草苁蓉环烯醚萜苷的肝损伤保护作用及抑瘤作用的研究
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摘要
第一部分
草苁蓉环烯醚萜苷对四氯化碳所致大鼠急性肝损伤的保护作用
目的：观察草苁蓉环烯醚萜苷(IGBR)对四氯化碳(CCl4)所致肝损伤的保护作用。
方法：1. IGBR的制备：将10 kg草苁蓉全草切碎后用90%乙醇提取,将提取液减压浓缩,获草苁蓉醇提取物1.86 kg。将醇提取物加水溶解,在等体积二氯甲烷中进行萃取分离。取其水层提取物,上MCI-gel CHP20P凝胶柱(Mitsubishi Chemical Co.),用10%-100%甲醇梯度洗脱,收集50%甲醇洗脱成分,经高效液相层析分离得到IGBR。2.建立CCI4肝损伤模型：大鼠腹腔内注射CCl4(1ml/kg),制备大鼠急性肝损伤模型。3.动物分组及处理：将雄性Wistar大鼠,重(160-180 g)50只,随机分成5组,每组10只,即正常对照组(groupⅠ)、模型组(groupⅡ)、IGBR低剂量组(groupⅢ)、IGBR高剂量组(groupⅣ),、益肝灵组(groupⅤ)。首先将IGBR和益肝灵溶于0.5%羧甲基纤维素中,然后IGBR低、高剂量组和益肝灵组分别灌胃IGBR (100、200mg/kg)和益肝灵(50mg/kg),正常对照组和模型组以等体积的0.5%羧甲基纤维素溶液灌胃,每天一次,连续7天。第7天最后一次给药后1小时,将50%CCL4橄榄油按1 ml/kg剂量注射至Ⅱ-Ⅴ组大鼠腹腔内,而Ⅰ组只注射同体积的橄榄油。腹腔注射CC1416h后,断头取血及肝组织,进行肝组织病理学观察。分离血清,测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白蛋白(ALB)、肿瘤坏死因子(TNF-α)、丙二醛(MDA)、脂氢过氧化物(LOOH)、谷胱甘肽(GSH),测肝组织超氧化物歧化酶(SOD)、铜锌超氧化物岐化酶(Cu/Zn-SOD)、二氧化锰超氧化物歧化酶(Mn-SOD)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽还原酶(GR)、过氧化氢酶(CAT)、丙二醛(MDA)、脂氢过氧化物(LOOH)、谷胱甘肽(GSH)和一氧化氮合酶(iNOS)、细胞色素P4502E1(CYP2E1)表达。
结果：HE染色结果表明,IGBR高、低剂量组和益肝灵组,大鼠CCl4所致肝损伤明显减轻。在大鼠血清中,与正常组相比,CC14肝损伤模型组的ALT、AST、TNF-α水平明显升高；与模型组比较,IGBR高、低剂量组和益肝灵组明显降低CCl4所致急性肝损伤大鼠血清ALT、AST(除IGBR低剂量组外)、TNF-α水平,IGBR高剂量组和益肝灵组血清ALB水平明显提高。在大鼠肝组织中,与正常组比较,CCl4肝损伤模型组的SOD、Cu/Zn-SOD、Mn-SOD、GPx、GR和CAT活性降低；与模型组比较,IGBR高剂量组和益肝灵组肝组织中的SOD、Mn-SOD、GPx(益肝灵组除外)、GR和CAT(包括IGBR低剂量组)活性升高。在血清及肝组织中,与模型组比较,IGBR高剂量组和益肝灵组MDA含量都明显降低,IGBR低剂量组的MDA只有在肝组织中明显降低,且IGBR高剂量组LOOH含量都明显降低,而益肝灵组的LOOH只有在血清中明显降低,IGBR高剂量组和益肝灵组中GSH的活性明显升高；与IGBR低剂量组比较,IGBR高剂量组GSH活性明显升高。与模型组比较,IGBR高剂量组和益肝灵组对CCl4诱导的肝组织iNOS的表达异常增高有显著降低作用,IGBR高剂量组对CCl4诱导的肝组织CYP2E1蛋白表达明显增高,IGBR高剂量组对CCl4诱导的肝组织CYP2E1-特异的单氧合酶的活性有明显增高作用,且同IGBR低剂量组比较,活性明显增高。说明IGBR高剂量组能抑制肝iNOS的表达,提高CYP2E1蛋白含量及恢复其功能。
结论：草苁蓉环烯醚萜苷对CC14所致的大鼠急性肝损伤具有保护作用。机制可能与草苁蓉环烯醚萜苷抑制脂质过氧化、增高抗氧化酶活力、抑制炎症反应、恢复CYP2E1功能和抑制iNOS的表达有关。
第二部分草苁蓉环烯醚萜苷对H22小鼠肝癌移植瘤的抑瘤作用
目的：目前草苁蓉提取物环烯醚萜苷的研究尚不多见,本研究通过建立H22肝癌移植瘤模型,探讨草苁蓉环烯醚萜苷对小鼠H22移植瘤的生长抑制作用。
方法：1. IGBR的制备：将10kg草苁蓉全草切碎后用90%乙醇提取,将提取液减压浓缩,获草苁蓉醇提取物1.86 kg。将醇提取物加水溶解,在等体积二氯甲烷中进行萃取分离。取其水层提取物,上MCI-gel CHP20P凝胶柱(Mitsubishi Chemical Co.),用10%-100%甲醇梯度洗脱,收集50%甲醇洗脱成分,经高效液相层析分离得到IGBR。2.建立H22皮下移植瘤模型：常规方法复苏H22小鼠肝癌细胞,取0.2mL细胞悬液注射到雄性昆明小鼠腹腔内,连续传两代。无菌操作取第2代腹水,用无菌生理盐水稀释,调整细胞密度为1×107个/mL取0.2mL瘤细胞悬液接种到雄性昆明小鼠的右腋窝皮下[4]。3.动物分组及处理：接种后第二天,随机分成模型组、IGBR高剂量组、中剂量组、低剂量组和5-氟尿嘧啶(5-Fu)组,每组各10只。IGBR高、中和低剂量组分别按400、200和100 mg/kg-d剂量ig给药,每日1次,共10次；5-Fu组按25 mg/kg·d剂量隔日ip给药,共5次；模型组则以生理盐水替代药物ig给药。各组动物给药第10 d停药,禁食不禁水16 h后处死。另设正常组10只,每天生理盐水ig给药,共10 d。取瘤、脾脏、胸腺并称重,计算抑癌率及瘤组织病理学观察。眼眶取血,分离血清TNF-α、IL-2、T-AOC和MDA含量。琼脂糖凝胶电泳技术分析药物对DNA的作用,测定微血管密度(MVD),免疫组织化学法检测瘤组织中VEGF、KDR和HIF-1α表达。
