本文選題：中樞神經(jīng)系統(tǒng) + 炎癥反應(yīng)�。� 參考：《華中科技大學(xué)》2014年博士論文

【摘要】：第一部分消退素D1對(duì)脂多糖誘導(dǎo)的小鼠中樞神經(jīng)系統(tǒng)炎癥反應(yīng)的影響 目的：研究消退素D1(resolvin D1, RvD1)對(duì)脂多糖(lipopolysaccharide,LPS)引起的小鼠中樞神經(jīng)系統(tǒng)炎癥介質(zhì)的表達(dá),小膠質(zhì)細(xì)胞激活,以及核因子κB (nuclear factor-kappaB,NF-κB)通路活化的影響,以探討RvD1對(duì)脂多糖誘導(dǎo)的小鼠中樞神經(jīng)系統(tǒng)炎癥的影響及可能的機(jī)制。 方法：健康雄性C57BL/6小鼠100只,8-12周,隨機(jī)分為五組：(1)對(duì)照組：腹腔和側(cè)腦室注射等體積生理鹽水；(2)LPS組：側(cè)腦室給予2μ1生理鹽水后；立即腹腔注射LPS250μg/kg (100μl);(3)RvD1小劑量組：側(cè)腦室給予RvD11ng (2μl)后立即腹腔注射LPS;(4) RvD1大劑量組：側(cè)腦室給予RvD110ng (2μl)后立即腹腔注射LPS；(5)拮抗劑組：側(cè)腦室給予甲酰肽受體(formyl-peptide receptor, FPR)拮抗劑Boc-21μg+RvD110ng (2μl)后立即腹腔注射LPS。4小時(shí)后,過量麻醉處死小鼠,免疫組化染色觀察小膠質(zhì)細(xì)胞標(biāo)志物離子鈣接頭蛋白分子-1(ionized calcium bindingadaptor molecule-1,Iba-1)陽(yáng)性細(xì)胞；ELISA法檢測(cè)小鼠血清、皮質(zhì)以及海馬中腫瘤壞死因子-α(tumor necrosis factor-alpha, TNF-α)和白介素-1β (interleukin-1beta, IL-1β)濃度；Western blot檢測(cè)皮質(zhì)以及海馬組織IKB-α和胞核內(nèi)NF-κB p65亞基的表達(dá)；電泳遷移率改變分析(electrophoretic mobility shift assays,EMSA)檢測(cè)皮質(zhì)和海馬組織中NF-κB DNA結(jié)合活性。 結(jié)果：與對(duì)照組相比,LPS組血清、皮質(zhì)以及海馬炎癥因子TNF-α和IL-1β濃度明顯增高,小膠質(zhì)細(xì)胞激活。側(cè)腦室注射RvDl對(duì)血清TNF-α和IL-1p濃度無顯著影響,但RvD110ng可以抑制LPS引起的皮質(zhì)和海馬TNF-a和IL-1p濃度升高和小膠質(zhì)細(xì)胞激活,RvD11ng無顯著效果。LPS還可引起皮質(zhì)和海馬IκBα蛋白降解,胞核NF-κB p65表達(dá)增加以及NFκB DNA結(jié)合活性的增加；RvD10ng側(cè)腦室給予可以抑制LPS引起的IκBα和NF-κB活化。FPR2拮抗劑Boc-2可以阻斷RvD1的作用。 結(jié)論：RvDl可降低LPS引起的神經(jīng)系統(tǒng)炎性因子TNFa和IL-1p的產(chǎn)生,其抗炎作用是通過與FPR2結(jié)合進(jìn)而抑制NF-κB信號(hào)通路實(shí)現(xiàn)的。 第二部分消退素D1對(duì)脂多糖誘導(dǎo)的小膠質(zhì)細(xì)胞M1極化的影響 目的：小膠質(zhì)細(xì)胞存在兩種極化的激活方式：M1極化(經(jīng)典活化)和M2極化(選擇性活化)。M1極化的小膠質(zhì)細(xì)胞表達(dá)促炎癥因子,如TNF-α、IL-1β和一氧化氮(nitric oxide, NO)。該部分研究RvD1對(duì)LPS誘導(dǎo)的小膠質(zhì)細(xì)胞M1極化及其相關(guān)信號(hào)通路的影響,以探討RvDl在神經(jīng)系統(tǒng)發(fā)揮抗炎作用的機(jī)制。 方法：體外培養(yǎng)的小鼠BV-2小膠質(zhì)細(xì)胞株,分為五組：(1)對(duì)照組；(2)RvD1組：用不同濃度的RvD1(0.1nM、1nM、10nM、100nM)處理;(3)LPS組：用100ng/ml LPS處理;(4)RvDl+LPS組：不同濃度的RvD1(0.1nM、1nM、10nM、100nM)處理后30min后加入LPS處理；(5)拮抗劑組：在用RvD1和LPS處理前用10μM/LBoc-2處理30mmino采用MTT法檢測(cè)細(xì)胞活力,ELISA檢測(cè)培養(yǎng)上清中TNF-α和IL-1p濃度；硝酸還原酶法檢測(cè)培養(yǎng)上清中NO濃度；Western blot檢測(cè)小膠質(zhì)細(xì)胞誘生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)表達(dá),胞核NF-κB p65亞基表達(dá),IκB-α蛋白的降解,細(xì)胞外調(diào)節(jié)蛋白激酶(extracellular regulated protein kinase, ERK)、p38以及c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)磷酸化水平；免疫熒光觀察p65細(xì)胞內(nèi)定位；EMSA檢測(cè)NF-κB以及激活蛋白-1(activator protein-1,AP-1)的DNA結(jié)合活性。 