经腹腔及阴囊途径注射GYY4137对大鼠睾丸缺血再灌注损伤保护作用的比较

Comparison of intraperitoneal and intratesticular GYY4137 therapy for the treatment of testicular ischemia reperfusion injury in rats

目的比较腹腔内与阴囊内注射GYY4137对大鼠睾丸缺血再灌注损伤的治疗作用.方法选取健康成年雄性SD大鼠40只随机分为4组,每组10只,对照组(A组)、睾丸缺血再灌注损伤组(B组)、睾丸缺血再灌注损伤+GYY4137腹腔给药组(C组)、睾丸缺血再灌注损伤+GYY4137阴囊给药组(D组).A组为假手术组,B、C、D组大鼠建立单侧睾丸扭转复位模型,C组和D组在复位前分别腹腔内和阴囊内注射GYY4137(100μmol/kg).复位4h后取左侧睾丸待测.苏木精-伊红(HE)染色观察睾丸组织病理改变;测定丙二醛(MDA)、超氧化物岐化酶(SOD)含量;以原位缺口末端标记法(TUNEL)检测生精细胞凋亡指数;采用免疫组织化学检测B细胞淋巴瘤/白血病-2相关X蛋白(bax)和半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3的表达水平;蛋白质印迹法(Westem blot)检测肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-1β蛋白表达水平;实时定量反转录聚合酶链反应(RT-qPCR)方法检测睾丸组织中bax、Caspase-3、TNF-α和IL-1β表达水平,应用SPSS20.0统计软件分析,计量资料以均值±标准差(Mean±SD)表示.结果B组可见生精细胞广泛脱落坏死,上皮排列紊乱.与B组比较,C、D组损伤明显改善;B组中MDA含量(5.13±0.26)与A组(1.21±0.20)比较显著上升(t=37.790,P<0.05),SOD含量(0.22±0.04)与A组(0.86±0.05)比较显著下降(t=30.710,P<0.05);与B组比较,C和D组MDA含量(3.26±0.23、2.42±0.24)明显下降(t=17.040、24.220,P<0.05),SDA含量(0.42±0.04、0.58±0.04)显著上升(t=11.450、20.070,P<0.05);B组凋亡指数(33.56±1.45)比A组(6.35±1.12)显著增高(t=46.960,P<0.05),C组和D组凋亡指数(22.58±1.67、17.86±1.65)与B组比较明显下降(t=15.700、22.600,P<0.05);免疫组化结果显示B组中bax和Caspase-3的表达水平较A组显著增加,C组和D组较之明显降低;B组TNF-α(0.774±0.042)和IL-1β(0.674±0.037)的表达水平与A组(0.147±0.022、0.136±0.018)比较明显增高(t=41.820、41.350,P<0.05),C组(0.425±0.029、0.417±0.030)和D组(0.331±0.027、0.308±0.025)较之明显降低(t=21.620、17.060、28.060、25.920,P<0.05);PCR结果显示,与B组(5.35±0.43、3.76±0.31、4.35±0.31、3.86±0.34)比较,C组(3.86±0.32、2.56±0.21、3.08±0.25、2.92±0.28)和D组(3.78±0.36、2.23±0.22、2.96±0.26、2.56±0.25)bax、Caspase-3、TNF-α和IL-1β的表达降低均有所下降(t=8.790、10.130、10.080、6.750、8.850、12.730、10.860、9.740,P<0.05),D组下降程度较显著.结论GYY4137对大鼠睾丸缺血再灌注损伤具有明显的保护作用,阴囊内给药的疗效与腹腔内给药比较更佳.

Objective To investigate the efficacy of intraperitoneal GYY4137 therapy and intratesticular GYY4137 therapy to testicular ischemia reperfusion injury in an experimental rat model.Methods 4 groups were set up as the sham-operation group(group A),torsion/detorsion(T/D)group(group B),torsion/detorsion plus intraperitoneal GYY4137(G-IP)group(group C),and torsion/detorsion plus intratesticular GYY4137(G-IT)group(group D).Group A was the control group.In order to form a testicular T/D model,the left testis was operated and the rotation reached 720 degrees clockwise which lasted 1 hour before reperfusion in group B,group C and group D.Group C accepted 100 pcmol/kg of GYY4137 intraperitoneally half an hour after testicular rotation,while group D was treated with the same dose by intratesticular injection.Four hours after detorsion,the testis was collected and subsequently assessed.The histopathological change of testis was observed with the help of hematoxylin and eosin(HE)staining;the expression of malondialdehyde(MDA)and superoxide dismutase(SOD)was measured;the apoptosis index was determined through TdT-mediated dUTP nick end labeling(TUNEL)methods;immunohistochemistry method was used to evaluate the expression of B cell lymphoma/Ieukemia-2 associated X protein(bax)and cysteinyl aspartate-specific protease(Caspase)-3;tumor necrosis factor-a(TNF-α)and interleukin(IL)-1 p expression was detected through Western blotting;real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to examine the expression of bax,Caspase-3,TNF-α and IL-1 p.Results Group B showed significant changes in histology and an enhancement in the level of oxidative stress and apoptosis compared to the group A,which was relatively ameliorated in group C and group D.Compared with group A(1.21±0.20,0.86±0.05),group B(5.13±0.26,0.22±0.04)showed an increase in the MDA level and a decrease in SOD expression while the changes seemed to be eased in group C(3.26±0.23,0.42±0.04)and group D(2.42±0.24,0.58±0.04,t=37.790,3.710,17.040,24.220,11.450,20.070,P<0.05).The expression of Caspase-3 and bax turned out to be strengthened by T/D in group B and relatively decreased with GYY4137 treatment in both group C and group D.Moreover,an enhancement of TNF-a and IL-1 p expression was found in group B(0.774±0.042,0.674±0.037)compared with group A(0.147±0.022,0.136±0.018)while an inhabitation by GYY4137 in group C(0.425±0.029,0.417±0.030)and group D(0.331±0.027,0.308±0.025)was detected(221.620,17.060,28.060,25.920,P<0.05).Compared with group B(5.35±0.43,3.76±0.31,4.35±0.31,3.86±0.34),the expression of bax,Caspase-3,TNF-α and IL-1 p was inhibited in group C(3.86±0.32,2.56±0.21,3.08±0.25,2.92±0.28)and group D(3.78±0.36,2.23±0.22,2.96±0.26,2.56±0.25).GYY4137,moderating these observed changes,displayed a more protective effect in group D compared to group C.The above results showed statistically significance(t=8.790,10.130,10.080,6.750,8.850,12.730,10.860,9.740,P<0.05).Conclusion Our study indicated that GYY4137 treatment has obvious protective effect on testicular ischemia reperfusion injury in rats,and the efficacy of intratesticular therapy with GYY4137 is greater than that of intraperitoneal therapy,which may provide a more valuable approach for testicular torsion therapy.