结果：1.IGBR对肝癌H22移植瘤小鼠抑癌率的影响：IGBR低、中、高剂量组小鼠肝癌H22移植瘤的生长有明显抑制作用。与模型组比较,IGBR低、中、高剂量组和5-Fu组肝癌H22移植瘤小鼠瘤重显著减轻。与5-Fu组比较,IGBR各剂量组小鼠瘤重均显著增高,IGBR各剂量组抑癌率明显低于5-Fu组。2. IGBR对小鼠试验后体重及免疫器官指数的影响：试验末期IGBR高剂量组小鼠脾指数与模型组比较差异显著,与IGBR低、中剂量组比较差异显著。5-Fu组小鼠体重、胸腺指数和脾指数均低于模型组,也低于IGBR各组。3. IGBR对血清TNF-a和IL-2水平的影响：与正常组比较,模型组小鼠TNF-a显著增高,IL-2显著降低。与模型组比较,IGBR低、高剂量组和5-Fu组TNF-a显著降低,而IGBR各剂量组和5-Fu组IL-2显著增高。4. IGBR对血清抗氧化指标的影响：与正常组比较,模型组小鼠MDA含量显著降低,但T-AOC无显著性差异。与模型组比较,IGBR各剂量组小鼠血清MDA含量显著降低,血清T-AOC显著提高；同时,IGBR各剂量组小鼠血清T-AOC也明显高于5-Fu组；IGBR高剂量组T-AOC与IGBR低、中剂量组比较差异显著。5. IGBR可激活核酸内切酶,在核小体间规则地切割DNA,而且各浓度的药物诱导核酸内切酶切割DNA小片断的大小相等。6.模型组小鼠肿瘤组织毛细血管大量增生,IGBR组和5-Fu组肿瘤边缘区毛细血管明显减少。与模型组比较,IGBR组平均血管密度均有明显减少,并呈现量-效关系。7.免疫印迹结果显示：与模型组组比较,在IGBR组、5-Fu组中血管生成及调控因子VEGF、KDR和HIF-1α的蛋白表达水平分别有明显差异,这些因子的表达水平随着IGBR浓度的增加而分别减少。
结论：IGBR对H22移植瘤具有明显的抑制作用,其作用可能与其抑制新生血管生成、增高机体免疫机能和抗氧化能力有关。
Objective:to observe the protective effect of iridoid glucosides from Boschniakia rossica (IGBR) against carbon tetrachloride (CCl4)-induced liver injury
Method:1. the iridoid glucoside(IGBR) extract:whole russian boschnai aki herb (10kg) were chopped and extracted with 90%EtOH, and evaporated in vacuo to give 1.86 kg of a dark brown powder which was dissolved by H2O,and then successively partitioned with CH2CL2 and H2O in equal capacity. The aqueous layer was subjected to column chromatography over MCI-gel CHP20P (Mitsubishi Chemical Co.), with aqueous MeOH in a decreasing polarity (10%,-100%MeOH.), The fraction of 50%MeOH was collected,the IGBR was obtained through high performance liquid chromatography.2. CCl4-induced liver injury model: CCL4 at a dose of 1 ml/kg of body weight was given intraperitoneally to rats to induce liver damage.3. Animals and study design:50 male Wistar rats weighing 160-180 g assigned to five groups randomly.10 rats each group, group I:normal control, group II:the model group, group III:the low-dose IGBR group, group IV:the high-dose IGBR group, group V:the silymarin group.First, IGBR or silymarin suspension for intra-gastric administration was prepared by suspending in 0.5%CMC, then the low-dose IGBR group with an i.g.100mg.kg IGBR, the high-dose IGBR group an i.g.200mg/kg IGBR, the silymarin group with an i.g.50mg/kg silymarin, the normal control rats and the model group with an i.g.0.5%carboxy methyl cellulose (CMC,vehicle) separately. once daily for seven consecutive days. At 1 hr after the last pre-treatment,50%CCL4 in oliver oil was given intraperitoneally to the rats of groups II-V at a dose of 1 ml/kg of body weight, while oliver oil (wehicle) was injected to groupⅠ. Sixteen hours the administra-tion of CCL4, the rats were killed by cervical decapitation. Blood and hepatic tissue were collected, histopathology observation were carried out. Blood was centrifuged to obtain the serum. To test the alanine aminotransferase (ALT), aminotransferase (AST), albumin (ALB), serum tumour necrosis factor-a(TNF-a), Malondi-aldehyde (MDA), Hepatic lipid hydroperoxide (LOOH), reducedglutathione (GSH), superox-ide dismutase (SOD), copper/zinc-superoxide dismutase (Cu/zn-SOD), manganese-superoxide dismutase(Mn-SOD), glutathione peroxidase(GPx), glutathione reductase (GR), catalase (CAT),Malondi-aldehyde (MDA), Hepatic lipid hydroperoxide (LOOH), reducedglutathione (GSH), inducible nitric oxide syn-thase (iNOS), cytochrome P450 2E1 (CYP2E1) protein expression.
