Effect and Mechanism of Puerarin on Experimental Autoimmune Encephalomyelitis in Mice
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摘要:
目的 探索葛根素(Puerarin)治疗小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis, EAE)的作用和机制。 方法 将C57BL/6小鼠随机分为对照组、EAE组、Puerarin处理EAE组(Puerarin组)。从诱导EAE当日开始,Puerarin组用葛根素(80 mg/kg)灌胃,其余2组用同体积生理盐水灌胃,每日1次。每天记录神经功能评分及体重变化;劳克坚劳蓝(luxol fast bBlue,LFB)髓鞘染色检测脊髓脱髓鞘情况;ELISA法检测血清中IL-6、IL-10、IL-17、IL-22和TGF-β的水平;Western 印迹法检测脊髓组织NF-κB、IκBα、IL-17、Foxp3等蛋白表达。 结果 Puerarin组小鼠神经功能评分及平均分均较EAE组明显降低(P < 0.01),而体重明显增加(P < 0.01)。应用葛根素后,EAE小鼠脱髓鞘面积(P < 0.01)及病理评分(P < 0.05)均明显下降。Puerarin组小鼠促炎因子IL-6、IL-17和IL-22水平显著降低(P < 0.01),抑炎因子IL-10和TGF-β显著增加(P < 0.05)。应用葛根素后:NF-κB及IL-17表达明显减少(P < 0.05);IκBα及Foxp3表达显著增加(P < 0.01)。 结论 葛根素可缓解EAE小鼠症状,减少脱髓鞘。作用机制是抑制NF-κB通路,调节IL17/Tregs平衡,实现对免疫功能的调节。 -
关键词:
- 实验性自身免疫性脑脊髓炎 /
- 葛根素 /
- 辅助性T细胞17 /
- 调节性T细胞 /
- NF-κB通路
Abstract:Objective To explore the effect and mechanism of Puerarin on experimental autoimmune encephalomyelitis (EAE) in mice. Methods C57BL / 6 mice were randomly divided into control group, EAE group and purearin treated EAE group. From the first day of EAE induction, puerarin group was gavaged with Puerarin (80 mg/kg), and the other two groups were given the same volume of normal saline once a day. The neurological function score and weight changes were recorded every day; the demyelination of spinal cord was detected by LFB myelination staining; the levels of IL-6, IL-10, IL-17, IL-22 and TGF-β in serum were detected by ELISA; the protein expression of NF-κB, IκBα, IL-17, Foxp3 in spinal cord tissue were detected by Western blot. Results Compared with EAE group, the neurological function score and average score of purearin group were significantly decreased (P < 0.01), while the body weight was significantly increased (P < 0.01). The demyelinating area (P < 0.01) and pathological score (P < 0.05) of EAE mice were significantly decreased after puerarin treatment. The levels of proinflammatory cytokines IL-6, IL-17 and IL-22 were significantly decreased in puerarin