。更多还原

【Abstract】 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.更多还原