Results:The results of HE dyeing indicated that the rats liver damage induced by CCL4 among the high or low-dose IGBR group or the silymarin group was significantly decreased. The level of serum ALT/AST/TNF-a in the CCl4 group were significant higher than those in the control; The level of serum ALT/AST(except the low-dose IGBR group)/TNF-a in the CCl4 group were significant lower than those in the model. The level of serum ALB was significantly elevated in the high-dose IGBR group and the silymarin group. In the hepatic tissue, The activities of SOD/Cu/Zn-SOD /Mn-SOD/GPx/GR/CAT were found to be decreased significantly in CCl4-induced model animals when compared with the control rats. The activities of SOD/Cu/Zn-SOD/Mn-SOD/GPx(except the silymarin group)/GR/CAT (include the low-dose IGBR group) were found to be elevated compared with the model group. In the serum and the hepatic tissue, the content of MDA significantly decreased in the high-dose IGBR group and the silymarin group compared with the model group, the MDA content of low-dose IGBR group decreased hepatic tissue, and the LOOH content of high-dose IGBR group significantly decreased,while the LOOH content of the silymarin group decreased only in the serum. The GSH activities significantly elevated in the high-dose IGBR group and the silymarin group.The GSH activities significantly elevated compared with the low -dose IGBR group. The CCL4-induced hepatic tissue iNOS abnormality expression was significantly decreased in the high-dose IGBR group and the silymarin group compared with the model group. in the high-dose IGBR group,a higher level of CYP2E1 protein expression was found than in the rats intoxicated with CCL4 alone. CYP2E1-specific monooxygenase activity significantly increased in the high-dose IGBR group, compared with the low-dose IGBR group, the CYP2E1-specific monooxygenase activity markedly increased. Illustrated that the high-dose IGBR group can suppress the liver iNOS expression, Elevate CYP2E1 protein level and recover its function.
Conclusion:IGBR had a protective effect on CCL4-Induced acute liver injury, by suppressing lipid peroxidation, heighten antioxidase activity, suppressing the inflammatory response, reducing Oxidative stress, recovering the CYP2E1 function and suppressing iNOS expression.
Part 2 Anti-tumor effect generated by iridoid glucosides from Boschniakia rossica in H22-bearing mice
Objective:now, the study about iridoid glucoside is less, To investigate the anti-tumor effect Generated by iridoid glucosides from Boschniakia rossica (IGBR) in H22-bearing mice.
