硝酸银和不同粒径纳米银在大鼠体内的急性毒性作用及生物分布研究

摘要

Objective To explore the acute toxic effect and the cumulative target organ of silver nitrate and nano-silver with two different particle diameters in rats.Methods Thirty-six adult SD rats were divided into small particle size nano-silver group (SNS),large particle size nano-silver group (LNS),silver nitrate group (SN),and control group (C) according to the random number table,with 9 rats in each group.The rats of the four groups were respectively injected with 10 mg/mL nano-silver solution (particle diameter of 20 nm,prepared by saline) in silver dose of 30 mg/kg by tail vein for once,10 mg/mL nano-silver solution (particle diameter of 100 nm,prepared by saline) in silver dose of 30 mg/kg,1.67 mg/mL silver nitrate solution (prepared by glucose solution) in silver dose of 3 mg/kg,and 30 mg/mL polyvinylpyrrolidone solution (prepared by saline) in dose of 90 mg/kg.(1) Toxicity test.The general observation was performed within 14 days after injection,and the deviation between value of body mass before injection and each of that on post injection day (PID) 1,7,and 14 were respectively recorded.On PID 1,7,and 14,3 rats of each group were harvested for determination of serum content of alanine aminotransferase (ALT),aspartate aminotransferase (AST),total protein,and albumin by fully automatic biochemical analyzer.Then the rats were sacrificed immediately,and heart tissue,liver tissue,spleen tissue,lung tissue,kidney tissue,and brain tissue were collected to calculate the organ coefficient.Organ samples with obvious changes in organ coefficient were collected for histopathological observation by HE staining,with 3 samples in each group at each time point.(2) Bio-distribution.The specimens of heart,liver,spleen,lung,and kidney of rats from groups SNS,LNS,and SN were collected for detection of silver content by inductively coupled plasma mass spectrometry,with 3 samples in each group at each time point.Data were processed with analysis of variance of factorial design,LSD test,and Dunnett's T3 test.Results (1) The general condition of rats in groups C and SN after injection were normal.The state of rats of groups SNS and LNS was poor with black secretion in the eye and other phenomena on PID 1,which recovered from PID 3 on.(2) The deviations between values of body mass before injection and that on PID 14 in rats of groups LNS and SN were significantly decreased as compared with deviation of group C (with P values below 0.01),but deviation of group SNS was not significantly changed (P ＞ 0.05).The deviations between values of body mass before injection and each of that on PID 1 and 7 in rats in the other three groups were similar to those in group C (with P values above 0.05).(3) Compared with those in group C,the serum content of total protein of rats in group SN on PID 1 was significantly decreased,and liver coefficient was significantly increased (with P values below 0.05).On PID 1,the serum content of ALT of rats in group LNS was (61.0 ± 8.7) U/L,which was significantly higher than that in group C [(40.0 ±4.6) U/L,P ＜0.01].Compared with those in group C [(126.0±3.5) U/Land4.05±0.23],the serum content of AST of rats in groups SNS and LNS on PID 1 [(249.7 ± 107.2) and (237.0 ± 38.3) U/L] was significantly increased,and liver coefficients (3.50 ± 0.38 and 3.31 ± 0.07) were significantly decreased,with P values below 0.05.Compared with those in group C [(69.2 ±4.8) U/L and 4.32 ± 0.39],the serum content of AST of rats in groups SNS and LNS on PID 7 [(181.0 ± 51.5) and (167.7 ± 16.5) U/L] was also significantly increased,and liver coefficients (3.55 ± 0.18 and 3.62 ± 0.21) were also significantly decreased,P ＜ 0.05 or P ＜ 0.01.On PID 14,the four liver biochemical indexes in serum and all organ coefficients of rats in the other three groups were similar to those in group C (with P values above 0.05).