灯盏细辛对大鼠肾冷缺血再灌注损伤中细胞凋亡的影响

目的 观察中药灯盏细辛对大鼠肾脏冷缺血再灌注损伤(IRI)中肾脏细胞凋亡及相关基因表达的影响.方法 封闭群SD大鼠36只,随机分为3组,每组12只.假手术组(A组),对照组(B组),实验组(C组).药物应用:C组术前15 min,灯盏细辛注射液按1.2 ml/100 g通过尾静脉注射,A、B组按相应剂量注射生理盐水.动物手术:A组,切除右肾.B、C组采用的是冷IRI模型,3组大鼠均在术后24h再次手术切除左肾进行检测.透射电镜检查肾组织形态学,免疫组织化学检测凋亡相关的基因bcl-2与bax的表达,原位末端标记法(TUNEL)检测细胞凋亡.结果 (1)超微结构检查(透射电镜):A组结构正常；B组细胞呈损伤形态:线粒体肿胀,微绒毛减少,胞质内空泡形成,部分细胞核可见凋亡迹象.C组病变较B组显著减轻.(2)免疫组织化学蛋白阳性染色指数(PI):缺血再灌注后B、C组的bcl-2表达分别为(21.21±1.18)％和(35.52±1.94)％,较A组(4.95±0.77)％均增多(P＜0.05),B组低于C组(P＜0.05).B、C组的bax表达分别为(58.55±2.90)％和(45.90±3.14)％,较A组(4.67±0.67)％增多(P＜0.05),而且B组高于C组(P＜0.05).A组的蛋白阳性染色指数的比值bcl-2/bax为(1.06±0.07)高于B组(0.35±0.03)和C组(0.78±0.07,P＜0.05),而且C组高于B组(P＜0.05).(3)细胞凋亡检测(TUNEL):细胞凋亡指数B组(28.57±3.58)％和C组(19.99±3.37)％均显著大于A组(2.33±0.42)％(P＜0.01),C 组小于B组(P＜0.01).结论 灯盏细辛减少大鼠肾脏冷IRI诱导的肾脏细胞的凋亡,与调节凋亡相关基因bcl-2与bax表达有关.

Effect of eirgeron breviscapus (vant) hand-mazz on apoptosis of renal cells during cold ischemia and reperfusion injury in rats

Objective To explore the effects of eirgeron breviscapus (vant) hand-mazz on apoptosis of renal cells and the expression of related genes in cold ischemia reperfusion injury (IRI) of rats.Methods Thirty-six SD rats were randomly assigned to three groups:sham-operation (A),control (B),and experimental (C).In group C,Eirgeron breviscapus (vant) hand-mazz was injected (1.2 ml/100 g)15 min before operation,and in groups A and B the rats were injected with NaCl ( 1.2 ml/100 g).Group A was only subjected to the removal of the right kidney,and cold ischemia reperfusion model was established in groups B and C.Twenty-four h after operation,left kidneys of all rats in three groups were resected.The morphological changes of the kidneys were observed under the electronic microscopy.The expression of bcl-2 and bax was detected by using immunohistochemistry.Apoptosis was examined by using terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL).Results ( 1 ) Ultrastructure ( electronic microscopy):A normal ultrastructure was observed in group A.The cells were injured in group B:mitochondria swollen,microvillus reduced and cytoplasm vacuolated,and apoptosis appeared in some nuclei.The degree of tubular epithelium injury in group C was milder than in group B; (2) Immunohistochemistry staining:The expression of bcl-2 in renal tubules in group A (4.95 ±0.77)％ was significantly lower than that in group B (21.21 ± 1.18 ) ％ and group C ( 35.52 ± 1.94 ) ％ ( P ＜ 0.05 ),meanwhile,there were fewer bcl-2 positive cells in group B than in group C ( P ＜ 0.05).The expression of bax in renal tubules in group A (4.67 ±0.67)％ was significantly lower than in group B (58.55 ±2.90)％ and group C (45.90 ±3.14)％ (P ＜0.05),meanwhile,there were more bax positive cells in group B than in group C (P ＜0.05).The ratio of bcl-2 I/bax I in group A ( 1.06 ±0.07) was higher than in group B (0.35 ±0.03) and group C (0.78 ±0.07,P ＜0.05),meanwhile,the ratio in group C was higher than in group B (P＜0.05); (3) There were more TUNEL positive cells in group B (28.57 ±3.58)％ and group C ( 19.99 ± 3.37) ％ than in group A (2.33 ± 0.42 ) ％ in tubular epithelium ( P ＜ 0.01 ).TUNEL positive cells in group B were significantly more than those in group C ( P ＜0.01 ).Conclusion Eirgeron breviscapus (vant) hand-mazz decreases apoptotis in tubular epithelium after cold ischemia-reperfusion injury by regulating the expressions of apoptosis-related genes bcl-2 and bax.