小活络丸的免疫抑制、抗氧化及抗炎镇痛作用(英文)

Immunosuppresive,antioxidative,anti-inflammatory and analgesic effect of Xiahuoluo pills

背景:小活络丸具有祛风除湿、活络通痹等功效,用于治疗风寒湿痹、肢体疼痛,麻木拘挛等症。目的:观察小活络丸对小鼠再次免疫应答、特异性免疫(包括细胞免疫和体液免疫)、非特异性免疫(包括补体C3、单核-巨噬细胞系统和红细胞黏附功能)和自由基损伤,以及对疼痛及多种炎症模型的药理作用。设计:随机对照,分层实验。单位:广州市中医中药研究所和广州市中医医院药剂科。材料:选用NIH小鼠和ICR小鼠共628只,鼠龄6～8周。小活络丸(成分:胆南星、制川乌、制草乌、地龙、乳香(制)等;广州陈李济药厂生产,生产批号:19980612)。兔抗小鼠IgG和补体C3抗血清试剂盒,由广州市医药卫生研究所药物室提供;超氧化物歧化酶活性和丙二醛含量测定试剂盒,南京建成生物工程研究所产品。方法:实验于1998-09/1999-12在广州市中医中药研究所药理研究室和广州市医药卫生研究所药物研究室动物室完成。①观察小活络丸抑制鸡红细胞诱导小鼠再次免疫应答作用:取ICR小鼠84只,雌雄各半,取出20只作为空白对照组;其余均腹腔注射环磷酰胺0.2g/kg,1次/d,1d。第4和12天,共2次腹膜内注射鸡红细胞诱导免疫增强病理模型探讨再次免疫应答。空白对照组(20只)腹腔注射等量生理盐水;动物于加强免疫后,按随机抽签法分为3组:环磷酰胺组20只(灌胃环磷酰胺40mg/kg),小活络丸组21只(灌胃小活络丸混悬液5.54g/kg),模型组20只(灌胃等量蒸馏水);均1次/d,连续灌胃给药7d。19d后分别按单向免疫扩散法测定血清IgG,补体C3含量,采用聚乙二醇沉淀法测定循环免疫复合物含量变化。②观察小活络丸抑制小鼠迟发型超敏反应作用:取ICR小鼠54只,雌雄各半。第1天皮下注射10g/L2,4-二硝基氟苯50μL/只致敏,第4天,将处理后动物按随机抽签法分为3组:泼尼松组(灌胃泼尼松0.01g/kg),小活络丸组(灌胃小活络丸混悬液5.54g/kg),模型组(灌胃等量蒸馏水),每组18只。均1次/d,连续灌胃给药7d。11d后各组小鼠均涂10g/L2,4-二硝基氟苯25μL/右耳,24h后计算肿胀度(右、左耳质量差值)。③观察小活络丸抑制小鼠红细胞免疫黏附功能:取NIH小鼠36只,雌雄各半,按随机抽签法分为3组:环磷酰胺组(灌胃环磷酰胺20mg/kg),小活络丸组(灌胃小活络丸5.54g/kg),空白对照组(灌胃等量蒸馏水),每组12只。均1次/d,连续灌胃给药7d。7d后取小鼠眼眶血,计算红细胞C3b受体花环率和红细胞免疫复合物花环率。④观察小活络丸抑制小鼠碳粒廓清作用:取ICR小鼠33只,雌雄不拘,将其按随机抽签法分为3组:泼尼松组(灌胃泼尼松20mg/kg),小活络丸组(灌胃小活络丸混悬液5.54g/kg),空白对照组(灌胃等量蒸馏水);均1次/d,连续灌胃7d。于给药第7天末次给药后2h,进行碳粒廓清试验,计算廓清指数K(K值=(LogA1-LogA2)/(T2-T1),式中A1和A2分别表示2和10min所取血样的吸光度,T1和T2表示给碳粒后5和10min取血时间;以K值高低,评价碳粒廓清作用。⑤观察小活络丸对鸡红细胞免疫小鼠溶血素、C3和丙二醛含量及超氧化物歧化酶活性的影响:取ICR小鼠80只,雌雄各半,取20只小鼠作为空白对照组,其余均腹腔注射2×108L-1鸡红细胞15mL/kg作免疫诱导,空白对照组均以等量生理盐水代替鸡红细胞,灌胃蒸馏水,1次/d,连续灌胃7d。按随机抽签法将免疫处理的动物分为3组:免疫对照组(灌胃给予蒸馏水),环磷酰胺组(灌胃环磷酰胺20mg/kg),小活络丸组(灌胃小活络丸混悬液5.54g/kg);均1次/d,连续灌胃7d;于第7天末次给药后2h,计算IgM型溶血素半数溶血值[(样品吸收度/鸡红细胞半数溶血时的吸收度)×稀释倍数]。按相应试剂盒操作说明书方法测定血清C3含量及丙二醛含量和超氧化物歧化酶活性。⑥观察小活络丸抑制小鼠琼脂肉芽组织增生作用:取NIH小鼠59只,皮下注射20g/L琼脂0.5mL/只,24h后,按随机抽签法分为3组:双氯芬酸组(灌胃双氯芬酸10mg/kg),小活络丸组(灌胃给予小活络丸混悬液5.54g/kg),模型组(灌胃等量蒸馏水);均1次/d,连续灌胃给药7d;第8天处死小鼠,以每千克体质量琼脂肉芽克数表示抑制增生作用。⑦观察小活络丸抑制小鼠醋酸性扭体反应作用:取NIH小鼠63只,按随机抽签法分为3组:双氯芬酸组(灌胃双氯芬酸50mg/kg),小活络丸组(灌胃小活络丸混悬液5.54g/kg),模型组(灌胃等量蒸馏水);均1次/d,连续灌胃给药2d。末次给药后2h,腹腔注射0.1mol/L醋酸0.2mL/只,计数20min内小鼠的扭体次数。⑧观察小活络丸对二甲苯、巴豆油和角叉菜胶诱发急性渗出性炎症及炎症渗出液中前列腺素E含量的影响:取NIH小鼠219只,双氯芬酸组(灌胃双氯芬酸50mg/kg),小活络丸组(灌胃小活络丸混悬液5.54g/kg),模型组(灌胃等量蒸馏水);均1次/d,共2d。末次给药后2h,①涂二甲苯25μL/右耳,20min后;②涂巴豆油25μL/右耳,4h后;计算耳肿胀度(右耳片重-左耳片重)。③用10g/L角叉菜胶20μL/右足皮内注射致炎,3h后,计算肿胀度(右足和左足间重量差值);并测致炎足渗出液中前列腺素E含量。组间计量资料差异比较采用t(方差不齐用t’)检验。主要观察指标:小活络丸对小鼠再次免疫应答、特异性免疫、非特异性免疫和自由基损伤,以及对疼痛及多种炎症模型的药理作用。结果:NIH小鼠和ICR小鼠628只均进入结果分析。①模型组小鼠IgG、循环免疫复合物含量明显高于其他3组(P<0.01),C3含量明显低于其他3组(P<0.05～0.01)。②泼尼松组和小活络丸组小鼠耳肿胀度明显低于空白对照组(P<0.01)。③空白对照组红细胞C3b受体花环率明显高于其他2组(P<0.01),红细胞免疫复合物花环率明显低于其他2组(P<0.01)。④泼尼松组和小活络丸组吞噬指数(K值)明显低于空白对照组(P<0.01)。⑤环磷酰胺组和小活络丸组IgM型溶血素半数溶血值和血清丙二醛含量明显低于免疫对照组(P<0.01),C3含量明显高于免疫对照组(P<0.01),血清超氧化物歧化酶活力与免疫对照组比较,差异不明显(P>0.05)。⑥双氯芬酸组和小活络丸组小鼠琼脂肉芽组织质量与体质量比值明显低于模型组(P<0.01)。⑦双氯芬酸组和小活络丸组小鼠20min内扭体次数明显少于模型组(P<0.01,0.05)。⑧双氯芬酸组二甲苯炎症模型和巴豆油炎症模型耳肿胀度、角叉菜胶急性炎症模型足肿胀度、炎症渗出液中前列腺素E含量明显小于或低于模型组(P<0.05～0.01),小活络丸组炎症渗出液中前列腺素E含量明显低于模型组(P<0.01)。结论:小活络丸既有免疫抑制的药理作用,又有抗增殖性炎症、镇痛、抗氧化药效学效应。

