2种莲必治注射液对大鼠的毒性作用

目的：研究2种莲必治注射液对大鼠的毒性作用,为探讨其不良反应机制提供实验依据。方法：2种莲必治注射液：莲必治A（含亚硫酸氢钠穿心莲内酯98.7%,其他相关物质1.3%）,莲必治B（含亚硫酸氢钠穿心莲内酯49.1%,其他相关物质50.9%）。SPF级雄性SD大鼠70只,随机分为7组：分别为莲必治A高剂量组（1600mg/kg）、中剂量组（800mg/kg）、低剂量组（400mg/kg）;莲必治B高剂量组（200mg/kg）、中剂量组（100mg/kg）、低剂量组（50mg/kg）和空白对照组（生理盐水）,每组10只。各组动物均按10mL/kg单次尾静脉注射给药。观察给药后3h内大鼠的表现;连续收集给药后0～6、7～12、13～18、19～24h各组大鼠尿液,测定尿酮体、尿潜血、尿素氮、尿肌酐、碱性磷酸酶（ALP）、乳酸脱氢酶（LDH）、N-乙酰-β-D氨基葡萄糖苷酶（NAG）和β2-微球蛋白（β2-MG）;给药后24h经心脏采血检测血液生化指标;最后处死大鼠取各脏器称重,计算脏器系数,行肾脏组织病理学检查。结果：莲必治B高剂量组给药后2只大鼠出现抽搐和毛发竖立,30min后恢复正常,其他组大鼠未见异常。莲必治A高、中、低剂量组与莲必治B高剂量组大鼠尿酮体阳性≥（＋2）的大鼠数多于空白对照组,莲必治A高、中剂量组和莲必治B高、中、低剂量组大鼠尿潜血阳性≥（＋2）的大鼠数多于空白对照组。莲必治A高剂量组大鼠尿素氮含量给药前和给药后7～12h分别为（319±108）mmol/L和（488±139）mmol/L,差异有统计学意义（P〈0.05）。莲必治A高、中、低剂量组和莲必治B高、中剂量组肌酐含量,给药前分别为（3338±1534）、（3502±1457）、（3428±1729）、（3305±922）和（3480±870）mol/L,给药后7～12h,分别增加至（6137±1544）、（5847±1319）、（5630±1622）、（5613±1968）和（5218±1496）mol/L,差异有统计学意义（均P〈0.05）。另外,莲必治A高、中剂量组的肌酐含量明显高于对照组（4326±576）mol/L]（P〈0.05）;肌酐含量的增加与亚硫酸氢钠穿心莲内酯间呈剂量相关（r=0.7909）。莲必治A高剂量组ALP含量给药后7～12h为（152±61）,高于对照组（99±37）U/L];莲必治B高剂量组ALP含量给药后7～12h为（143±42U/L）,高于给药前（94±42）U/L]和对照组,差异均有统计学意义（均P〈0.05）。莲必治A高、中、低剂量组与莲必治B高、中剂量组LDH含量给药前分别为（19±7）、（18±11）、（18±8）、（17±5）和（17±10）U/L,给药后0～6h增加至（88±56）、（80±27）、（50±17）、（57±16）和（28±6）U/L;莲必治A高、中、低剂量组与莲必治B高剂量组LDH含量给药后7～12h分别增加至（44±21）、（36±17）、（34±13）和（31±7）U/L;差异均有统计学意义（均P〈0.05）。另外,莲必治A高、中、低剂量组与莲必治B高、中剂量组的LDH含量给药后0～6h高于相同时间段的空白对照组（18±10）U/L],莲必治A高、中、低剂量组与莲必治B高剂量组的LDH含量给药后7～12h高于相同时间段的空白对照组（23±7）U/L],差异有统计学意义（均P〈0.05）;LDH含量增加与亚硫酸氢钠穿心莲内酯剂量相关（r=0.8990）。莲必治A高、中剂量组NAG含量给药后0～6h分别为（0.30±0.04）和（0.31±0.04）U/L;莲必治A高、中、低剂量组NAG含量给药后7～12h分别为（0.45±0.07）、（0.40±0.18）和（0.37±0.14）U/L;莲必治A高、中、低剂量组和莲必治B高、中、低剂量组NAG含量给药后13～18h分别为（0.35±0.09）、（0.31±0.06）、（0.39±0.18）、（0.60±0.09）、（0.57±0.06）和（0.33±0.08）U/L;莲必治A中、低剂量组和莲必治B高、中、低剂量组NAG含量给药后19～24h分别为（0.32±0.03）、（0.39±0.14）、（0.32±0.07）、（0.34±0.05）和（0.29±0.08）U/L;均高于相同时间段的空白对照组（0.26±.0.05）、（0.22±0.06）、（0.24±0.07）和（0.24±0.06）U/L],差异有统计学意义（均P〈0.05）。莲必治B高剂量组β2-MG含量给药后19～24h为（1.03±0.45）mg/L,对照组β2-MG含量为（0.54±0.24）mg/L,差异有统计学意义（P〈0.05）;NAG和β2-MG增加均与其他相关物质的含量有关（r=0.8749,r=0.9819）。各给药组大鼠血生化检测结果与对照组比较差异无统计学意义,各给药组大鼠主要脏器外观、组织病理和脏器系数均未见明显改变。结论：莲必治注射液对大鼠的潜在肾毒性与其纯度和剂量有关,相关物质含量高以及给药剂量大均可增加肾损害程度。

Toxic effects of two kinds of Lianbizhi injections on rats

Objective：To study the toxic effects of two kinds of Lianbizhi injections on rats in order to provide experimental evidence for the causes of adverse reactions.Methods：Two kinds of Lianbizhi injections were used in this experiment as follows：Lianbizhi A containing andrographolide sodium bisulfis 98.7% and other related substance 1.3%,Lianbizhi B containing andrographolide sodium Bisulfis 49.1% and other related substance 50.9%.Seventy SPF male SD rats were randomly divided into 7 groups：the low-dose（400 mg/kg）,moderate-dose（800 mg/kg）,and high-dose（1 600mg/kg） Lianbizhi A groups,the low-dose（50 