摘要
目的观察姜黄素对小鼠实验性自身免疫性脑脊髓炎(EAE)的抗炎作用,并初步探究其作用机制。方法40只雌性C57BL/6小鼠,随机分为EAE+DMSO组、EAE+姜黄素组、DMSO组、姜黄素组4组,每组10只,其中EAE+DMSO组、EAE+姜黄素组建立EAE模型;DMSO组作为EAE+DMSO组的溶剂对照组,此两组小鼠自免疫日起1周后每日腹腔注射20%DMSO溶液0.2 ml;姜黄素组及EAE+姜黄素组小鼠自免疫日起1周后每日腹腔注射姜黄素溶液10 mg/kg。自免疫日起,每天对各组小鼠进行临床症状评分;于EAE发病高峰期(第21天)将小鼠麻醉后处死,取脊髓组织进行HE染色;ELISA法测定各组小鼠血清INF-γ、IL-17的浓度;脾脏淋巴细胞进行CD4^+T细胞分选;RT-PCR技术测定小鼠脊髓组织Atg-5 mRNA及LC3-ⅡmRNA水平;免疫组化染色评估脊髓组织LC3-Ⅱ的表达水平。结果在小鼠EAE模型成功建立的基础上,DMSO组及姜黄素组小鼠均无明显神经功能损伤症状,脊髓组织HE染色未见炎症反应;EAE+DMSO组临床症状评分(3.312±0.347)与DMSO组比较明显升高(P<0.05),脊髓组织HE染色提示大量炎性细胞浸润及局部血管袖套样改变;与EAE+DMSO组相比,EAE+姜黄素组小鼠临床症状评分(1.562±0.463)明显降低(P<0.05),脊髓组织HE染色可见炎性细胞浸润明显减轻,未见典型血管袖套样改变。DMSO组及姜黄素组小鼠CD4^+T细胞百分比(分别为39.76%±6.51%、41.90%±2.53%)、血清INF-γ[分别为(35.86±15.54) pg/ml、(40.36±18.55) pg/ml]、IL-17[分别为(18.39±5.96) pg/ml、(20.56±5.52) pg/ml]差异均无统计学意义(P>0.05);与DMSO组相比,EAE+DMSO组血清INF-γ[(210.56±47.74) pg/ml]、IL-17[(218.09±35.33) pg/ml]明显升高(P<0.01),脾脏CD4^+T淋巴细胞(67.53%±7.78%)增加(P<0.05);EAE+姜黄素组血清INF-γ[(70.34±17.02) pg/ml]、IL-17[(29.30±11.73) pg/ml]较EAE+DMSO组明显下调(P<0.01),脾脏CD4^+T淋巴细胞(46.46%±2.41%)减少(P<0.05)。DMSO组及姜黄素组Atg-5(分别为1.00±0.00、0. 9 7±0. 1 2)、 L C 3-Ⅱ(分别为1. 0 0±0. 0 0、 0. 9 7±0. 1 2)基因表达水平以及L C 3-Ⅱ免疫组化阳性细胞计数[分别为(16.33±1.24)个/HP、(14.67±1.25)个/HP]无明显差异(P>0.05);与DMSO组相比,EAE+DMSO组Atg-5(0.70±0.06)、LC3-Ⅱ(0.67±0.10)基因表达水平以及LC3-Ⅱ免疫组化阳性细胞计数[(8.33±2.05)个/HP]均明显下调(P<0.05);EAE+姜黄素组小鼠脊髓Atg-5(1.56±0.08)、LC3-Ⅱ(1.99±0.11)基因表达水平以及LC3-Ⅱ免疫组化阳性细胞计数[(19.67±1.70)个/HP]与EAE+DMSO组比较明显上调(P<0.05)。结论姜黄素可缓解EAE病程中的神经功能损伤,下调EAE病程中外周及中枢炎症反应,同时上调中枢Atg-5、LC3-Ⅱ的表达乃至中枢神经细胞的自噬水平,并可能因此限制EAE病程中CNS的炎症损伤过程。
Objective To investigate the anti-inflammator y effect of curcumin on the mouse with experimental autoimmune encephalomyelitis(EAE),and explore its mechanism of action.Methods Forty female C57 BL/6 mice were randomly divided into EAE+DMSO group,EAE+curcumin group,DMSO group and curcumin group with10 mice in each group.Mice in EAE+DMSO group and EAE+curcumin group were used to establish the EAE model.DMSO group was the solvent control of EAE+DMSO group,and 0.2 ml 20% DMSO solution daily were intraperitoneally injected in the both groups a week after immunization;and mice in curcumin group and EAE+curcumin group were daily intraperitoneally injected with 10 mg/kg curcumin solution a week after immunization.From the date of immunization,the mice in each group were evaluated for clinical symptoms every day.Mice were anesthetized and sacrificed at the peak period of EAE(21 d),their spinal cord tissues were taken for HE staining,serum concentrations of INF-γ and IL-17 were measured by ELISA,spleen lymphocytes were sorted for CD4^+ T.The expression levels of Atg-5 mRNA and LC3-Ⅱ mRNA in spinal cord tissue were determined by RT-PCR,and of LC3-Ⅱ was estimated by immunohistochemical staining.Results Based on the successful establishment of mouse EAE model,there were no obvious neurological injury symptoms in DMSO group and curcumin group,and no inflammatory