(4) The liver of rats in group SN had slight degeneration on PID 1,the liver cells around the central vein of liver of rats in group SNS had slight degeneration on PID 7,and the liver cells got severely eosinophilic degeneration in liver of rats in group LNS on PID 7.There was no significant pathological change in the liver of rats in each group at the rest time points.(5) The silver content of lung and kidney in rats of group SNS on PID 1,that of spleen and kidney in rats of group LNS on PID 1,and that of heart and kidney in rats of groups LNS and SNS on PID 7 was significantly less than that of group SN (with P values below 0.05).The silver content of liver and spleen in rats of group SNS on PID 14 was significantly more than that of group SN (with P values below 0.05).Compared with that of group SN,the silver content of lung on PID 1 and liver on PID 7 in rats of group LNS was significantly increased (with P values below 0.05).On PID 14,there was no significant change in the silver content of all organs of rats between group SN and group LNS (with P values above 0.05).The silver content of heart,lung,and kidney on PID 1 and heart on PID 7 in rats of group LNS was significantly more than that of group SNS (with P values below 0.05).On PID 14,the silver content of each organ of rats in group SNS was close to that in group LNS (with P values above 0.05).Conclusions Silver nitrate and nanosilver with two different particle diameters have a short acute toxic effect on the liver of rats,and the liver has certain ability of self-healing.Nano-silver is mainly accumulated in the liver.The distribution of nano-silver with large particle diameter in organs is more widely than that of nano-silver with small particle diameter.%目的 探讨硝酸银和2种粒径纳米银对大鼠的急性毒性作用及蓄积靶器官.方法选取36只成年SD大鼠,按照随机数字表法分为小粒径纳米银组、大粒径纳米银组、硝酸银组和对照组,每组9只.上述4组大鼠分别经尾静脉一次性注射10 mg/mL纳米银溶液(纳米银颗粒直径为20 nm,生理盐水配制),银注射剂量为30 mg/kg;10 mg/mL纳米银溶液(纳米银颗粒直径为100 nm,生理盐水配制),银注射剂量为30 mg/kg;1.67 mg/mL硝酸银溶液(葡萄糖溶液配制),银注射剂量为3 mg/kg;30 mg/mL聚乙烯吡咯烷酮溶液(生理盐水配制),注射剂量为90 mg/kg.(1)毒性实验.注射后14d内每日行大体观察,记录注射前分别与注射后1、7、14d的体质量差值.于注射后1、7、14d每组各取3只大鼠,采用全自动生化分析仪检测血清中ALT、AST、总蛋白、白蛋白含量;之后立即处死大鼠,取心脏、肝脏、脾脏、肺脏、肾脏和大脑组织,计算各个脏器系数.选取脏器系数变化明显的脏器标本,每组各时相点3个样本,行HE染色观察组织病理学变化.(2)生物分布实验.选取小粒径纳米银组、大粒径纳米银组、硝酸银组大鼠的心脏、肝脏、脾脏、肺脏、肾脏标本,每组各时相点3个样本,采用电感耦合等离子体质谱仪检测各脏器银含量.对数据行析冈设计方差分析、LSD检验、Dunnett's T3检验.结果 (1)对照组和硝酸银组大鼠注射后大体情况无异常;2个纳米银组大鼠注射后1d出现状态不佳、眼部有黑色分泌物等现象,注射后3d起恢复.(2)与对照组比较,硝酸银组、大粒径纳米银组大鼠注射后14 d与注射前的体质量差值明显减少(P值均小于0.01);小粒径纳米银组大鼠该指标则与之接近(P＞0.05).与对照组比较,另3组大鼠注射后1、7d与注射前的体质量差值无明显变化(P值均大于0.05).(3)与对照组比较,注射后1d硝酸银组大鼠血清中总蛋白含量下降、肝脏系数明显上升(P值均小于0.05).注射后1d,大粒径纳米银组大鼠血清中ALT含量为(61.0 ±8.7)U/L,明显高于对照组的(40.0±4.6)U/L,P＜0.01.与对照组的(126.0±3.5)U/L、4.05 ±0.23比较,注射后1d小粒径纳米银组、大粒径纳米银组大鼠血清中AST含量明显上升[(249.7±107.2)、(237.0±38.3) U/L],肝脏系数(3.50±0.38、3.31 ±0.07)明显下降,P值均小于0.05.与对照组的(69.2±4.8)U/L、4.32 ±0.39比较,注射后7d小粒径纳米银组、大粒径纳米银组大鼠血清中AST含量[(181.0 ±51.5)、(167.7 ±16.5)U/L]亦明显上升,肝脏系数(3.55 ±0.18、3.62±0.21)亦明显下降,P＜0.05或P＜0.01.与对照组比较,注射后14d另3组大鼠血清中4项肝脏生化指标、各个脏器系数均无明显变化(P值均大于0.05).(4)注射后1d,硝酸银组大鼠肝脏有轻微变性;注射后7d,小粒径纳米银组大鼠肝脏中央静脉周围肝细胞轻微变性,大粒径纳米银组大鼠肝脏肝细胞重度嗜酸性变.其余时相点各组大鼠的肝脏无明显病理改变.(5)注射后1d,小粒径纳米银组大鼠肺脏、肾脏与大粒径纳米银组大鼠脾脏、肾脏及注射后7d2个纳米银组大鼠心脏、肾脏的银含量明显少于硝酸银组(P值均小于0.05),注射后14 d小粒径纳米银组大鼠肝脏、脾脏的银含量明显多于硝酸银组(P值均小于0.05).与硝酸银组比较,大粒径纳米银组大鼠注射后1d肺脏及注射后7d肝脏的银含量明显增多(P值均小于0.05),注射后14 d各脏器银含量无明显变化(P值均大于0.05).大粒径纳米银组大鼠注射后1d心脏、肺脏、肾脏及注射后7d心脏的银含量明显多于小粒径纳米银组(P值均小于0.05),注射后14d2组大鼠各脏器银含量相近(P值均大于0.05).结论 硝酸银和2种粒径的纳米银对大鼠的肝脏有短暂的急性毒性作用,肝脏对这种毒性作用有一定的自愈能力.纳米银主要蓄积于肝脏,大粒径纳米银在脏器中的分布较小粒径纳米银更广.