mg/kg）,moderate-dose（100 mg/kg）,and high-dose（200mg/kg） Lianbizhi B groups,and the empty control group（normal saline）.Each group comprised 10 rats.All rats received respectively a single intravenous injection of Lianbizhi 10 mL/kg via caudal vein.The presentation of rats was observed within 3 hours after drug administration.The rats＇urine was collected within 0～6,7～12,13～18 and 19～24 hours after drug administration.The levels and values of acetone bodies,occult blood,BUN,creatinine（Crea）,alkaline phosphatase（ALP）,lactate dehydrogenase（LDH）,N-acetyl-β-D-glucosaminidase（NAG）,and β2 microglobulin（β2-MG） were measured.The blood was collected from rat＇KG-1/3]s hearts,and the biochemical indexes were measured.Finally,the rats were sacrificed.Their organs were weighed,organ coefficient was calculated,and histopathological changes in the kidney were observed.Results：Two rats in the high-dose Lianbizhi B group developed convulsion and erect hair,and they recovered 30 minutes later.No abnormal manifestation was found in rats in other groups.The number of rats having positive ketones in urine≥（＋2）] in high-,moderate-,and low-dose Lianbizhi A groups and high-dose Lianbizhi B group was more than that in the control group.The number of rats having positive occult blood in urine≥（＋2）] in the high-,moderate-dose Lianbizhi A groups and high-,moderate-,and low-dose Lianbizhi B groups was more than that in the control group.The BUN levels in the high-dose Lianbizhi A group were（319±108） mmol/L before drug administration and increased to（488±139） mmol/L within 7～12 hours after drug administration.The difference was statistically significant（P0.05）.The Crea levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-and moderate-dose Lianbizhi B groups were respectively（3 338±1534）,（3502±1457）,（3428±1 729）,（3 305±922）,and（3 480±870） mol/L before drug ministration and increased respectively to（6 137±1 544）,（5 847±1 319）,（5 630±1 622）,（5 613±1 968）,and（5 218±1 496） mol/L within 7～12 hours after drug administration.The differences were statistically significant（all P0.05）.In addition,the Crea levels in high-and moderate-dose Lianbizhi A groups were markedly higher than that （4 326±576） mol/L] in the control group（P0.05）.The increase in Crea levels was related to the dose of andrographolide sodium bisulfis（r=0.790 9）.The ALP level in the high-dose Lianbizhi A group was（152±61） U/L within 7～12 hours after drug administration,which was higher than that （99±37）U/L] in the control group.The ALP level in the high-dose Lianbizhi B group was（143±42）U/L within 7～12 hours after drug administration,which was higher than that （94±42）U/L] before drug administration and that in the control group.The differences were statistically significant（all P0.05）.The LDH levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-and