Method:1. the iridoid glucoside(IGBR) extract:whole russian boschnai aki herb (10 kg) were chopped and extracted with 90%EtOH, and evaporated in vacuo to give 1.86 kg of a dark brown powder which was dissolved by H2O,and then successively partitioned with CH2CL2 and H2O in equal capacity. The aqueous layer was subjected to column chromatography over MCI-gel CHP20P (Mitsubishi Chemical. Co.), with aqueous MeOH in a decreasing polarity (10%,-100 %MeOH), The fraction of 50%MeOH was collected,the IGBR was obtained through high performance liquid chromatography.2. Establish H22-bearing mice model:Anabiosis H22 mice hepatoma carcinoma cell(HCC) with routine method, 0.2mL cell suspension was given intraper-itoneally to male mice of Kunming, two successive generation were propagated. The hydroperitoneum of second generation was diluted with stroke-physiological saline solution under the sterile operation. Modulated cell density to 1×10/mL, another 0.2mL cell suspension was given subcutaneouly to male mice right armpit of Kunming.3. Animals and study design:sequenti luce after inoculation, mice were assigned to five groups randomly. 10 rats each group, the model group, the high-dose IGBR group, the medium-dose IGBR group, the low-dose IGBR group, five Efudex (5-Fu) group, the high-dose IGBR group with an i.g.400mg/kg.d IGBR, the medium-dose IGBR group, with an i.g.200mg/kg.d IGBR, the low-dose IGBR group, with an i.g.100mg/kg.d IGBR, once daily for ten consecutive days.5-Fu group with an i.p.25mg.kg.d 5-Fu every other day, five times all, while the model group with an i.g liquor natrii chloridi isotonicus replace medicine. All animals were discontinuated in the tenth day, At 16 hr after absolute diet but water, the mice were killed. Another ten mice as control group, with an i.g liquor natrii chloridi isotonicus for ten days.tumors, spleen, thymus were collected and weighed, calculate the tumor growth inhibition ratios and to observe the pathobiology of tumor tissue. Collect blood from fossa orbitalis, separate serum, detect the content of tumor necrosis factor-a(TNF-a), interlekin-2 (IL-2), the total antioxidant capacity (T-AOC) and malondialdehyde (MDA). Analyze the effect of medicine on deoxyribonucleic acid(DNA) with agarose gel electrophoresis. microvessel density (MVD) was determined, blood vessel endothelium growth factor(VEGF), KDR, histoplasma tissue inhibitory factor-la(HIF-la) express were detected with immunohistochemical method.
Results:l.The administration with 400,200 and 100 mg/kg-d IGBR inhibited the growth of transplanted tumor with the inhibitory ratios, the tumor growth inhibition ratios effect of IGBR on H22-bearing mice:The administration with 400,200 and 100 mg/kg-d IGBR have manifest inhibit effect on H22-bearing mice. The administration with 400,200 and 100 mg/kg·d IGBR and 5-Fu group the weight of H22-bearing mice lessen obviously compared with model group. The administration with IGBR all the mice tumor weight significant increase compared with 5-Fu group, the inhibitory ratios of all IGBR groups more lower than 5-Fu group.2. The effect of IGBR on mice weight and immune organ index number after the experiment:the spleen index number of high-dose IGBR group have significant difference compared with model group, as well as the low, medium, high-dose IGBR group.while the weight, thymus index number and spleen index number of 5-Fu group not only lower than model group but also lower than each IGBR group.3. The effect of IGBR on mice serum level of TNF-a and IL-2:compared with control group, the level of serum TNF-a significant increase, the level of serum IL-2 significant degrade. The level of serum TNF-a of low-dose IGBR group, high-dose IGBR group and 5-Fu group significant degrade compared with model group,while all IGBR group and 5-Fu group TNF-a level significant increase.4. The effect of IGBR on serum antioxidate index numbenthe mice MDA content of model group significant degrade,but there is no significant difference in T-AOC. Meanwhile,the level of T-AOC of all IGBR groups significant higher than 5-Fu group, the level of T-AOC has significant difference between high-dose IGBR group and low-dose IGBR group or medium-dose IGBR group.5. IGBR can activate endonuclease, and regular incise DNA in the nucleosome, furthermore all dose medicine induce endonuclease incise DNA in equal fragment. 6.a great quantity micrangium hyperplasia in the mice tumor tissue of model group, the tumor marginal zone micrangium of IGBR group and 5-Fu group obviously decrease.The average blood vessel density of IGBR group obviously decrease, presented dose-effect relationship.7. immunoblotting result indicate:compared with model group, the level of protein expression of VEGF, KDR and HIF-1αof the Angiogenesis and regulating factor has obviously difference between the IGBR and 5-Fu group. These factors express level decreased accompany with the density increase
Conclusion:IGBR possess apparente depressant effect on the growth of transplanted H22 tumor, may be related with suppress newly born Angiogenesis, improve immune function and antioxdative capability.