結(jié)果：使用濃度的LPS和RvDl對(duì)BV-2細(xì)胞活力無顯著影響。LPS可引起培養(yǎng)上清中TNF-α、IL-1β、NO濃度增高,iNOS表達(dá)增加,RvD11nM、10nM、100nM可降低TNF-α和IL-1p濃度。100nM RvD1可減少LPS誘導(dǎo)產(chǎn)生的NO和iNOS。100nMRvD1還可以抑制LPS刺激引起的IκB-α降解,p65核轉(zhuǎn)位,抑制絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)ERK1/2、p38、JNK蛋白的磷酸化,并抑制LPS引起的NF-κB以及AP-1DNA結(jié)合活性的增加。Boc-2可阻斷RvDl的作用。 結(jié)論：RvD1可抑制LPS誘導(dǎo)的小膠質(zhì)細(xì)胞M1極化而發(fā)揮抗炎作用,該作用可能與抑制NF-κB和MAPK/AP-1通路激活有關(guān)。 第三部分消退素D1對(duì)白介素-4誘導(dǎo)的小膠質(zhì)細(xì)胞M2極化的影響 目的：小膠質(zhì)細(xì)胞存在兩種極化的激活方式：M1極化(經(jīng)典活化)和M2極化(選擇性活化)。M2小膠質(zhì)細(xì)胞主要介導(dǎo)炎癥消退以及組織修復(fù)。本部分主要研究RvDl對(duì)白介素-4(interleukin-4,IL-4)誘導(dǎo)的小膠質(zhì)細(xì)胞M2極化及其相關(guān)信號(hào)通路的影響,以探討RvD1在神經(jīng)系統(tǒng)中發(fā)揮抗炎促消退作用的機(jī)制。 方法：體外培養(yǎng)的小鼠BV-2小膠質(zhì)細(xì)胞株,分為五組：(1)對(duì)照組；(2)RvD1組：用100nM RvD1處理；(3)IL-4組：用10ng/ml IL-4處理;(4)RvD1+IL-4組：100nMRvDl處理30min后加入終濃度為10ng/ml的IL-4；(5)拮抗劑組：在用RvD1和IL-4處理前用10μMBoc-2或100μM來氟米特或1μMGW9662處理30min。采用Western blot和免疫熒光檢測(cè)小膠質(zhì)細(xì)胞M2標(biāo)志物精氨酸酶-1(Arginase1,Arg1)和幾丁質(zhì)酶-3樣蛋白-3(Chitinase3-like3,Ym1)蛋白表達(dá)；免疫熒光觀察胞核過氧化物酶增殖活化受體-gamma(peroxisome proliferator-activated receptor gamma,PPARy)細(xì)胞內(nèi)定位；Western blot檢測(cè)信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子-6(signal transducer and activator of transcription-6,STAT6)磷酸化水平以及PPARγ表達(dá)水平,EMSA檢測(cè)STAT6和PPARγ的DNA結(jié)合活性。為進(jìn)一步確定FPR2、STAT6和PPARγ在RvD1和IL-4誘導(dǎo)的小膠質(zhì)細(xì)胞選擇性活化中的作用,我們使用Boc-2或來氟米特或GW9662阻斷相應(yīng)的受體或者通路,觀察BV-2細(xì)胞Argl和Yml表達(dá)。 結(jié)果：IL-4刺激引起小膠質(zhì)細(xì)胞M2標(biāo)志物Argl和Yml表達(dá)增多,STAT6磷酸化,胞核PPARγ表達(dá)增加以及STAT6和PPARγ DNA結(jié)合活性增加。RvD1預(yù)處理可進(jìn)一步增加Argl和Yml表達(dá),提高STAT6磷酸化水平、細(xì)胞核內(nèi)PPARγ表達(dá)水平以及STAT6和PPARγ的DNA結(jié)合活性。Boc-2可拮抗RvDl的作用。阻斷STAT6可抑制RvD1和IL-4引起的胞核PPARγ表達(dá)和DNA結(jié)合活力增加。阻斷STAT6或PPARγ可消除RvD1和IL-4誘導(dǎo)的M2標(biāo)志物Argl和Yml表達(dá)。 結(jié)論：RvDl可促進(jìn)IL-4誘導(dǎo)的小膠質(zhì)細(xì)胞M2極化,從而發(fā)揮抗炎促消退作用,其作用是通過與FPR2結(jié)合進(jìn)而促進(jìn)STAT6/PPARγ信號(hào)通路激活實(shí)現(xiàn)的。
[Abstract]:Effect of the first part of melatonin D1 on the inflammatory response of the central nervous system of mice induced by lipopolysaccharide