moderate-dose Lianbizhi B groups were respectively（19±7）,（18±11）,（18±8）,（17±5）,and（17±10）U/L before drug administration,and then increased respectively to（88±56）,（80±27）,（50±17）,（57±16）,and（28±6）U/L within 0～6 hours after drug administration.The LDH levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-dose Lianbizhi B groups increased respectively to（44±21）,（36±17）,（34±13）,and（31±7）U/L within 7～12 hours after drug administration.The differences were statistically significant（all P0.05）.In addition,the LDH levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-and moderate-dose Lianbizhi B groups were higher than that （18±10）U/L] at the same time periods in the control group,the LDH levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-dose Lianbizhi B groups were higher than that （23±7）U/L] at the same time periods in the control group.The differences were statistically significant（all P0.05）.The increase in LDH levels was related to the dose of andrographolide sodium bisulfis（r=0.899 0）.The NAG levels in the high-and moderate-dose Lianbizhi A groups were respectively（0.30±0.04） and（0.31±0.04） U/L within 0～6 hours after drug administration;the NAG levels in the high-,moderate-,and low-dose Lianbizhi A groups were respectively（0.45±0.07）,（0.40±0.18）,and（0.37±0.14） U/L within 7～12 hours after drug administration;the NAG levels in the high-,moderate-,and low-dose Lianbizhi A groups as well as high-,moderate-,and low-dose Lianbizhi B groups were respectively（0.35±0.09）,（0.31±0.06）,（0.39±0.18）,（0.60±0.09）,（0.57±0.06）,and（0.33±0.08）U/L within 13～18 hours after drug administration;the NAG levels in the moderate-and low-dose Lianbizhi A groups as well as high-,moderate-,and low-dose Lianbizhi B groups were respectively（0.32±0.03）,（0.39±0.14）,（0.32±0.07）,（0.34±0.05）,and（0.29±0.08）U/L within 19～24 hours after drug administration;which were higher than those （0.26±0.05）,（0.22±0.06）,（0.24±0.07）,and（0.24±0.06）U/L] in the control group.The differences were statistically significant（P0.05）.The β2-MG levels in the high-dose Lianbizhi B group was（1.03±0.45） mg/L within 19～24 hours after drug administration,and it was（0.54±0.24）mg/L in the control group;the difference was statistically significant（P0.05）.The increase in NAG and β2-MG levels was associated with the amount of other related substance（r=0.874 9,r=0.981 9）.There were no statistically significant differences in biochemical results in rats between the drug-exposed groups and the control group.No marked changes in the appearance of rat＇KG-1/3]s organs,histopathology,and organ coefficient were found.Conclusion：The potential nephrotoxic effects of Lianbizhi injection on rats is related to its purity and dosage;high amount of other related substance and high dosage of the drug could increase renal damage.