草苁蓉环烯醚萜苷对四氯化碳所致大鼠急性肝损伤的保护作用
目的：观察草苁蓉环烯醚萜苷(IGBR)对四氯化碳(CCl4)所致肝损伤的保护作用。
方法：1. IGBR的制备：将10 kg草苁蓉全草切碎后用90%乙醇提取,将提取液减压浓缩,获草苁蓉醇提取物1.86 kg。将醇提取物加水溶解,在等体积二氯甲烷中进行萃取分离。取其水层提取物,上MCI-gel CHP20P凝胶柱(Mitsubishi Chemical Co.),用10%-100%甲醇梯度洗脱,收集50%甲醇洗脱成分,经高效液相层析分离得到IGBR。2.建立CCI4肝损伤模型：大鼠腹腔内注射CCl4(1ml/kg),制备大鼠急性肝损伤模型。3.动物分组及处理：将雄性Wistar大鼠,重(160-180 g)50只,随机分成5组,每组10只,即正常对照组(groupⅠ)、模型组(groupⅡ)、IGBR低剂量组(groupⅢ)、IGBR高剂量组(groupⅣ),、益肝灵组(groupⅤ)。首先将IGBR和益肝灵溶于0.5%羧甲基纤维素中,然后IGBR低、高剂量组和益肝灵组分别灌胃IGBR (100、200mg/kg)和益肝灵(50mg/kg),正常对照组和模型组以等体积的0.5%羧甲基纤维素溶液灌胃,每天一次,连续7天。第7天最后一次给药后1小时,将50%CCL4橄榄油按1 ml/kg剂量注射至Ⅱ-Ⅴ组大鼠腹腔内,而Ⅰ组只注射同体积的橄榄油。腹腔注射CC1416h后,断头取血及肝组织,进行肝组织病理学观察。分离血清,测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白蛋白(ALB)、肿瘤坏死因子(TNF-α)、丙二醛(MDA)、脂氢过氧化物(LOOH)、谷胱甘肽(GSH),测肝组织超氧化物歧化酶(SOD)、铜锌超氧化物岐化酶(Cu/Zn-SOD)、二氧化锰超氧化物歧化酶(Mn-SOD)、谷胱甘肽过氧化物酶(GPx)、谷胱甘肽还原酶(GR)、过氧化氢酶(CAT)、丙二醛(MDA)、脂氢过氧化物(LOOH)、谷胱甘肽(GSH)和一氧化氮合酶(iNOS)、细胞色素P4502E1(CYP2E1)表达。
结果：HE染色结果表明,IGBR高、低剂量组和益肝灵组,大鼠CCl4所致肝损伤明显减轻。在大鼠血清中,与正常组相比,CC14肝损伤模型组的ALT、AST、TNF-α水平明显升高；与模型组比较,IGBR高、低剂量组和益肝灵组明显降低CCl4所致急性肝损伤大鼠血清ALT、AST(除IGBR低剂量组外)、TNF-α水平,IGBR高剂量组和益肝灵组血清ALB水平明显提高。在大鼠肝组织中,与正常组比较,CCl4肝损伤模型组的SOD、Cu/Zn-SOD、Mn-SOD、GPx、GR和CAT活性降低；与模型组比较,IGBR高剂量组和益肝灵组肝组织中的SOD、Mn-SOD、GPx(益肝灵组除外)、GR和CAT(包括IGBR低剂量组)活性升高。在血清及肝组织中,与模型组比较,IGBR高剂量组和益肝灵组MDA含量都明显降低,IGBR低剂量组的MDA只有在肝组织中明显降低,且IGBR高剂量组LOOH含量都明显降低,而益肝灵组的LOOH只有在血清中明显降低,IGBR高剂量组和益肝灵组中GSH的活性明显升高；与IGBR低剂量组比较,IGBR高剂量组GSH活性明显升高。与模型组比较,IGBR高剂量组和益肝灵组对CCl4诱导的肝组织iNOS的表达异常增高有显著降低作用,IGBR高剂量组对CCl4诱导的肝组织CYP2E1蛋白表达明显增高,IGBR高剂量组对CCl4诱导的肝组织CYP2E1-特异的单氧合酶的活性有明显增高作用,且同IGBR低剂量组比较,活性明显增高。说明IGBR高剂量组能抑制肝iNOS的表达,提高CYP2E1蛋白含量及恢复其功能。
结论：草苁蓉环烯醚萜苷对CC14所致的大鼠急性肝损伤具有保护作用。机制可能与草苁蓉环烯醚萜苷抑制脂质过氧化、增高抗氧化酶活力、抑制炎症反应、恢复CYP2E1功能和抑制iNOS的表达有关。
第二部分草苁蓉环烯醚萜苷对H22小鼠肝癌移植瘤的抑瘤作用
目的：目前草苁蓉提取物环烯醚萜苷的研究尚不多见,本研究通过建立H22肝癌移植瘤模型,探讨草苁蓉环烯醚萜苷对小鼠H22移植瘤的生长抑制作用。
方法：1. IGBR的制备：将10kg草苁蓉全草切碎后用90%乙醇提取,将提取液减压浓缩,获草苁蓉醇提取物1.86 kg。将醇提取物加水溶解,在等体积二氯甲烷中进行萃取分离。取其水层提取物,上MCI-gel CHP20P凝胶柱(Mitsubishi Chemical Co.),用10%-100%甲醇梯度洗脱,收集50%甲醇洗脱成分,经高效液相层析分离得到IGBR。2.建立H22皮下移植瘤模型：常规方法复苏H22小鼠肝癌细胞,取0.2mL细胞悬液注射到雄性昆明小鼠腹腔内,连续传两代。无菌操作取第2代腹水,用无菌生理盐水稀释,调整细胞密度为1×107个/mL取0.2mL瘤细胞悬液接种到雄性昆明小鼠的右腋窝皮下[4]。3.动物分组及处理：接种后第二天,随机分成模型组、IGBR高剂量组、中剂量组、低剂量组和5-氟尿嘧啶(5-Fu)组,每组各10只。IGBR高、中和低剂量组分别按400、200和100 mg/kg-d剂量ig给药,每日1次,共10次；5-Fu组按25 mg/kg·d剂量隔日ip给药,共5次；模型组则以生理盐水替代药物ig给药。各组动物给药第10 d停药,禁食不禁水16 h后处死。另设正常组10只,每天生理盐水ig给药,共10 d。取瘤、脾脏、胸腺并称重,计算抑癌率及瘤组织病理学观察。眼眶取血,分离血清TNF-α、IL-2、T-AOC和MDA含量。琼脂糖凝胶电泳技术分析药物对DNA的作用,测定微血管密度(MVD),免疫组织化学法检测瘤组织中VEGF、KDR和HIF-1α表达。
