2. 南京晓庄学院食品科学学院,江苏 南京 211117
2. School of Food Technology, Nanjing Xiaozhuang University, Nanjing 211117, China
脑卒中是危害人类健康的重大疾病[1]。磷酸川芎嗪 (tetramethylpyrazine phosphate,TMPP) 的抗脑缺血作用证实与抗自由基、减少炎症反应有关[2]。我们在前期研究发现,冰片对不同脑区具有不同的脑靶效应,即存在脑区特异性,这种特异性和冰片不同剂量密切相关[3]。已有研究证实冰片和川芎联用在脑缺血的疗效上优于川芎单用[4]。但目前还未见冰片-TMPP配伍在不同脑区抗脑缺血作用的报道,也未见冰片剂量对这一作用的干预研究。
1 材料与方法 1.1 药品和试剂冰片 (江苏省中医院);TMPP (利民制药厂);尼莫地平 (拜耳医药公司),总蛋白、SOD和MDA试剂盒 (南京建成生物工程研究所);IL-1β、IL-6和TNF-α试剂盒 (北京华英生物技术研究所)。
1.2 仪器酶标仪 (BIOTEC,Synergy),微循环记录仪 (美国BIOPAC,MP100公司),组织分散器 (宁波新芝生物科技公司,XHF-D)。
1.3 分组、造模、给药和检测方法麻醉大鼠[♂,250~300 g,浙江省实验动物中心,合格证号SCXK (浙) 2014-0001],分离双侧颈总动脉。颈部正中切口,暴露第一颈椎横突翼孔,电凝双侧椎动脉。24 h后夹闭双侧颈总动脉20 min,然后再灌注。将造模成功的大鼠分为10组,每组6只:模型组、T组 (给予13.3 mg·kg-1的TMPP) 和TB Ⅰ~Ⅷ组 (13.3 mg·kg-1的TMPP分别配伍0.01、0.02、0.04、0.08、0.16、0.32、0.64、1.28 g·kg-1冰片),另制备假手术组6只。模型组和假手术组给予NS。连续灌胃给药7 d后进行皮层、海马、下丘脑和纹状体的微循环、IL-1β、IL-6、TNF-α、SOD、MDA含量的检测。
1.4 统计学分析所有数据分析由SPSS13.0完成,各组数据用x±s表示,组间比较采用t检验。
2 结果TMPP单用可明显增加皮层微循环、皮层和海马SOD,降低皮层IL-1β、IL-6、TNF-α和海马IL-1β,其他指标未见明显改善。合用冰片后,则能产生新的明显改善,包括海马区微循环、IL-6、TNF-α含量;下丘脑区IL-1β、IL-6、TNF-α、SOD和MDA含量;纹状体区微循环、IL-1β、IL-6、TNF-α、SOD和MDA含量。提示TMPP与冰片合用效果优于单用。结果见Tab 1~4。
Group | Microcirculation/BPU | IL-1β/ng·L-1 | IL-6/ng·L-1 | TNF-α/ng·L-1 | SOD/kU·g-1 Pro | MDA/μmol·g-1 Pro |
Sham | 87.94±22.20 | 182.62±5.14 | 230.58±13.09 | 164.41±18.06 | 69.86±14.13 | 2.61±0.69 |
Model | 50.24±8.02## | 218.33±1.65## | 327.88±26.10## | 216.81±10.60## | 53.52±8.39# | 4.80±1.00## |
T | 68.37±8.70** | 205.29±10.77* | 291.57±27.56* | 196.88±11.46* | 64.54±2.14* | 3.66±0.57* |
TB Ⅰ | 73.46±9.67** | 209.69±7.13* | 282.86±38.61* | 197.59±12.84* | 65.07±8.54* | 3.73±0.54* |
TB Ⅱ | 70.60±19.22* | 208.99±8.21* | 288.67±18.45* | 199.68±14.84* | 64.65±8.30* | 3.71±0.63* |
TB Ⅲ | 64.86±11.67* | 209.01±7.95* | 277.85±25.01** | 197.71±13.88* | 64.18±4.92* | 3.66±0.62* |
TB Ⅳ | 65.28±13.67* | 206.06±7.94** | 264.88±15.06** | 201.95±11.88* | 68.03±11.61* | 3.59±0.42* |
TB Ⅴ | 70.09±7.32** | 204.36±9.04** | 271.36±18.80** | 196.14±13.17* | 63.95±5.68* | 3.53±0.71* |
TB Ⅵ | 66.81±12.17* | 209.66±4.30** | 281.11±9.06** | 191.33±16.28** | 58.26±5.95 | 3.44±0.53* |
