康 峰

(河南中醫(yī)藥大學(xué)，河南鄭州 450000)

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靈芝多糖對(duì)長期運(yùn)動(dòng)大鼠巨噬細(xì)胞吞噬功能及NO和IL-1β表達(dá)的影響

康 峰

(河南中醫(yī)藥大學(xué)，河南鄭州 450000)

探討靈芝多糖(GLP)對(duì)長期運(yùn)動(dòng)大鼠巨噬細(xì)胞吞噬功能及NO、IL-1β表達(dá)的影響。分別用低劑量(4.5 mg/mL)、中劑量(9 mg/mL)、高劑量(18 mg/mL)的GLP灌胃大鼠模型并給予持續(xù)30d游泳運(yùn)動(dòng)訓(xùn)練，正常組和對(duì)照組用生理鹽水灌胃，正常組不進(jìn)行運(yùn)動(dòng)訓(xùn)練。檢測貼壁巨噬細(xì)胞分泌NO、IL-1β水平，Western blot檢測巨噬細(xì)胞中iNOS水平，并檢測巨噬細(xì)胞吞噬雞紅細(xì)胞能力及巨噬細(xì)胞活性。結(jié)果表明，對(duì)照組中NO含量和巨噬細(xì)胞吞噬率顯著低于正常組(P<0.05)，中劑量組和高劑量組NO水平顯著高于對(duì)照組(P<0.05)；對(duì)照組巨噬細(xì)胞iNOS水平極顯著低于正常組(P<0.01)，低、中、高劑量組巨噬細(xì)胞iNOS水平極顯著高于對(duì)照組(P<0.01)；低、中劑量組巨噬細(xì)胞吞噬率顯著高于對(duì)照組(P<0.05)，高劑量組巨噬細(xì)胞吞噬率顯著高于對(duì)照組(P<0.01)；中劑量組巨噬細(xì)胞吞噬指數(shù)極顯著高于對(duì)照組(P<0.01)；對(duì)照組巨噬細(xì)胞活性極顯著低于正常組(P<0.01)，低、中、高劑量組巨噬細(xì)胞活性均極顯著高于對(duì)照組(P<0.01)。說明GLP能促進(jìn)巨噬細(xì)胞分泌NO、IL-1β，提高巨噬細(xì)胞吞噬功能和巨噬細(xì)胞活性。

巨噬細(xì)胞；靈芝多糖；吞噬功能；一氧化氮

機(jī)體的免疫能力是機(jī)體抵抗外界致病因素的重要組成部分，與身體體質(zhì)密切相關(guān)。運(yùn)動(dòng)對(duì)機(jī)體免疫能力的作用較為復(fù)雜[1]。運(yùn)動(dòng)時(shí)間、運(yùn)動(dòng)強(qiáng)度等都可以引起免疫功能的改變[2]。巨噬細(xì)胞屬于免疫細(xì)胞，在免疫系統(tǒng)中發(fā)揮重要作用。巨噬細(xì)胞可以吞噬細(xì)胞殘片和病原體，促進(jìn)機(jī)體康復(fù)[3]。不同運(yùn)動(dòng)強(qiáng)度和運(yùn)動(dòng)環(huán)境影響巨噬細(xì)胞的吞噬功能[4]。

靈芝多糖(GLP)廣泛存在于靈芝屬真菌的子實(shí)體和菌絲體中，屬于葡聚糖。靈芝多糖具有提高免疫力、抗腫瘤、抗衰老、保護(hù)心血管系統(tǒng)等多種功效[5-6]。為了明確靈芝多糖對(duì)長期運(yùn)動(dòng)大鼠巨噬細(xì)胞功能的影響，本研究構(gòu)建長期運(yùn)動(dòng)大鼠模型，同時(shí)給予靈芝多糖灌胃，用ELISA、Western blot等方法檢測靈芝多糖對(duì)巨噬細(xì)胞吞噬功能、細(xì)胞活性和NO、IL-1β表達(dá)的影響。

1 材料與方法

1.1 材料

1.1.1 實(shí)驗(yàn)動(dòng)物 Wistar雄性大鼠55只，2月齡，體重160 g～230 g，購自于鄭州大學(xué)醫(yī)學(xué)實(shí)驗(yàn)室。