group (P < 0.01), while IL-10 and TGF-β were significantly increased (P < 0.05). After puerarin treatment, the expressions of NF-κB and IL-17 significantly decreased (P < 0.05), and the expressions of IκBα and Foxp3 significantly increased (P < 0.01). Conclusion Puerarin can alleviate the symptoms of EAE mice and reduce demyelination. The mechanism of action is to inhibit NF-κB pathway, to balance of IL-17/Tregs, and to regulate of immune function. -
Key words:
- Experimental autoimmune encephalomyelitis /
- Puerarin /
- Th17 /
- Treg /
- NF-κB pathway
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近年来,课题组对姜科姜花属植物萜类成分进行了系统研究[1-6],从滇姜花、圆瓣姜花和毛姜花中分离得到一系列对多种肿瘤细胞具有显著体外细胞毒活性的萜类化合物[7-9]。一些姜科植物中的二萜类成分具有抗菌、抗肿瘤等活性[10-11]。滇姜花(Hedychium yunnanense Gangep)中的呋喃二萜Coronarin E含量较高,该成分没有细胞毒活性和抗菌活性,将其通过光敏氧化反应制备具有生物活性的丁烯酸内酯结构的二萜衍生物[12],有产率较高、选择性高、绿色环保的特点。课题组对Coronarin E经二氧化硒氧化、酰化、光敏氧化三步反应,制备两个衍生物,对其抗菌活性(抑菌圈、MIC和联合用药)及体外抗肿瘤活性进行较为深入的研究,进一步验证了二萜衍生物的生物活性,为寻找较好的药物前体提供了理论基础和科学依据。
1. 材料与方法
1.1 仪器
78-1型磁力加热搅拌器(杭州仪器电机厂);分析天平(上海第二天平仪器厂);AM-500型核磁共振波谱仪((瑞士BRUKER公司);LED灯(上海一恒科学仪器有限公司);OSB-2100 旋转蒸发仪(上海爱朗仪器有限公司);ES-315 高压蒸汽灭菌锅,(TOMY 公司);恒温培养箱(上海一恒科学仪器有限公司);SW-CJ-2FD 超净工作台(AIRTECH公司);电热恒温鼓风干燥箱(上海一恒科技有限公司)。
1.2 试剂
Coronarin E由本课题组从姜科植物滇姜花中分离得到。所用有机试剂(化学纯)和化学试剂均购自昆明市医药公司化学试剂玻璃仪器采供站,柱色谱硅胶均为青岛海洋化工厂产品,培养基配料均购自雅云生物科技有限公司。
1.3 实验方法
1.3.1 Coronarin E的SeO2氧化反应
取400 mg coronarin E、168 mg SeO2溶于5 mL干燥的二氯甲烷中,加入368 mg过氧叔丁醇,在常温下搅拌反应2 h,TLC检测原料反应完全。反应液经200~300目硅胶柱色谱分离纯化,石油醚-乙酸乙酯(80∶1~40∶1)洗脱,得到化合物1(无色油状物,见图1)300 mg,产率为71%。
1.3.2 化合物1的酰化反应
取87 mg 1-萘甲酸、0.12 mL N,N-二异丙基碳二亚胺(DIC),0.5 mg对二甲氨基吡啶(DMAP),溶于5 mL干燥过的二氯甲烷,常温搅拌10 min后,加入79 mg化合物1,搅拌反应2 h后,TLC检测反应完全,加入0.2 mL水及50 mL石油醚继续搅拌10 min,超声30 min。产物用150 mL石油醚与100 mL 70%甲醇分配,后者再用100 mL石油醚萃取,合并两次萃取的石油醚层,浓缩后经200-300目硅胶柱色谱分离,石油醚-氯仿(10∶1~5∶1)洗脱。得到化合物2(白色固体,见图2)52.7 mg,产率为44.1%。
1.3.3 化合物2的光敏氧化反应
取52.7 mg化合物2溶于10 mL吡啶中,加入1.0 mg四苯基卟啉(TPP),通入氧气并搅拌,在LED灯照射下反应2 h,TCL检测原料反应完全。溶剂蒸干,经硅胶色谱分离纯化,石油醚-乙酸乙酯(4∶1~1∶1)洗脱,得到化合物3(白色固体)和4(白色固体),见图3,分别为12 mg 和8 mg,产率分别为21.3%和14.1%。
1.3.4 化合物的抗菌活性筛选