结果：1.IGBR对肝癌H22移植瘤小鼠抑癌率的影响：IGBR低、中、高剂量组小鼠肝癌H22移植瘤的生长有明显抑制作用。与模型组比较,IGBR低、中、高剂量组和5-Fu组肝癌H22移植瘤小鼠瘤重显著减轻。与5-Fu组比较,IGBR各剂量组小鼠瘤重均显著增高,IGBR各剂量组抑癌率明显低于5-Fu组。2. IGBR对小鼠试验后体重及免疫器官指数的影响：试验末期IGBR高剂量组小鼠脾指数与模型组比较差异显著,与IGBR低、中剂量组比较差异显著。5-Fu组小鼠体重、胸腺指数和脾指数均低于模型组,也低于IGBR各组。3. IGBR对血清TNF-a和IL-2水平的影响：与正常组比较,模型组小鼠TNF-a显著增高,IL-2显著降低。与模型组比较,IGBR低、高剂量组和5-Fu组TNF-a显著降低,而IGBR各剂量组和5-Fu组IL-2显著增高。4. IGBR对血清抗氧化指标的影响：与正常组比较,模型组小鼠MDA含量显著降低,但T-AOC无显著性差异。与模型组比较,IGBR各剂量组小鼠血清MDA含量显著降低,血清T-AOC显著提高；同时,IGBR各剂量组小鼠血清T-AOC也明显高于5-Fu组；IGBR高剂量组T-AOC与IGBR低、中剂量组比较差异显著。5. IGBR可激活核酸内切酶,在核小体间规则地切割DNA,而且各浓度的药物诱导核酸内切酶切割DNA小片断的大小相等。6.模型组小鼠肿瘤组织毛细血管大量增生,IGBR组和5-Fu组肿瘤边缘区毛细血管明显减少。与模型组比较,IGBR组平均血管密度均有明显减少,并呈现量-效关系。7.免疫印迹结果显示：与模型组组比较,在IGBR组、5-Fu组中血管生成及调控因子VEGF、KDR和HIF-1α的蛋白表达水平分别有明显差异,这些因子的表达水平随着IGBR浓度的增加而分别减少。
结论：IGBR对H22移植瘤具有明显的抑制作用,其作用可能与其抑制新生血管生成、增高机体免疫机能和抗氧化能力有关。
Objective:to observe the protective effect of iridoid glucosides from Boschniakia rossica (IGBR) against carbon tetrachloride (CCl4)-induced liver injury
Method:1. the iridoid glucoside(IGBR) extract:whole russian boschnai aki herb (10kg) were chopped and extracted with 90%EtOH, and evaporated in vacuo to give 1.86 kg of a dark brown powder which was dissolved by H2O,and then successively partitioned with CH2CL2 and H2O in equal capacity. The aqueous layer was subjected to column chromatography over MCI-gel CHP20P (Mitsubishi Chemical Co.), with aqueous MeOH in a decreasing polarity (10%,-100%MeOH.), The fraction of 50%MeOH was collected,the IGBR was obtained through high performance liquid chromatography.2. CCl4-induced liver injury model: CCL4 at a dose of 1 ml/kg of body weight was given intraperitoneally to rats to induce liver damage.3. Animals and study design:50 male Wistar rats weighing 160-180 g assigned to five groups randomly.10 rats each group, group I:normal control, group II:the model group, group III:the low-dose IGBR group, group IV:the high-dose IGBR group, group V:the silymarin group.First, IGBR or silymarin suspension for intra-gastric administration was prepared by suspending in 0.5%CMC, then the low-dose IGBR group with an i.g.100mg.kg IGBR, the high-dose IGBR group an i.g.200mg/kg IGBR, the silymarin group with an i.g.50mg/kg silymarin, the normal control rats and the model group with an i.g.0.5%carboxy methyl cellulose (CMC,vehicle) separately. once daily for seven consecutive days. At 1 hr after the last pre-treatment,50%CCL4 in oliver oil was given intraperitoneally to the rats of groups II-V at a dose of 1 ml/kg of body weight, while oliver oil (wehicle) was injected to groupⅠ. Sixteen hours the administra-tion of CCL4, the rats were killed by cervical decapitation. Blood and hepatic tissue were collected, histopathology observation were carried out. Blood was centrifuged to obtain the serum. To test the alanine aminotransferase (ALT), aminotransferase (AST), albumin (ALB), serum tumour necrosis factor-a(TNF-a), Malondi-aldehyde (MDA), Hepatic lipid hydroperoxide (LOOH), reducedglutathione (GSH), superox-ide dismutase (SOD), copper/zinc-superoxide dismutase (Cu/zn-SOD), manganese-superoxide dismutase(Mn-SOD), glutathione peroxidase(GPx), glutathione reductase (GR), catalase (CAT),Malondi-aldehyde (MDA), Hepatic lipid hydroperoxide (LOOH), reducedglutathione (GSH), inducible nitric oxide syn-thase (iNOS), cytochrome P450 2E1 (CYP2E1) protein expression.