1.1.2 主要試劑 胎牛血清購自于杭州四季青生物科技有限公司；RPMI1640培養(yǎng)基購自于美國Sigma；青鏈霉素購自于北京索萊寶生物科技有限公司；IL-1β、iNOS、GAPDH單克隆抗體購自于美國CST公司；GLP購自于杭州眾芝康菇生物技術(shù)有限公司；ELISA檢測試劑盒、BCA蛋白濃度檢測試劑盒購自于上海紀(jì)寧生物科技有限公司。

1.1.3 主要儀器 MK3酶標(biāo)儀購自于美國Thermo公司；BXF-88顯微鏡購自于上海炳宇光學(xué)儀器有限公司。

1.2 方法

1.2.1 動(dòng)物分組及運(yùn)動(dòng)訓(xùn)練 所有實(shí)驗(yàn)動(dòng)物隨機(jī)分為5組，每組11只大鼠。分別為正常組、對(duì)照組和低、中、高劑量組。低、中、高劑量組分別灌胃GLP濃度為4.5 mg/mL、9 mg/mL、18 mg/mL(每只大鼠每天每千克體重灌胃10 mL)。正常組、對(duì)照組用等量的生理鹽水灌胃。除正常組以外，其他4組大鼠均在水溫32℃的游泳池中游泳訓(xùn)練，第1天游泳時(shí)間為25 min，此后每天增加5 min，增加至40 min后不再增加訓(xùn)練時(shí)間，此后每天游泳訓(xùn)練40 min，持續(xù)30 d。游泳訓(xùn)練和GLP灌胃同時(shí)進(jìn)行。

1.2.2 巨噬細(xì)胞分離 大鼠運(yùn)動(dòng)訓(xùn)練及灌胃30 d后，腹腔注射RPMI1640培養(yǎng)液5 mL后，輕輕揉其腹部，眼球放血，頸椎脫臼處死大鼠。用注射器從大鼠側(cè)腹部吸出腹腔內(nèi)的液體。轉(zhuǎn)移到離心管中，1 500 r/min、4℃離心10 min，棄掉上清液，加入適量含有10% FBS的RPMI1640細(xì)胞培養(yǎng)液，調(diào)整細(xì)胞濃度為3×106個(gè)/mL，將細(xì)胞接種到96孔細(xì)胞培養(yǎng)板中，放置于37℃、體積分?jǐn)?shù)為5% CO2培養(yǎng)箱中培養(yǎng)3.5 h，棄上清液，加入冰預(yù)冷的含有10% FBS的RPMI1640細(xì)胞培養(yǎng)液洗滌后，貼壁細(xì)胞即為巨噬細(xì)胞。

1.2.3 巨噬細(xì)胞分泌NO水平檢測 取貼壁的巨噬細(xì)胞培養(yǎng)48 h，取上清液100 μL。加入濃度為300 g/L的硫酸鋅5 μL充分混合后，5 000 r/min離心10 min，將上清液轉(zhuǎn)移到EP管中，加入等體積的Griess溶液，混合均勻后，放置于室溫下緩慢震蕩10 min，用濾光片酶標(biāo)儀檢測550 nm處每孔中的吸光度，Griess溶液能與檢測樣品相互作用生成亞硝酸鹽，NO的含量與亞硝酸鹽含量相同。根據(jù)亞硝酸鹽標(biāo)準(zhǔn)品計(jì)算待測樣品中的NO水平。

1.2.4 iNOS表達(dá)水平檢測 收集培養(yǎng)48 h的巨噬細(xì)胞，用冰預(yù)冷的PBS洗滌細(xì)胞2次，在細(xì)胞中加入細(xì)胞裂解液，放置于冰上裂解細(xì)胞35 min，用移液槍轉(zhuǎn)移細(xì)胞裂解液至離心管中，4℃、14 000 r/min離心15 min，吸取蛋白上清液至EP管中。蛋白濃度測定按照BCA蛋白濃度檢測試劑盒檢測。Western blot檢測iNOS的表達(dá)水平，步驟同1.2.5。

1.2.5 巨噬細(xì)胞分泌IL-1β含量檢測 取貼壁的巨噬細(xì)胞培養(yǎng)48 h，取上清液，一部分按照ELISA檢測試劑盒說明書檢測上清液中的IL-1β水平。一部分按照Western blot方法檢測IL-1β含量。方法為上清液與Loading buffer充分混合后，放在100℃的孵育器上煮沸5 min。SDS-PAGE凝膠電泳，每孔加入50 μL的變性蛋白樣品。80 V電壓電泳30 min后，調(diào)整電壓至120 V至電泳結(jié)束。取出蛋白凝膠4℃轉(zhuǎn)印至PVDF膜上，經(jīng)50 g/L脫脂奶粉封閉2 h后，依次與一抗(400倍稀釋)、二抗(1000倍稀釋)孵育反應(yīng)后，PBST洗滌3次。轉(zhuǎn)移到暗室中，滴加顯色液，顯影、定影后，曝光。分析蛋白表達(dá)水平。