Objective : To study the effects of melatonin D1 ( RvD1 ) on the expression of inflammatory mediators in the central nervous system of mice induced by lipopolysaccharide ( LPS ) , the activation of microglial cells , and the activation of nuclear factor - kappa B ( NF - 魏B ) pathway .

Methods : 100 healthy male C57BL / 6 mice , 8 - 12 weeks , were randomly divided into five groups : ( 1 ) control group : abdominal cavity and lateral ventricle were injected with equal volume physiological saline ;
( 2 ) LPS group : the lateral ventricle was given 2.1 normal saline ;
LPS250 渭g / kg ( 100 渭l ) was injected intraperitoneally immediately ; ( 3 ) RvD1 small dose group : LPS was injected intraperitoneally immediately after the lateral ventricle was given RvD11ng ( 2渭l ) ; ( 4 ) RvD1 bolus group : LPS was injected intraperitoneally immediately after administration of RvD110ng ( 2 渭l ) in the lateral ventricle .
( 5 ) antagonist group : LPS was injected intraperitoneally immediately after administration of Boc - 21渭g + RvD110ng ( 2渭l ) to the lateral ventricle . After 4 hours of injection , the mice were killed by excessive anesthesia . Immunohistochemical staining was used to observe the molecular - 1 ( Iba - 1 ) positive cells of the small glial cell marker ion calcium joint protein - 1 ( Iba - 1 ) .
The levels of tumor necrosis factor - alpha ( TNF - 偽 ) and interleukin - 1尾 ( IL - 1尾 ) in serum , cortex and hippocampus of mice were detected by ELISA .
Western blot was used to detect the expression of NF - 魏B in the cortex and hippocampus .
electrophoretic mobility shift assays ( EMSA ) were used to detect NF - 魏B DNA binding activity in cortex and hippocampus .

Results : Compared with the control group , the levels of TNF - 偽 and IL - 1尾 in the serum , cortex and hippocampus of LPS group were significantly higher than those in control group . The concentration of TNF - 偽 and IL - 1p in the cortex and hippocampus was not significantly affected by injection of RvD110ng , but RvD110ng could inhibit the increase of TNF - a and IL - 1p in the cortex and hippocampus induced by LPS .
RvD10ng lateral ventricle administration can inhibit the activation of I魏B 偽 and NF - 魏B induced by LPS . FPR2 antagonist Boc - 2 can block the effect of RvD1 .