BACKGROUND： Xiaohuoluo pill can expel pathogenic wind, dampness and activate collaterals. It is used for treatment of Bi-syndrome due to wind-cold-dampness, pain and numbness in limbs. OBJECTIVE： To observe the pharmacological effect of Xiaohuoluo pills on secondary immune response, specific immunity （including cellular immunity and humoral immunity）, non-specific immunity [including complement 3（C3）, mononuclear phagocyte system （MPS） and red blood cell （RBC） adhesion function] and free radical injury as well as pain and many other inflammations in mice. DESIGN： A randomized controlled stratified trial. SETTING： Guangzhou Institute of Traditional Chinese Medicine and Chinese Materia Medica; Department of Pharmacy, Guangzhou Hospital of Traditional Chinese Medicine. MATERIALS： Totally 628 NIH and ICR mice of 6 to 8 weeks were involved in this trial. Xiaohuoluo pills （components： Dannanxing, Zhichuanwu, Zhicaowu, Dilong, Ruxiang and so on; Chenli Pharmaceutical Factory, Guangzhou; Batch No. 19980612） were used in this trial. Rabbit antimouse immunoglobulin G （IgG） and C3 antiserum reagent kit （Guangzhou Institute of Medicine and Health） and reagent kit for measuring the antioxidizing activity of superoxide dismutase （SOD） and the level of malondialdehyde （MDA） （Jiancheng Institute of Bioengineering, Nanjing） were used. METHODS： This trial was carried out in the Guangzhou Institute of Traditional Chinese Medicine and Chinese Materia Medica; Department of Pharmacy, Guangzhou Institute of Medicine and Health during September 1998 to December 1999. ① To observe the suppressive effect of Xiaohu.oluo pills on cock red blood cell （CRBC）-induced secondary immune response： Eight-four ICR mice, male and female in half, were selected. Twenty of 84 mice served as blank controls; The other 64 mice were intraperitoneally injected with cyclophos-phamide （CY） of 0.2 g/kg once. On the 4^th and 12^th days, CRBC was intraperitoneally injected into the mice twice to induce immunoenhancing pathological models to form secondary immune response. Mice served as blank controls were intraperitoneally injected with the same volume of normal saline; The immunoenhanced mice were assigned into 3 groups by a lot： CY group （n=20, CY, 40 mg/kg, intragastric administration, i.g.）, Xiaohuoluo pills （n=21, Xiaohuoluo pills suspension, 5.54 g/kg, i.g.） and model group （n=20, distilled water, the same volume as other groups, i,g,）; once a day within 7 successive days, 19 