采用滤纸片扩散法测试化合物3和4的抑菌圈直径[13-14];采用微量倍比稀释法[15-16]测定化合物3和4的最小抑菌浓度(MIC);化合物4的联合用药测试采用棋盘法,测试该成分分别与万古霉素、氨苄西林、卡那霉素3种抗生素联合用药的最小抑菌浓度[17]。
1.3.5 体外细胞毒活性测试
采用MTT法[18],测定化合物3和4对五个肿瘤细胞株的体外细胞毒活性(阳性对照采用顺铂)。
2. 结果
2.1 化合物3和4的NMR波谱数据
化合物3:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.98(3H,s,H-19),1.19(3H,s,H-20),3.05(1H,d,J = 10.5 Hz,H-9),5.87(1H,t,J = 3.0 Hz,H-7),4.95(1H,br.s,H-17a),5.34(1H,br.s,H-17b),6.67(1H,dd,J = 16.5,10.5 Hz,H-11),5.94(1H,s,H-14),6.45(1H,d,J = 16.5 Hz,H-12),6.44(1H,s,H-16),8.98(1H,m,H-9′),7.80(1H,m,H-8′),7.36(1H,br.s,H-4′),8.51(1H,d,J = 8.1 Hz,H-3′),8.26(1H,d,J = 8.2 Hz,H-7′),8.23(1H,d,J = 8.2 Hz,H-5′),7.88(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.12(t,C-1),19.38(t,C-2),42.54(t,C-3),33.90(s,C-4),48.99(d,C-5),28.87(t,C-6),76.54(d,C-7),146.57(s,C-8),58.83(d,C-9),40.05(s,C-10),141.53(d,C-11),124.80(d,C-12),162.25(s,C-13),116.77(d,C-14),171.25(s,C-15),98.50(d,C-16),115.57(t,C-17),33.47(q,C-18),21.79(q,C-19),14.56(q,C-20),166.82(s,C-1′),128.88(s,C-2′),130.68(d,C-3′),125.68(d,C-4′),134.30(d,C-5′),132.00(s,C-6′),129.52(d,C-7′),126.39(d,C-8′),128.41(d,C-9′),127.15(d,C-10′),134.86(s,C-11′)。
化合物4:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.96(3H,s,H-19),1.19(3H,s,H-20),2.97(1H,d,J = 10.0 Hz,H-9),5.81(1H,t,J = 2.5 Hz,H-7),4.89(1H,br.s,H-17a),5.32(1H,br.s,H-17b),6.97/6.99(1H,dd,J = 15.5,10.0 Hz,H-11),6.23(1H,d,J = 15.5 Hz,H-12),7.15/7.16(1H,s,H-14),6.15/6.16(1H,s,H-15),8.98(1H,m,H-9′),7.16(1H,br.s,H-4′),7.64(1H,m,H-8′),8.01(1H,d,J = 8.2 Hz,H-3′),8.16(1H,d,J = 8.2 Hz,H-7′),8.22(1H,d,J = 8.2 Hz,H-5′),7.87(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.18(t,C-1),19.97(t,C-2),42.59(t,C-3),33.81(s,C-4),48.99(d,C-5),29.07(t,C-6),76.95(d,C-7),146.73(s,C-8),58.78(d,C-9),39.98(s,C-10),136.91(d,C-11),122.79(d,C-12),131.00(s,C-13),144.94(d,C-14),97.21(d,C-15),170.67(s,C-16),115.20(t,C-17),33.54(q,C-18),21.94(q,C-19),14.69(q,C-20),166.71(s,C-1′),128.98(s,C-2′),130.64(d,C-3′),125.66(d,C-4′),134.93(d,C-5′),131.97(s,C-6′),129.52(d,C-7′),126.35(d,C-8′),128.29(d,C-9′),127.16(d,C-10′),134.88(s,C-11′)。
2.2 化合物3和4的抗菌活性
化合物3对两种MRSA病原菌具有一定的抗菌活性,化合物4对多种革兰氏阳性菌、革兰氏阴性菌具有明显的抗菌活性,见表1。