Results:The results of HE dyeing indicated that the rats liver damage induced by CCL4 among the high or low-dose IGBR group or the silymarin group was significantly decreased. The level of serum ALT/AST/TNF-a in the CCl4 group were significant higher than those in the control; The level of serum ALT/AST(except the low-dose IGBR group)/TNF-a in the CCl4 group were significant lower than those in the model. The level of serum ALB was significantly elevated in the high-dose IGBR group and the silymarin group. In the hepatic tissue, The activities of SOD/Cu/Zn-SOD /Mn-SOD/GPx/GR/CAT were found to be decreased significantly in CCl4-induced model animals when compared with the control rats. The activities of SOD/Cu/Zn-SOD/Mn-SOD/GPx(except the silymarin group)/GR/CAT (include the low-dose IGBR group) were found to be elevated compared with the model group. In the serum and the hepatic tissue, the content of MDA significantly decreased in the high-dose IGBR group and the silymarin group compared with the model group, the MDA content of low-dose IGBR group decreased hepatic tissue, and the LOOH content of high-dose IGBR group significantly decreased,while the LOOH content of the silymarin group decreased only in the serum. The GSH activities significantly elevated in the high-dose IGBR group and the silymarin group.The GSH activities significantly elevated compared with the low -dose IGBR group. The CCL4-induced hepatic tissue iNOS abnormality expression was significantly decreased in the high-dose IGBR group and the silymarin group compared with the model group. in the high-dose IGBR group,a higher level of CYP2E1 protein expression was found than in the rats intoxicated with CCL4 alone. CYP2E1-specific monooxygenase activity significantly increased in the high-dose IGBR group, compared with the low-dose IGBR group, the CYP2E1-specific monooxygenase activity markedly increased. Illustrated that the high-dose IGBR group can suppress the liver iNOS expression, Elevate CYP2E1 protein level and recover its function.
Conclusion:IGBR had a protective effect on CCL4-Induced acute liver injury, by suppressing lipid peroxidation, heighten antioxidase activity, suppressing the inflammatory response, reducing Oxidative stress, recovering the CYP2E1 function and suppressing iNOS expression.
Part 2 Anti-tumor effect generated by iridoid glucosides from Boschniakia rossica in H22-bearing mice
Objective:now, the study about iridoid glucoside is less, To investigate the anti-tumor effect Generated by iridoid glucosides from Boschniakia rossica (IGBR) in H22-bearing mice.