1.2.6 巨噬細(xì)胞吞噬功能檢測 將制備的巨噬細(xì)胞用RPMI1640培養(yǎng)基懸浮，充分混合后，加入Hanks液后，與適量的雞紅細(xì)胞混合均勻，調(diào)整巨噬細(xì)胞與雞紅細(xì)胞數(shù)量比例約為1∶600，放置于37℃孵育反應(yīng)0.5 h，期間每隔10 min震蕩混勻1次。1 000 r/min離心10 min，吸取細(xì)胞懸浮液滴加到載玻片上，制作細(xì)胞懸液涂片。用丙酮和甲醇混合液固定后，滴加Giemsa染液孵育8 min。顯微鏡下觀察100個(gè)巨噬細(xì)胞中吞噬雞紅細(xì)胞的巨噬細(xì)胞數(shù)目。計(jì)算吞噬率，吞噬率=100%×100個(gè)巨噬細(xì)胞中吞噬雞紅細(xì)胞的巨噬細(xì)胞數(shù)量/100。吞噬指數(shù)=100%×100個(gè)巨噬細(xì)胞總共吞噬的雞紅細(xì)胞數(shù)目/1001.2.7 MTT法檢測巨噬細(xì)胞活性 取濃度為3×106個(gè)/mL的巨噬細(xì)胞，接種到96孔細(xì)胞培養(yǎng)板中，每孔中加入100 μL細(xì)胞懸液，每組設(shè)置5個(gè)復(fù)孔，同時(shí)設(shè)置空白組，空白組不加入細(xì)胞懸浮液，用等量的DMSO溶液代替，放置于37℃、體積分?jǐn)?shù)為5% CO2培養(yǎng)箱中培養(yǎng)2 h，觀察細(xì)胞貼壁后，棄掉細(xì)胞培養(yǎng)液及未貼壁的細(xì)胞，加入Hanks液洗滌細(xì)胞2次，在細(xì)胞中加入適量的細(xì)胞培養(yǎng)液，放在37℃環(huán)境下反應(yīng)2 h后，在細(xì)胞中加入MTT溶液10 μL，MTT溶液濃度為5 mg/mL，充分混合后，放置于37℃孵育4 h，棄掉上清液，加入DMSO溶液100 μL，振蕩混合40 s，酶標(biāo)儀檢測579 nm吸光度。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 巨噬細(xì)胞分泌 NO水平檢測結(jié)果

對(duì)照組NO含量顯著低于正常組(P<0.05)，中、高劑量組NO水平顯著高于對(duì)照組(P<0.05)。說明長期運(yùn)動(dòng)能降低巨噬細(xì)胞分泌NO的水平，靈芝多糖能有效提高巨噬細(xì)胞分泌NO水平。

對(duì)照組巨噬細(xì)胞iNOS水平顯著低于正常組(P<0.01)，低、中、高劑量組巨噬細(xì)胞iNOS水平顯著高于對(duì)照組(P<0.01)(圖1)。

2.2 巨噬細(xì)胞分泌IL-1β水平檢測結(jié)果

對(duì)照組IL-1β含量顯著低于正常組(P<0.05)，中、高劑量組IL-1β含量顯著高于對(duì)照組(P<0.05)；對(duì)照組IL-1β水平顯著低于正常組(P<0.01)，低、中劑量組IL-1β水平顯著高于對(duì)照組(P<0.05)，高劑量組IL-1β水平極顯著高于對(duì)照組(P<0.01)(圖2)。

2.3 巨噬細(xì)胞吞噬功能檢測結(jié)果

對(duì)照組巨噬細(xì)胞吞噬率顯著低于正常組(P<0.05)，低、中劑量組巨噬細(xì)胞吞噬率顯著高于對(duì)照組(P<0.05)；高劑量組巨噬細(xì)胞吞噬率極顯著高于對(duì)照組(P<0.01)；中劑量組巨噬細(xì)胞吞噬指數(shù)極顯著高于對(duì)照組(P<0.01)(圖3)。