TB Ⅶ | 69.50±11.01** | 212.73±5.72* | 283.97±16.27** | 188.84±14.18** | 55.07±3.44 | 3.48±0.27* |
TB Ⅷ | 69.33±7.83** | 211.19±6.52* | 294.79±23.93* | 193.84±20.40* | 53.74±3.95 | 3.86±0.21* |
#P < 0.05, ##P < 0.01 vs sham; *P < 0.05, **P < 0.01 vs model group, the below were same |
Group | Microcirculation/BPU | IL-1β/ng·L-1 | IL-6/ng·L-1 | TNF-α/ng·L-1 | SOD/kU·g-1 Pro | MDA/μmol·g-1 Pro |
Sham | 95.97±15.49 | 177.75±21.62 | 233.54±16.63 | 165.02±13.73 | 73.33±10.65 | 2.70±0.81 |
Model | 59.09±5.28## | 237.90±24.32## | 341.90±28.43## | 219.02±17.42## | 57.83±6.21# | 4.81±1.04## |
T | 66.59±11.95 | 212.59±10.53* | 325.54±44.02 | 207.74±24.24 | 64.61±3.50* | 3.77±0.16* |
TB Ⅰ | 65.25±7.78 | 208.31±19.59* | 323.82±43.67 | 203.25±11.33 | 65.56±3.79* | 3.75±0.46* |
TB Ⅱ | 65.59±10.11 | 208.21±18.88* | 324.00±17.32 | 205.34±12.90 | 65.94±1.85* | 3.49±0.93* |
TB Ⅲ | 68.60±9.56 | 201.37±26.78* | 307.41±26.26 | 203.15±18.12 | 69.44±8.97* | 3.46±0.94* |
TB Ⅳ | 70.41±8.06* | 199.04±27.03* | 297.93±36.45* | 192.05±20.23* | 69.17±5.08* | 3.46±0.60* |
TB Ⅴ | 72.87±6.99** | 192.63±20.39** | 299.77±17.54* | 188.57±11.48** | 70.17±7.33* | 3.49±0.66* |
TB Ⅵ | 69.14±8.23* | 201.25±26.58* | 288.09±39.24* | 186.19±11.72** | 70.39±6.91** | 3.18±0.48** |
TB Ⅶ | 69.40±9.76* | 205.31±17.32* | 310.08±21.91 | 193.34±20.31* | 67.89±5.76* | 3.52±0.53* |
TB Ⅷ | 64.64±9.33 | 215.44±16.77 | 313.49±38.22 | 192.45±13.87* | 66.33±5.80* | 3.91±0.66 |
Group | Microcirculation/BPU | IL-1β/ng·L-1 | IL-6/ng·L-1 | TNF-α/ng·L-1 | SOD/kU·g-1 Pro | MDA/μmol·g-1 Pro |
Sham | 86.15±21.34 | 178.62±26.79 | 228.49±20.61 | 165.02±15.59 | 75.61±8.98 | 2.84±0.75 |
Model | 58.34±9.24# | 240.05±26.28## | 303.28±25.53## | 203.28±13.31## | 59.06±9.06## | 4.89±0.87## |
T | 64.20±13.99 | 216.25±32.59 | 286.12±10.60 | 188.15±18.47 | 62.06±3.43 | 4.77±0.93 |
TB Ⅰ | 60.79±13.34 | 219.21±22.30 | 290.61±30.91 | 186.83±21.35 | 62.56±4.04 | 4.47±1.22 |
TB Ⅱ | 64.61±11.83 | 209.98±19.83* | 290.98±27.86 | 187.81±19.90 | 63.00±3.69 | 4.17±0.57 |