表 1 化合物3和4对病原菌株抑菌活性筛选结果(抑菌圈直径:mm)Table 1. The result of antimicrobial activities against pathogens ( diameter of inhibition zone:mm)病原菌株 化合物3 化合物4 金黄色葡萄球菌29213 − 10.1 MRSA 1450 8.2 10.5 MRSA 1505 10.1 10.3 MRSA 2024 − 8.2 MRSA I-20 − 10.0 MRSA I-67 − 9.8 MRSA 1957 − 8.1 MRSA 28299 − 10.5 克雷伯氏菌13883 − 10.8 粪肠球菌29212 − 9.4 白色葡萄球菌1029 − 12.0 铜绿假单胞菌PA01 − 10.2 大肠杆菌25922 − 10.0 鼠伤寒沙门氏菌χ 8956 − 17.0 鲍曼不动杆菌19606 − 13.2 枯草芽孢杆菌6633 − − 注:表中抑菌圈直径为三次测量的平均值;“−”表示无抑菌圈。革兰氏阳性菌:金黄色葡萄球菌(Staphylococcus aureus ATCC 29213),7个耐甲氧西林金黄色葡萄球菌(MRSA 1450、1505、2024、1957、28299、I-20、I-67),白色葡萄球菌(Staphylococcus albus 1029);革兰氏阴性菌:鼠伤寒沙门氏菌(Salmonella typhimurium χ 8956),铜绿假单胞菌(Pseudomonas aeruginosa PA01),大肠杆菌(Escherichia coil ATCC 25922),枯草芽孢杆菌(Bacillus subtilis ATCC 6633),鲍曼不动杆菌(Acinetobacter baumanii ATCC 19606),肺炎克雷伯氏菌(Klebsiella pneumonia ATCC 13883),粪肠球菌(Enterococcus faecalis ATCC 29212)。 2.3 化合物与抗生素的联合用药
化合物4与三种抗生素联合使用时,对MRSA病原菌株抑制活性有不同程度的协同或相加作用,见表2。
表 2 化合物4与三种抗生素的联合用药测试结果Table 2. Combination test of compound 4 with three antibiotics菌株 药物 MIC(μg/ml) 最佳抑菌点(化合物4∶抗生素) FICI 作用方式 鼠伤寒沙门氏菌χ8956 化合物4 0.25 万古霉素 0.25 0.125∶0.0625 0.75 + 氨苄西林 2 0.125∶1 1 + 卡那霉素 2 0.0625∶1 0.75 + 鲍曼不动杆菌19606 化合物4 0.5 万古霉素 0.25 0.125∶0.125 0.75 + 氨苄西林 8 0.25∶8 1.5 − 卡那霉素 4 0.25∶4 1.5 − 白色葡萄球菌1029 化合物4 0.5 万古霉素 0.125 0.125∶0.03125 0.5 ++ 氨苄西林 0.5 0.25∶0.125 0.75 + 卡那霉素 4 0.125∶1 0.5 ++ 注:1、FICI = 甲药MIC联合/甲药MIC单用 + 乙药MIC联合/乙药MIC单用,其中甲药代表化合物4,乙药代表抗生素。FICI > 1,表示两药有无关作用;0.5 < FICI≤1,表示两药有相加作用;FICI≤0.5,表示两药有协同作用。2、以“++”表示协同作用,“+”表示相加作用,“−”表示无关。 2.4 化合物3和4对5种肿瘤细胞株的细胞毒活性
化合物3对5种人类肿瘤细胞株具有显著的体外细胞毒活性;化合物4具有较弱的体外肿瘤生长抑制活性,见表3。
表 3 产物对五种肿瘤细胞株的半数生长抑制浓度IC50(μM)Table 3. The IC50 value of 3 and 4 against five tumor cell lines (μM)化合物编号 白血病HL-60 肝癌SMMC-7721 肺癌A-549 乳腺癌MCF-7 结肠癌SW480 3 2.55 2.77 1.17 2.49 1.37 4 15.71 15.62 26.49 25.13 22.87 顺铂 5.00 4.33 2.17 9.18 13.19 评价标准:无效IC50 > 40 μM;有效IC50 < 40 μM;标示下划线的为活性高于阳性对照顺铂。 3. 讨论