1.正常組；2.對(duì)照組；3.低劑量組；4.中劑量組；5.高劑量組；A.NO含量；B.Western blot檢測結(jié)果；C.iNOS蛋白表達(dá)率*P<0.05 vs 正常組；#P<0.05 vs 對(duì)照組；##P<0.01vs 對(duì)照組1.Normal group; 2.Control group; 3.Low dose group; 4.Middle dose group; 5.High dose group; A.NO content; B.Western blot test results; C.iNOS protein expression rate

Note:*P<0.05 vs normal group; #P<0.05 vs control group; ##P<0.01vs control group

圖1 巨噬細(xì)胞分泌NO含量檢測結(jié)果

Fig.1 Detection results of NO in macrophages

2.4 巨噬細(xì)胞活性檢測結(jié)果

對(duì)照組巨噬細(xì)胞活性極顯著低于正常組(P<0.01)，低、中、高劑量組巨噬細(xì)胞活性均極顯著高于對(duì)照組(P<0.01)(圖4)。

3 討論

巨噬細(xì)胞參與機(jī)體的特異性和非特異性免疫系統(tǒng)。當(dāng)機(jī)體出現(xiàn)受損組織和細(xì)胞時(shí)，巨噬細(xì)胞能夠吞噬清除損壞的細(xì)胞和組織碎片，促進(jìn)機(jī)體恢復(fù)[7-9]。巨噬細(xì)胞吞噬功能與運(yùn)動(dòng)有關(guān)[10]。GLP具有扶正固本、增強(qiáng)機(jī)體免疫力的作用[11]。癌癥晚期服用GLP可以增加患者的免疫功能[12]。GLP參與改善運(yùn)動(dòng)后的巨噬細(xì)胞吞噬功能的恢復(fù)[13]。NO在生物體內(nèi)發(fā)揮重要作用，是一種效應(yīng)分子。NO廣泛存在于神經(jīng)系統(tǒng)、免疫系統(tǒng)、心血管系統(tǒng)等組織系統(tǒng)中[14]。在一氧化氮合酶(iNOS)的作用下，精氨酸被催化生成NO。NO含量異常會(huì)引起機(jī)體免疫系統(tǒng)和炎癥反應(yīng)異常[15]。NO水平下調(diào)，巨噬細(xì)胞吞噬功能減弱。研究表明，運(yùn)動(dòng)會(huì)對(duì)機(jī)體內(nèi)NO含量產(chǎn)生影響，NO含量的變化與運(yùn)動(dòng)時(shí)間和運(yùn)動(dòng)強(qiáng)度有關(guān)[16]。高強(qiáng)度的運(yùn)動(dòng)會(huì)導(dǎo)致機(jī)體內(nèi)NO水平下降[17]。一次性竭力運(yùn)動(dòng)小鼠NO含量明顯高于靜息狀態(tài)下NO水平[18]。本研究結(jié)果表明，長期運(yùn)動(dòng)可以明顯降低巨噬細(xì)胞分泌NO水平，用中劑量和高劑量的GLP灌胃后，巨噬細(xì)胞分泌NO水平逐漸恢復(fù)。說明GLP可以恢復(fù)巨噬細(xì)胞分泌NO。

1.正常組；2.對(duì)照組；3.低劑量組；4.中劑量組；5.高劑量組；A.ELISA法檢測結(jié)果；B.Western blot結(jié)果圖；C.IL-1β蛋白表達(dá)率

*P<0.05 vs 正常組；**P<0.01 vs 正常組；#P<0.05 vs 對(duì)照組；##P<0.01 vs 對(duì)照組

1.Normal group; 2.Control group; 3.Low dose group; 4.Middle dose group; 5.High dose group; A.ELISA test results; B.Western blot results; C.IL-1β protein expression rate

*P<0.05 vs normal group;**P<0.01 vs normal group; #P<0.05 vs control group; ##P<0.01 vs control group

圖2 巨噬細(xì)胞分泌IL-1β水平檢測結(jié)果

Fig.2 The detection results of macrophage secreting IL-1β

1.正常組；2.對(duì)照組；3.低劑量組；4.中劑量組；5.高劑量組；A.巨噬細(xì)胞吞噬率；B.巨噬細(xì)胞吞噬指數(shù) *P<0.05 vs 正常組；#P<0.05 vs 對(duì)照組；##P<0.01 vs 對(duì)照組1.Normal group; 2.Control group; 3.Low dose group; 4.Middle dose group; 5.High dose group; A.Macrophage phagocytic rate; B.Macrophage phagocytic index