Method:1. the iridoid glucoside(IGBR) extract:whole russian boschnai aki herb (10 kg) were chopped and extracted with 90%EtOH, and evaporated in vacuo to give 1.86 kg of a dark brown powder which was dissolved by H2O,and then successively partitioned with CH2CL2 and H2O in equal capacity. The aqueous layer was subjected to column chromatography over MCI-gel CHP20P (Mitsubishi Chemical. Co.), with aqueous MeOH in a decreasing polarity (10%,-100 %MeOH), The fraction of 50%MeOH was collected,the IGBR was obtained through high performance liquid chromatography.2. Establish H22-bearing mice model:Anabiosis H22 mice hepatoma carcinoma cell(HCC) with routine method, 0.2mL cell suspension was given intraper-itoneally to male mice of Kunming, two successive generation were propagated. The hydroperitoneum of second generation was diluted with stroke-physiological saline solution under the sterile operation. Modulated cell density to 1×10/mL, another 0.2mL cell suspension was given subcutaneouly to male mice right armpit of Kunming.3. Animals and study design:sequenti luce after inoculation, mice were assigned to five groups randomly. 10 rats each group, the model group, the high-dose IGBR group, the medium-dose IGBR group, the low-dose IGBR group, five Efudex (5-Fu) group, the high-dose IGBR group with an i.g.400mg/kg.d IGBR, the medium-dose IGBR group, with an i.g.200mg/kg.d IGBR, the low-dose IGBR group, with an i.g.100mg/kg.d IGBR, once daily for ten consecutive days.5-Fu group with an i.p.25mg.kg.d 5-Fu every other day, five times all, while the model group with an i.g liquor natrii chloridi isotonicus replace medicine. All animals were discontinuated in the tenth day, At 16 hr after absolute diet but water, the mice were killed. Another ten mice as control group, with an i.g liquor natrii chloridi isotonicus for ten days.tumors, spleen, thymus were collected and weighed, calculate the tumor growth inhibition ratios and to observe the pathobiology of tumor tissue. Collect blood from fossa orbitalis, separate serum, detect the content of tumor necrosis factor-a(TNF-a), interlekin-2 (IL-2), the total antioxidant capacity (T-AOC) and malondialdehyde (MDA). Analyze the effect of medicine on deoxyribonucleic acid(DNA) with agarose gel electrophoresis. microvessel density (MVD) was determined, blood vessel endothelium growth factor(VEGF), KDR, histoplasma tissue inhibitory factor-la(HIF-la) express were detected with immunohistochemical method.
Results:l.The administration with 400,200 and 100 mg/kg-d IGBR inhibited the growth of transplanted tumor with the inhibitory ratios, the tumor growth inhibition ratios effect of IGBR on H22-bearing mice:The administration with 400,200 and 100 mg/kg-d IGBR have manifest inhibit effect on H22-bearing mice. The administration with 400,200 and 100 mg/kg·d IGBR and 5-Fu group the weight of H22-bearing mice lessen obviously compared with model group. The administration with IGBR all the mice tumor weight significant increase compared with 5-Fu group, the inhibitory ratios of all IGBR groups more lower than 5-Fu group.2. The effect of IGBR on mice weight and immune organ index number after the experiment:the spleen index number of high-dose IGBR group have significant difference compared with model group, as well as the low, medium, high-dose IGBR group.while the weight, thymus index number and spleen index number of 5-Fu group not only lower than model group but also lower than each IGBR group.3. The effect of IGBR on mice serum level of TNF-a and IL-2:compared with control group, the level of serum TNF-a significant increase, the level of serum IL-2 significant degrade. The level of serum TNF-a of low-dose IGBR group, high-dose IGBR group and 5-Fu group significant degrade compared with model group,while all IGBR group and 5-Fu group TNF-a level significant increase.4. The effect of IGBR on serum antioxidate index numbenthe mice MDA content of model group significant degrade,but there is no significant difference in T-AOC. Meanwhile,the level of T-AOC of all IGBR groups significant higher than 5-Fu group, the level of T-AOC has significant difference between high-dose IGBR group and low-dose IGBR group or medium-dose IGBR group.5. IGBR can activate endonuclease, and regular incise DNA in the nucleosome, furthermore all dose medicine induce endonuclease incise DNA in equal fragment. 6.a great quantity micrangium hyperplasia in the mice tumor tissue of model group, the tumor marginal zone micrangium of IGBR group and 5-Fu group obviously decrease.The average blood vessel density of IGBR group obviously decrease, presented dose-effect relationship.7. immunoblotting result indicate:compared with model group, the level of protein expression of VEGF, KDR and HIF-1αof the Angiogenesis and regulating factor has obviously difference between the IGBR and 5-Fu group. These factors express level decreased accompany with the density increase
Conclusion:IGBR possess apparente depressant effect on the growth of transplanted H22 tumor, may be related with suppress newly born Angiogenesis, improve immune function and antioxdative capability.
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