二萜coronarin E经三步衍生化反应,制备具有丁烯酸内酯结构单元的二萜衍生物3和4。对3和4的生物活性测试表明:化合物4对多种革兰氏阳性菌、革兰氏阴性菌有明显的抗菌活性,化合物3对两种MRSA具有一定的抗菌活性。化合物4对鼠伤寒沙门氏菌(Salmonella typhimurium χ8956)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、白色葡萄球菌(Staphylococcus albus 1029)的抗菌效果显著。化合物4对鼠伤寒沙门氏菌的抗菌活性接近万古霉素,高于氨苄西林、卡那霉素;对鲍曼不动杆菌的抗菌活性高于氨苄西林、卡那霉素;对白色葡萄球菌的抗菌活性接近氨苄西林,高于卡那霉素。化合物4与三种抗生素联用时,对鼠伤寒沙门氏菌抑制活性均具有相加作用。化合物4与万古霉素、卡那霉素联用时对白色葡萄球菌抑制活性具有协同作用,与氨苄西林联用时具有相加作用。化合物4与万古霉素联用时对鲍曼不动杆菌抑制活性具有相加作用。
化合物3对5种人类肿瘤细胞株(白血病细胞株HL-60、肝癌细胞株SMMC-7721、肺癌细胞株A-549、乳腺癌细胞株MCF-7和结肠癌细胞株SW-480)均具有显著的体外细胞毒活性,超过阳性对照顺铂;化合物4具有较弱的体外肿瘤生长抑制活性。
由此可见,以姜科二萜为原料,经结构改造制备丁烯酸内酯结构单元的二萜衍生物,并从中寻找有苗头的抗菌、抗癌活性成分或先导化合物,可作为未来抗菌、抗肿瘤药物研究与开发的一个方向。
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图 4 各组小鼠脊髓组织NF-κB信号通路及IL-17、Foxp3表达的变化
A:各组小鼠Western印迹蛋白表达;B:核转录因子κB相对表达水平;C:核转录因子κB抑制蛋白α相对表达水平;D:核转录因子κB与核转录因子κB抑制蛋白α表达比值;E:转录因子叉头蛋白3相对表达水平;F:白介素-17相对表达水平;G:转录因子叉头蛋白3与白介素-17表达比值。与对照组比较,#P < 0.05,##P < 0.01,###P < 0.001,####P < 0.0001;与EAE组比较,#P < 0.05,##P < 0.01,###P < 0.001,####P < 0.0001。
Figure 4. Changes of NF - κ B signaling pathway and expression of IL-17 and Foxp3 in spinal cord of mice in each group
表 1 各组小鼠血清IL-6、IL-17、IL-22、IL-10和TGF-β水平的变化(
$ \bar x \pm s$ )(pg/mL)Table 1. Changes of serum levels of IL-6,IL-17,IL-22,IL-10 and TGF-β in mice (
$\bar x \pm s $ )(pg/mL)项目 对照组 EAE 葛根素 IL-6 87.4 ± 15.4 179.3 ± 17.4# 151.7 ± 14.5##△ IL-17 42.7 ± 8.4 110.0 ± 15.5# 60.1 ± 9.9##△ IL-22 209.2 ± 54.8 507.4 ± 57.3# 429.4 ± 64.2##△ IL-10 110.4 ± 12.5 50.5 ± 7.5# 68.7 ± 9.5##△ TGF-β 12.02 ± 1.71 5.10 ± 0.56# 6.39 ± 0.73##△ 与对照组相比较,#P < 0.05,##P < 0.01;与EAE组比较,△P < 0.05。 表 2 各组小鼠脊髓组织NF-κB信号通路及IL-17、Foxp3表达的变化(
$\bar x \pm s $ )(蛋白相对表达水平)Table 2. Changes of NF-κB signaling pathway and expression of IL-17 and Foxp3 in spinal cord of mice (
$\bar x \pm s $ )(Relative protein expression level)项目 对照组 EAE 葛根素 NF-κB 1.15 ± 0.06 2.27 ± 0.36# 1.64 ± 0.13△ IκBα 1.09 ± 0.09 1.43 ± 0.07# 2.08 ± 0.10##△ NF-κB/IκBα 1.08 ± 0.13 1.98 ± 0.20# 0.58 ± 0.09##△ IL-17 1.03 ± 0.03 2.19 ± 0.09# 1.62±0.21##△ Foxp3 1.20 ± 0.08 0.49 ± 0.10# 0.41±0.09##△ IL-17/Foxp3 0.97 ± 0.11 3.21 ± 0.34# 与对照组比较,#P < 0.05,##P < 0.01;与EAE组比较,△P < 0.05。 -
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