reaction by HE staining in spinal cord tissue.The clinical symptom score in EAE+DMSO group(3.312±0.347) was significantly higher than that in DMSO group(P<0.05),and HE staining of spinal cord tissue indicated massive inflammatory cell infiltration and local vascular cuff appearance.Compared with the EAE+DMSO group,the clinical symptom score of EAE+curcumin group(1.562±0.463) was significantly lower(P<0.05),and the infiltration of inflammatory cells in HE stained spinal cord tissue was reduced significantly without typical vascular cuff like changes.For mice in DMSO group and curcumin group,the percentages of CD4^+T cells(39.76%±6.51%,41.90%±2.53%,respectively),ser um concentrations of INF-γ[(35.86±15.54) pg/ml,(40.36±18.55) pg/ml,respectively] and IL-17[(18.39±5.96) pg/ml,(20.56±5.52) pg/ml,respectively] showed no significant difference(P>0.05).The serum concentrations of INF-γ and IL-17 were significantly higher in EAE+DMSO group(210.56±47.74 and 218.09±35.33) than in DMSO group(P<0.01),and spleen CD4^+T lymphocyte(67.53%±7.78%) were significantly higher(P<0.05).The serum concentrations of INF-γand IL-17 were down-regulated significantly in EAE+curcumin group(70.34±17.02 and 29.30±11.73) than in EAE+DMSO group(P<0.01),and spleen CD4^+T lymphocyte(46.46%±2.41%) were decreased obviously(P<0.05).There was no significant difference between DMSO group and curcumin group in Atg-5 level(1.00±0.00 and 0.97±0.12,respectively),the LC3-Ⅱ gene expression level(1.00±0.00 and 0.97±0.12,respectively) and LC3-Ⅱ immunohistochemical positive cell count [(16.33 ±1.24)/HP and(14.67±1.25)/HP,respectively](P>0.05);Compared with the DMSO group,the Atg-5 level(0.70±0.06),LC3-Ⅱ gene expression level(0.67±0.10) and LC3-Ⅱ immunohistochemical positive cell count [(8.33±2.05)/HP] were down-regulated significantly in EAE+DMSO group(P<0.05);the Atg-5 level(1.56±0.08),LC3-Ⅱ gene expression level(1.99±0.11) and LC3-Ⅱimmunohistochemical positive cell count [(19.67±1.70)/HP] raised significantly in EAE+curcumin group when compared with that in EAE+DMSO group(P<0.05).Conclusions Curcumin may relieve the clinical nerve function injury in the course of EAE,downgrade the peripheral and central inflammatory response,meanwhile up-regulate the expression levels of Atg-5 and LC3-Ⅱ and upgrade the autophagy level of central nerve cells.Thus the inflammatory injury process in EAE course may be limited.
作者
鲍彩彩
原铂尧
孙梦娇
王满侠
亓斐
牛彦霞
BAO Cai-cai;YUAN Bo-yao;SUN Meng-jiao;WANG Man-xia;QI Fei;NIU Yan-xia(Department of Neurology, Second Hospital of Lanzhou University, Lanzhou 730000, China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2019年第7期593-599,共7页
Medical Journal of Chinese People's Liberation Army
基金
甘肃省科技厅国际合作项目(1604WKCA015)
神经病学疾病精准诊疗甘肃省国际科技合作基地项目(20180095)~~
关键词
实验性自身免疫性脑脊髓炎
姜黄素
自噬
炎症反应
experimental autoimmune encephalomyelitis
curcumin
autophagy
inflammatory reaction
