富氢盐水对重度烫伤延迟复苏大鼠肝脏的作用

Effects of hydrogen-rich saline on liver of severely scalded rats with delayed resuscitation

目的 探讨富氢盐水对重度烫伤延迟复苏大鼠肝脏的影响. 方法 取24只SD大鼠,应用台式控温烫伤仪制成40％ TBSAⅢ度烫伤模型,按随机数字表法分为富氢盐水组、乳酸钠林格液组,每组12只.伤后6h开始,2组大鼠分别采用富氢盐水（自制,浓度为0.6 mmol/L）、乳酸钠林格液,均按4 mL＆#183; kg-1＆#183;％TBSA-1进行腹腔注射补液,持续到伤后48 h,伤后第2个24 h补液量为伤后第1个24 h的一半.伤后6（补液前）、12、24、48 h,每组分别取3只大鼠行心脏采血,处死后取肝组织.HE染色观察肝组织病理学变化,细胞计数板行肝组织中性粒细胞计数,全自动生化分析仪检测血清AST、ALT水平,ELISA法检测肝组织中TNF-α、IL-1β、IL-6以及8羟基脱氧鸟苷（8-OHdG）的含量,分光光度计检测肝组织中丙二醛的吸光度值并定量.对数据行重复测量方差分析和LSD-t检验. 结果 伤后48 h,与乳酸钠林格液组比较,富氢盐水组大鼠肝组织炎性细胞浸润减少,肝细胞充血减轻.伤后12、24、48 h,富氢盐水组大鼠肝组织中性粒细胞数分别为（25.3±1.8） ×10^5、（19.6±0.6） ×10^5、（14.1±3.2）×10^5个/mL,显著低于乳酸钠林格液组的（31.9±2.0）×10^5、（30.9±2.2） ×10^5、（23.8±3.0） ×10^5个/mL（t值分别为5.6、7.6、8.7,P值均小于0.05）.伤后6、12h,2组大鼠血清AST、ALT水平以及肝组织TNF-α、IL-1 β、IL-6含量均相近（t值分别0.3 ～3.9与0.9 ～3.8,P值均大于0.05）.伤后24、48 h,富氢盐水组大鼠血清AST与ALT水平分别为（308 ±24）、（210±15） U/L与（93±7）、（70 ±5） U/L,显著低于乳酸钠林格液组的（541 ±39）、（505±18）U/L与（156 ±9）、（166 ±21） U/L（t值分别为17.5、16.7与30.3、6.9,P值均小于0.05）.伤后24、48 h,富氢盐水组大鼠肝组织TNF-α、1L-1 β、IL-6含量分别为（20.7±1.6）、（13.7±1.5）pg/mg,（7.7±1.5）、（6.3±1.2）pg/mg与（8.7±1.2）、（6.0±2.0）pg/mg,显著低于乳酸钠林格液组的（32.7±5.0）、（25.7±4.0）pg/mg,（16.3±2.5）、（12.0±2.7） pg/mg与（14.7±2.1）、（13.3±1.5） pg/mg（t值分别为5.2、5.7,4.7、4.7与10.4、4.4,P值均小于0.05）.2组大鼠伤后6h肝组织丙二醛水平及伤后6、12h肝组织8-OHdG水平均相近（t值分别为0.1与0.7、4.3,P值均大于0.05）.富氢盐水组大鼠伤后12、24、48 h肝组织丙二醛水平分别为（15.3±1.5）、（8.7±1.2）、（6.7±1.5） mmol/mg,显著低于乳酸钠林格液组的（27.3±4.7）、（20.3±1.5）、（14.0±1.0） mmol/mg（t值分别为5.2、5.7、5.1,P值均小于0.05）;伤后24、48 h肝组织8-OHdG水平分别为（124±12）、（79±10） pg/mg,显著低于乳酸钠林格液组的（191±10）、（136±15） pg/mg（t值分别为8.0与8.1,P值均小于0.05）. 结论 富氢盐水能够显著减轻重度烫伤延迟复苏大鼠肝脏中性粒细胞浸润,降低肝脏炎症因子水平,有效减轻肝脏氧化应激反应,进而保护大鼠肝脏.

