消退素D1对小鼠中枢神经系统炎症反应以及小胶质细胞极化的影响

发布时间：2018-05-04 07:55

  本文选题：中枢神经系统 + 炎症反应　； 参考：《华中科技大学》2014年博士论文

【摘要】：第一部分消退素D1对脂多糖诱导的小鼠中枢神经系统炎症反应的影响 目的：研究消退素D1(resolvin D1, RvD1)对脂多糖(lipopolysaccharide,LPS)引起的小鼠中枢神经系统炎症介质的表达,小胶质细胞激活,以及核因子κB (nuclear factor-kappaB,NF-κB)通路活化的影响,以探讨RvD1对脂多糖诱导的小鼠中枢神经系统炎症的影响及可能的机制。 方法：健康雄性C57BL/6小鼠100只,8-12周,随机分为五组：(1)对照组：腹腔和侧脑室注射等体积生理盐水；(2)LPS组：侧脑室给予2μ1生理盐水后；立即腹腔注射LPS250μg/kg (100μl);(3)RvD1小剂量组：侧脑室给予RvD11ng (2μl)后立即腹腔注射LPS;(4) RvD1大剂量组：侧脑室给予RvD110ng (2μl)后立即腹腔注射LPS；(5)拮抗剂组：侧脑室给予甲酰肽受体(formyl-peptide receptor, FPR)拮抗剂Boc-21μg+RvD110ng (2μl)后立即腹腔注射LPS。4小时后,过量麻醉处死小鼠,免疫组化染色观察小胶质细胞标志物离子钙接头蛋白分子-1(ionized calcium bindingadaptor molecule-1,Iba-1)阳性细胞；ELISA法检测小鼠血清、皮质以及海马中肿瘤坏死因子-α(tumor necrosis factor-alpha, TNF-α)和白介素-1β (interleukin-1beta, IL-1β)浓度；Western blot检测皮质以及海马组织IKB-α和胞核内NF-κB p65亚基的表达；电泳迁移率改变分析(electrophoretic mobility shift assays,EMSA)检测皮质和海马组织中NF-κB DNA结合活性。 结果：与对照组相比,LPS组血清、皮质以及海马炎症因子TNF-α和IL-1β浓度明显增高,小胶质细胞激活。侧脑室注射RvDl对血清TNF-α和IL-1p浓度无显著影响,但RvD110ng可以抑制LPS引起的皮质和海马TNF-a和IL-1p浓度升高和小胶质细胞激活,RvD11ng无显著效果。LPS还可引起皮质和海马IκBα蛋白降解,胞核NF-κB p65表达增加以及NFκB DNA结合活性的增加；RvD10ng侧脑室给予可以抑制LPS引起的IκBα和NF-κB活化。FPR2拮抗剂Boc-2可以阻断RvD1的作用。 结论：RvDl可降低LPS引起的神经系统炎性因子TNFa和IL-1p的产生,其抗炎作用是通过与FPR2结合进而抑制NF-κB信号通路实现的。 第二部分消退素D1对脂多糖诱导的小胶质细胞M1极化的影响 目的：小胶质细胞存在两种极化的激活方式：M1极化(经典活化)和M2极化(选择性活化)。M1极化的小胶质细胞表达促炎症因子,如TNF-α、IL-1β和一氧化氮(nitric oxide, NO)。该部分研究RvD1对LPS诱导的小胶质细胞M1极化及其相关信号通路的影响,以探讨RvDl在神经系统发挥抗炎作用的机制。 方法：体外培养的小鼠BV-2小胶质细胞株,分为五组：(1)对照组；(2)RvD1组：用不同浓度的RvD1(0.1nM、1nM、10nM、100nM)处理;(3)LPS组：用100ng/ml LPS处理;(4)RvDl+LPS组：不同浓度的RvD1(0.1nM、1nM、10nM、100nM)处理后30min后加入LPS处理；(5)拮抗剂组：在用RvD1和LPS处理前用10μM/LBoc-2处理30mmino采用MTT法检测细胞活力,ELISA检测培养上清中TNF-α和IL-1p浓度；硝酸还原酶法检测培养上清中NO浓度；Western blot检测小胶质细胞诱生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)表达,胞核NF-κB p65亚基表达,IκB-α蛋白的降解,细胞外调节蛋白激酶(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；免疫荧光观察p65细胞内定位；EMSA检测NF-κB以及激活蛋白-1(activator protein-1,AP-1)的DNA结合活性。 结果：使用浓度的LPS和RvDl对BV-2细胞活力无显著影响。