days later, the levels of serum IgG and C3 were measured with single immunodiffusion method, and the level of circulating immune compound （CIC） was measured with polyethylene glycol precipitation method. ② To observe the suppressive effect of Xiaohuoluo pills on delayed type hypersensitivity （DTH） ： Fifty-four ICR mice, male and female in half, were selected. On the 1^st day, the mice were sensitized by subcutaneous injection of 10 g/L 2,4-dinitrofluorobenzene （DNFB） of 50μL for each. On the 4^th day, the sensitized mice were assigned into 3 groups by a lot： Prednisone group （n=18, prednisone, 0.01 g/kg, i.g. ）, Xiaohuoluo pills （n=18, Xiaohuoluo pills suspension, 5.54 g/kg, i.g.）, model group （n=18, distilled water, the same volume as other groups, i.g.）, all once a day within 7 successive days. 11 days later, 10 g/L DNFB of 25 p.L was spread on the right ear of each mouse in each group. The swelling degree was calculated 24 hours later （The mass difference between right ear and left ear）. ③ To observe the suppressive effect of Xiaohuoluo pills on immune adhesion function of RBC of mouse： Thirty-six NIH mice, male and female in half, were selected and assigned into 3 groups by a lot： CY group （n=12, CY, 20 mg/kg, i.g.）, Xiaohuoluo pills （n=12, Xiaohuoluo pills suspension, 5.54 g/kg, i.g.） and blank control group （n=12, distilled water, the same volume as other groups, i.g.）, once a day within 7 successive days. 7 days later, blood was taken from the orbit of mice for calculating the rosette rate of RBC-C3b receptor and the rosette rate of RBC immune compound. ④ To observe the suppressive effect of Xiaohuoluo pills on carbon expurgatory： Thirty-three ICR mice, of either gender, were selected and assigned into 3 groups by. a lot： Prednisone group （prednisone , 20 mg/kg, i.g. ）, Xiaohuoluo pills （Xiaohuoluo pills suspension, 5.54 g/kg, i.g.）, blank control group （distilled water, the same volume as other groups, i.g.）, once a day within 7 successive days. Two hours after the last administration on the 7^th day, carbon expurgatory test was conducted to calculate expurgatory index K [K=（LogA1- LogA2）/（T2-T1）], in which, A, and A2 represented the absorbance of blood sample taken at 2 and 10 minutes respectively , and T1 and T2 represented blood taking time at 5 and 10 minutes after carbon treatment; The value of K was used to evaluate the effect of carbon expurgatory.