Objective To explore the effects of hydrogen-rich saline （HS） on liver of severely scalded rats with delayed resuscitation.Methods Twenty-four SD rats were inflicted with 40% TBSA fullthickness scald using a temperature-controlled scalding apparatus.The injured rats were divided into lactated Ringer＆#39;s solution （RS） and HS groups according to the random number table,with 12 rats in each group.Rats in groups RS and HS were respectively resuscitated with an intraperitoneal injection of 4 mL ＆#183; kg-1 ＆#183; % TBSA-1 of RS or HS （self-prepared,with concentration of hydrogen 0.6 mmol/L） 6 hours after injury up to 48 hours after scald.The infusion volume of the second 24 hours after injury was a half of that of the first 24 hours.At post scald hour （PSH） 6 （before resuscitation）,12,24,and 48,blood was collected from the heart of 3 rats in each group,and then the rats were sacrificed for harvesting liver tissue.The pathological change in liver tissue was observed with HE staining.The number of hepatic neutrophils was counted with a hematocytometer.Serum levels of AST and ALT were determined with full-automatic biochemical analyzer.Contents of TNF-α,IL-1β,IL-6,and 8-hydroxy-2＇ -deoxyguanosine （8-OHdG） in liver tissue were determined with ELISA.Absorbance value of malondialdehyde （MDA） in liver tissue was detected and quantified with spectrophotometer.Data were processed with analysis of variance of repeated measurement and LSD-t test.Results At PSH 48,moderate infiltration of inflammatory cells and hepatic hyperemia were observed in rats of group HS as compared with group RS.At PSH 12,24,and 48,the number of neutrophils in group HS was respectively （25.3 ± 1.8） ×10^5,（19.6 ± 0.6） ×10^5,and （14.1 ± 3.2） ×10^5 cells per mililitre,and they were significantly lower than those in group RS [（31.9 ± 2.0） ×10^5,（30.9 ± 2.2） ×10^5,and （23.8 ±3.0） ×10^5cells per mililitre,with t values respectively 5.6,7.6,and 8.7,P values below 0.05].At PSH 6 and 12,the serum levels of AST and ALT and the levels of TNF-α,IL-1β,and IL-6 in liver tissue were close between the two groups （with t values respectively 0.3-3.9 and 0.9-3.8,P values above 0.05）.At PSH 24 and 48,the serum levels of AST and ALT in group HS were respectively （308 ±24） and （210 ± 15） U/L and （93 ±7） and （70 ±5） U/L,which were significantly lower than those in group RS [（541 ±39） and （505 ± 18） U/L,with t values respectively 17.5 and 16.7,P values below 0.05; （156 ± 9） and （166 ± 21） U/L,with t values respectively 30.3 and 6.9,P values below 0.05].At PSH 24 and 48,the levels of TNF-α,IL-1 β,and IL-6 in liver tissue in group HS were respectively （20.7 ± 1.6） and （13.7 ±1.5） pg/mg,（7.7±1.5） and （6.3±1.2） pg/mg,and （8.7±1.2） and （6.0±2.0） pg/mg,which were significantly lower than those in group RS [（32.7 ± 5.0） and （25.7 ± 4.0） pg/mg,with t values respectively 5.2 and 5.7,P values below 0.05 ; （16.3 ± 2.5） and （12.0 ± 2.7） pg/mg,with t values both as 4.7,P values below 0.05 ; （14.7 ± 2.1） and （13.3 ± 1.5） pg/mg,with t values respectively 10.4 and 4.4,P values below 0.05].The level of MDA at PSH 6 and levels of 8-OHdG at PSH 6 and 12 in liver tissue were close between the two groups （with t values respectively 0.1,0.7,and 4.3,P values above 0.05）.In group HS,the levels of MDA in liver tissue at PSH 12,24,and 48 were respectively （15.3 ± 1.5）,（8.7 ± 1.2）,and （6.7 ± 1.5） mmol/mg,and the levels of hepatic 8-OHdG at PSH 24 and 48 were respectively （124 ± 12） and （79 ± 10） pg/mg,which were significantly lower than those in group RS [（27.3±4.7）,（20.3±1.5）,and （14.0±1.0） mmol/mg,with t values respectively 5.2,5.7,and 5.1,P values below 0.05 ; （191 ± 10） and （136 ± 15） pg/mg,with t values respectively 8.0 and 8.1,P values below 0.05].Conclusions Resuscitation with HS could protect liver of severely scalded rats with delayed resuscitation possibly by reducing infiltration of neutrophils,thus lowering the content of inflammatory cytokines,and effectively alleviating oxidative stress.