Conclusion : RvDl can reduce the inflammatory factors TNFa and IL - 1p induced by LPS , and its anti - inflammatory effect is achieved by binding to FPR2 and inhibiting NF - 魏B signal pathway .

Effect of the second part of melatonin D1 on the polarization of the lipopolysaccharide - induced microglial cell M1

Objective : There are two types of polarization activation in microglial cells : M1 polarization ( classical activation ) and M2 polarization ( selective activation ) . M1 - polarized microglial cells express pro - inflammatory factors , such as TNF - 偽 , IL - 1尾 and nitric oxide ( NO ) . This part studies the effects of RvD1 on the M1 polarization of microglial cells induced by LPS and related signal pathways to investigate the mechanism of RvD1 on the anti - inflammatory effect of RvD1 in the nervous system .

Methods : Mouse BV - 2 microglial cell line cultured in vitro was divided into five groups : ( 1 ) control group ;
( 2 ) RvD1 group : treated with different concentrations of RvD1 ( 0.1 nM , 1 nM , 10 nM , 100 nM ) ; ( 3 ) LPS group : treated with 100 ng / ml LPS ; ( 4 ) RvDl + LPS group : RvD1 ( 0.1 nM , 1 nM , 10 nM , 100 nM ) at different concentrations were treated with LPS after 30 min ;
( 5 ) antagonist group : MTT assay was used to detect the concentration of TNF - 偽 and IL - 1p in culture supernatant by MTT assay before treatment with 10 渭M / L Boc - 2 before treatment with RvD1 and LPS .
NO concentration in culture supernatant was detected by nitrate reductase method .
Western blot was used to detect the expression of inducible nitric oxide synthase ( iNOS ) , the expression of NF - 魏B , the degradation of I魏B - 偽 protein , extracellular regulated protein kinase ( ERK ) , p38 and c - Jun N - terminal kinase , and the phosphorylation of c - Jun N - terminal kinase .
Immunofluorescence was used to observe the intracellular localization .
EMSA detected the DNA binding activity of NF - 魏B and activator protein - 1 ( AP - 1 ) .

RESULTS : The concentration of LPS and RvDl had no significant effect on BV - 2 cell viability . LPS could increase the levels of TNF - 偽 , IL - 1尾 , NO in culture supernatant , increase the expression of iNOS , RvD11nM , 10nM , 100nM decrease the phosphorylation of NF - 魏B and IL - 1p in LPS - induced LPS - induced NF - 魏B and IL - 1DNA binding activity , and inhibit the increase of NF - 魏B and AP - 1DNA binding activity induced by LPS . Boc - 2 could block the role of RvDl .

Conclusion : RvD1 can inhibit the polarization of LPS - induced microglial cells M1 and play an anti - inflammatory effect , which may be related to the inhibition of NF - 魏B and MAPK / AP - 1 pathway activation .

Effect of the third part of melatonin D1 on the polarization of IL - 4 - induced microglial cell M2

Objective : To investigate the effects of RvD1 on the role of RvD1 on the anti - inflammatory and anti - inflammatory effects of interleukin - 4 ( IL - 4 ) induced by interleukin - 4 ( IL - 4 ) in the nervous system .

Methods : Mouse BV - 2 microglial cell line cultured in vitro was divided into five groups : ( 1 ) control group ;
( 2 ) RvD1 group : treated with 100 nM RvD1 ;
( 3 ) IL - 4 group : treated with 10 ng / ml IL - 4 ; ( 4 ) RvD1 + IL - 4 group : 100 nMRvDl treated for 30 min and then added IL - 4 with a final concentration of 10ng / ml ;
( 5 ) The antagonist group : 30 min before treatment with RvD1 and IL - 4 with 10.mu . MBoc - 2 or 100.mu . M or 100.mu . M of MBoc - 2 or 100.mu . M . The expression of M2 - marker - 1 ( Arginase1 , Arg1 ) and chitinase - 3 - like protein - 3 ( Chitinase3 - like 3 , Ym1 ) was detected by Western blot and immunofluorescence .
Immunofluorescence was used to observe the intracellular localization of activated receptor - gamma ( PPARy ) cells .
Western blot媯，

本文編號(hào)：1842216