⑤ To observe the effect of Xiaohuoluo pills on the levels of hemolysin, C3 and MDA and the activity of SOD in CRBC immune mice： Eighty ICR mice, male and female in half, were selected. Twenty of them served as blank controls, and the other mice were given immune induction by injection of 2×10^8 L^-1 CRBB of 15 mL/kg. Mice served as blank controls were given intragastric administration of the same volume of normal saline, once a day within 7 successive days. The immune-treated mice were assigned into 3 groups by a lot： immune control group （distilled water, i.g.）, CY group （CY, 20 mg/kg, i.g.）, Xiaohuoluo pills group （Xioohuoluo pills suspension, 5.54 g/kg, i.g.） , once a day within 7 successive days; ｜gM-type hemolytic concentration （HC50） was measured at 2 hours after the last administration on the 7^th day [ （Sample absorption / Absorption at HC50 of CRBC） xdiluted time]. The levels of serum C3 and MDA and the activity of SOD were measured according to the method from corresponding reagent kit.⑥ To observe the suppressive effect of Xiaohuoluo pills on agar granulation tissue hyperplasia in mice： Fifty-nine NIH mice were selected and given subcutaneous injection of 20 g/L agar of 0.5 mL for each. 24 hours later, the mice were assigned into 3 groups by a lot： diclofenac group （diclofenac, 10 mg/kg, i.g..）, Xiaohuoluo pills group （Xioohuoluo pills suspension, 5.54 g/kg, i.g.） and model group （distilled water, the same volume as other groups, /.g.）, once a day within 7 successive days; On the 8＇h day, the mice were sacrificed. The hyperplasiainhibiting effect was presented in the form of the mass of agar granulation tissue in one kilogram body mass ⑦ To observe the suppressive effect of Xiaohuoluo pills on acetic distortion reaction： Sixty-three NIH mice were se letted and assigned into 3 groups by a lot： diclofenac group （diclofenac, 50 mg/kg, i.g）, Xiaohuoluo pills group （Xiaohuoluo pills suspension, 5.54 g/kg, i.g.） and model group （distilled water, the same volume of other groups, i.g.）, once a day within 2 successive days. At 2 hours after the last administration, the mice were given intraperitoneal injection of 0.1 mol/L acetic acid of 0.2 mL for each one. The times of distortion of mice within 20 minutes were counted. ⑧ To observe the effect of Xioohuoluo pills on the acute exudative inflammation evoked by dimethylbenzene, croton oil and carrageenan, and the ievel of prostaglandin E in the inflammatory exudates：Totally 219 NIH mice were selected and assigned into 3 groups by a lot： diclofenac group （diclofenac, 50 mg/kg, i.g.）, Xiaohuoluo pills group （Xiaohuoluo pills suspension, 5.54 g/kg, i.g.） and model group （distilled water, the same volume of other groups, i.g.） once a day within 2 successive days. At 2 hours after the last administration, dimethylbenzene of 25μL was spread on the right ear for 20 minutes, or croton oil of 25 μL was also spread on the right ear, 4 hours later, the swelling of right ear was calculated （mass of right ear-mass of left ear）. 