TB Ⅲ | 64.80±10.02 | 201.68±29.37* | 289.50±26.95 | 181.11±11.75* | 67.50±4.56 | 3.86±0.71* |
TB Ⅳ | 67.41±11.88 | 183.20±13.92** | 281.87±26.18 | 180.06±12.50* | 67.11±4.88 | 4.06±0.78 |
TB Ⅴ | 65.37±7.38 | 189.87±14.69** | 275.78±15.01* | 172.98±6.53** | 71.44±8.34* | 4.07±0.51 |
TB Ⅵ | 64.88±8.28 | 196.14±13.55** | 262.11±16.46** | 171.67±6.66** | 69.50±7.26 | 4.06±0.88 |
TB Ⅶ | 68.68±12.14 | 206.78±13.03* | 275.72±27.02 | 177.10±14.30** | 64.11±5.60 | 5.13±0.62 |
TB Ⅷ | 64.81±5.78 | 202.78±26.00* | 274.92±24.70 | 178.81±11.98** | 63.67±7.97 | 4.47±0.87 |
Group | Microcirculation/BPU | IL-1β/ng·L-1 | IL-6/ng·L-1 | TNF-α/ng·L-1 | SOD/kU·g-1 Pro | MDA/μmol·g-1 Pro |
Sham | 82.01±7.84 | 164.16±13.76 | 202.29±11.54 | 165.02±15.59 | 70.22±10.50 | 3.00±0.82 |
Model | 64.71±6.50## | 237.96±23.71## | 299.34±34.86## | 211.15±24.10 | 56.39±9.87# | 4.47±0.67## |
T | 70.55±9.87 | 215.09±19.97 | 305.86±46.52 | 186.52±29.04 | 60.17±11.04 | 4.42±0.91 |
TB Ⅰ | 72.08±9.77 | 209.76±23.05 | 286.06±53.57 | 185.57±26.76 | 59.89±13.01 | 3.99±0.99 |
TB Ⅱ | 73.13±17.05 | 209.73±28.89 | 276.71±35.21 | 179.26±21.21 | 62.72±9.33 | 3.94±0.65 |
TB Ⅲ | 77.61±11.20* | 209.30±18.17* | 256.11±18.11* | 175.20±17.87 | 64.44±10.05 | 3.82±0.81 |
TB Ⅳ | 76.76±17.25 | 196.20±16.36** | 241.49±16.88** | 163.58±6.31 | 67.11±5.03* | 3.66±0.82 |
TB Ⅴ | 70.75±10.60 | 203.40±25.58* | 253.71±30.93* | 171.88±15.90 | 68.28±6.88* | 3.55±0.74* |
TB Ⅵ | 74.19±11.22 | 207.71±17.75* | 270.68±7.24 | 177.88±20.53 | 66.22±5.50 | 3.96±0.97 |
TB Ⅶ | 71.18±5.68 | 211.52±24.36 | 269.51±5.49 | 178.09±21.03 | 64.22±6.58 | 3.93±0.98 |
TB Ⅷ | 73.11±12.69 | 212.13±32.04 | 273.69±12.66 | 181.08±18.87 | 64.89±7.60 | 4.10±0.86 |
冰片的抗脑缺血作用已被国内外学者证实[5]。脑病治疗的区域特异性也已受广泛关注。本次研究发现川芎和冰片配伍治疗后,皮层、海马、下丘脑和纹状体组织的微循环、IL-1β、IL-6、TNF-α、SOD和MDA的改善均优于TMPP单用。此外,对各脑区、各指标的冰片最佳剂量进行统计,其最佳剂量排序是0.08 g·kg-1 > 0.16 g·kg-1 > 0.32 g·kg-1 > 0.04 g·kg-1。以上分析提示,TMPP和冰片配伍可以对缺血脑组织产生脑区特异性改善,而且这一改善作用与冰片剂量密切相关,剂量过大或过小均不利于脑缺血的治疗。
( 致谢: 本实验在南京中医药大学江苏省中药药效与安全性评价重点实验室/部省共建中药药理实验室完成。 )
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