*P<0.05 vs normal group; #P<0.05 vs control group; ##P<0.01 vs control group

圖3 巨噬細(xì)胞吞噬功能檢測結(jié)果

Fig.3 Results of phagocytic function of macrophage

1.正常組；2.對(duì)照組；3.低劑量組；4.中劑量組；5.高劑量組；**P<0.01 vs 正常組；##P<0.01 vs 對(duì)照組

1.Normal group; 2.Control group; 3.Low dose group; 4.Middle dose group; 5.High dose group; vs **P<0.01vs normal group; ##P<0.01 vs control group

圖4 巨噬細(xì)胞活性檢測結(jié)果

Fig.4 Detection results of macrophage activity

IL-1β由巨噬細(xì)胞產(chǎn)生，在抗原呈遞、細(xì)胞生長、炎性反應(yīng)等過程中發(fā)揮重要作用。局部低濃度的IL-1β可以促進(jìn)T細(xì)胞的活化，誘導(dǎo)B細(xì)胞的增殖和抗體分泌[19]。本研究結(jié)果表明，長期運(yùn)動(dòng)訓(xùn)練能夠造成IL-1β水平的降低，巨噬細(xì)胞吞噬功能及活性下降；灌胃GLP后，巨噬細(xì)胞分泌IL-1β的水平得到恢復(fù)。巨噬細(xì)胞與雞紅細(xì)胞混合后，長期運(yùn)動(dòng)大鼠巨噬細(xì)胞吞噬功能下降，而灌胃GLP后的巨噬細(xì)胞吞噬功能得到恢復(fù)。

活細(xì)胞內(nèi)產(chǎn)生的琥珀酸脫氫酶能夠與MTT結(jié)合生成復(fù)合物。利用MTT可以有效檢測巨噬細(xì)胞活性。細(xì)胞活性越大，OD值越大。本研究結(jié)果表明，長期運(yùn)動(dòng)能夠降低巨噬細(xì)胞的活性，而GLP可以恢復(fù)巨噬細(xì)胞的活性。 綜上所述，長期運(yùn)動(dòng)能夠降低巨噬細(xì)胞的吞噬功能和活性，抑制巨噬細(xì)胞分泌NO和IL-1β，GLP可提高長期運(yùn)動(dòng)大鼠巨噬細(xì)胞吞噬功能和細(xì)胞活性，促進(jìn)巨噬細(xì)胞分泌NO和IL-1β。

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Effects of GLP on Phagocytic Function of Macrophages and Expressions of NO and IL-1β in Rat Model of Long Term Exercise

KANG Feng
(HenanUniversityofTraditionalChineseMedicine,Zhengzhou,Henan,450000,China)

To investigate the effects ofGanodermalucidumpolysaccharides (GLP) on the phagocytic function of macrophages and the expressions of NO and IL-1 in rats with long-term exercise,the rats were administered with low dose (4.5 mg/mL),middle dose (9 mg/mL) and high dose (18 mg/mL) of GLP,and the rats were administered with swimming training for 30 days.At the same time,the normal group and control group were set,the normal group and control group were given normal saline to fill the stomach,and the normal group did not exercise.NO and IL-1 levels were detected in the supernatant of cultured 48 h cells.Western blot was used to detect the iNOS level of macrophages,and the ability of macrophage phagocytosis of chicken red blood cells and the activity of macrophages were also detected.Results showed that the content of NO in the control group was significantly lower than that in the normal group (P<0.05); The levels of NO in the middle dose group and high dose group were significantly higher than that in the control group (P<0.05).The levels of iNOS in the control group were significantly lower than those in the normal group (P<0.01),and the iNOS levels in the low,medium and high dose groups were significantly higher than those in the control group (P<0.01).The phagocytosis rate of low dose group was significantly higher than that of the control group (P<0.05); High dose group of macrophages were significantly higher than the control group (P<0.01).The phagocytic index of macrophage in the middle dose group was significantly higher than that in the control group (P<0.01).The activity of macrophages in control group was significantly lower than that in normal group (P<0.01); the activities of macrophages in low,medium and high dose group were significantly higher than that in control group (P<0.01).In conclusions,GLP can promote the secretion of NO and IL-1β,and improve the phagocytic function and activity of macrophages.

macrophage; GLP; phagocytic function; NO

2016-10-21

康 峰(1980-)，男，河南鞏義人，碩士，講師，主要從事動(dòng)物運(yùn)動(dòng)康復(fù)訓(xùn)練。

S852.4；852.2

A

1007-5038(2017)06-0061-05