10 g/L carrageenan of 20μL was subcutaneously injected into the right foot, 3 hours later, the swelling degree was calculated （The difference of right foot and left foot）; and the level of prostaglandin E in the inflammatory exudates was measured.⑨t test （t＇ test for heteroscedasticity） was used for comparing the difference in measurement data among groups. MAIN OUTCOME MEASURES： Pharmacological effect of Xiaohuoluo pills on secondary immune response, specific immunity, non-specific immunity and free radical injury as well as pain and many other inflammations in mice. RESULTS： Totally 628 NIH and ICR mice were involved in result analysis. ① The level of IgG and CIC of mice in the model group was significantly higher than that in the other 3 groups respectively （P 〈 0.01）, while the level of Cs was significantly lower than that in the other 3 groups （P 〈 0.05 to 0.01）. ② The swelling degree of mice in the diclofenac group and Xiaohuoluo pills group was significantly lower than that in the blank control group respectively （ both P 〈 0.01）. ③ The rosette rate of RBC-C3b receptor and RBC immune compound in the blank control group was significantly higher than that in the other 2 groups respectively （P 〈 0.01）. ④ The phagocytic index （K value ）in the diclofenac group and Xiaohuoluo pills group was significantly lower than that in the blank control group, respectively （both P 〈 0.01）.⑤ IgM-type HC50 and the level of serum MDA of CY group and Xiaohuoluo pills group were obviously lower than those in the immune control group （P 〈 0.01）, while the level of C3 was higher than that of immune control group, there was no significant difference in the activity of serum SOD between CY group or Xiaohuoluo pills group and immune control group （P 〉 0.05）. ⑥ The ratio of agar granulation tissue mass to body mass in the diclofenac group or Xiaohuoluo pills group was significantly lower than that in the model group （P 〈 0.01）. ⑦ The times of distortion of mice within 20 minutes in the diclofenac group or Xiaohuoluo pills group were significantly less than those of model group （P 〈 0.01, 0.05）. ⑧The ear swelling degree of dimethylbenzene-induced inflammatory models and croton oil-induced inflammatory models, and foot swelling degree of carrageenan-induced acute inflammatory models as well as the level of prostaglandin E in the inflammatory exudates in the diclefenac group were significantly milder or lower than those in the model group （P 〈 0.05 to 0.01）, and the level of prostaglandin E in the inflammatory exudates in the Xiaohuoluo pills group wassignificantly lower than that in the model group （P 〈 0.01）. CONCLUSION： Xiaohuoluo pills possess pharmacological effects of immunosuppression, anti-proliferative inflammation, analgesia and antioxidation.