LPS可引起培养上清中TNF-α、IL-1β、NO浓度增高,iNOS表达增加,RvD11nM、10nM、100nM可降低TNF-α和IL-1p浓度。100nM RvD1可减少LPS诱导产生的NO和iNOS。100nMRvD1还可以抑制LPS刺激引起的IκB-α降解,p65核转位,抑制丝裂原活化蛋白激酶(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结合活性的增加。Boc-2可阻断RvDl的作用。 结论：RvD1可抑制LPS诱导的小胶质细胞M1极化而发挥抗炎作用,该作用可能与抑制NF-κB和MAPK/AP-1通路激活有关。 第三部分消退素D1对白介素-4诱导的小胶质细胞M2极化的影响 目的：小胶质细胞存在两种极化的激活方式：M1极化(经典活化)和M2极化(选择性活化)。M2小胶质细胞主要介导炎症消退以及组织修复。本部分主要研究RvDl对白介素-4(interleukin-4,IL-4)诱导的小胶质细胞M2极化及其相关信号通路的影响,以探讨RvD1在神经系统中发挥抗炎促消退作用的机制。 方法：体外培养的小鼠BV-2小胶质细胞株,分为五组：(1)对照组；(2)RvD1组：用100nM RvD1处理；(3)IL-4组：用10ng/ml IL-4处理;(4)RvD1+IL-4组：100nMRvDl处理30min后加入终浓度为10ng/ml的IL-4；(5)拮抗剂组：在用RvD1和IL-4处理前用10μMBoc-2或100μM来氟米特或1μMGW9662处理30min。采用Western blot和免疫荧光检测小胶质细胞M2标志物精氨酸酶-1(Arginase1,Arg1)和几丁质酶-3样蛋白-3(Chitinase3-like3,Ym1)蛋白表达；免疫荧光观察胞核过氧化物酶增殖活化受体-gamma(peroxisome proliferator-activated receptor gamma,PPARy)细胞内定位；Western blot检测信号转导和转录激活因子-6(signal transducer and activator of transcription-6,STAT6)磷酸化水平以及PPARγ表达水平,EMSA检测STAT6和PPARγ的DNA结合活性。为进一步确定FPR2、STAT6和PPARγ在RvD1和IL-4诱导的小胶质细胞选择性活化中的作用,我们使用Boc-2或来氟米特或GW9662阻断相应的受体或者通路,观察BV-2细胞Argl和Yml表达。 结果：IL-4刺激引起小胶质细胞M2标志物Argl和Yml表达增多,STAT6磷酸化,胞核PPARγ表达增加以及STAT6和PPARγ DNA结合活性增加。RvD1预处理可进一步增加Argl和Yml表达,提高STAT6磷酸化水平、细胞核内PPARγ表达水平以及STAT6和PPARγ的DNA结合活性。Boc-2可拮抗RvDl的作用。阻断STAT6可抑制RvD1和IL-4引起的胞核PPARγ表达和DNA结合活力增加。阻断STAT6或PPARγ可消除RvD1和IL-4诱导的M2标志物Argl和Yml表达。 结论：RvDl可促进IL-4诱导的小胶质细胞M2极化,从而发挥抗炎促消退作用,其作用是通过与FPR2结合进而促进STAT6/PPARγ信号通路激活实现的。
[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 - 伪 ) 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 - 伪 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 - 伪 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 伪 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 - 伪 , 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 - 伪 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 - 伪 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 - 伪 , 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妫，

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