基于RIP1/RIP3探究西地那非对勃起功能障碍大鼠睾丸的影响

Exploring the effects of sildenafil on testicular of rats with erectile dysfunction based on RIP1/RIP3

目的探究程序性坏死通路在勃起功能障碍(ED)大鼠睾丸组织损伤中的作用及西地那非干预的机制。方法用高脂饲料喂养的方法建立ED大鼠模型,将其随机分为模型组、实验组、白细胞介素18组和实验+白细胞介素18组;另随机取10只大鼠为正常对照组。实验组灌胃20 mg·kg^(-1)西地那非;白细胞介素18组腹腔注射0.2μg·kg^(-1)白细胞介素18重组蛋白;实验组+白细胞介素18组灌胃20 mg·kg^(-1)西地那非、腹腔注射0.2μg·kg^(-1)白细胞介素18重组蛋白;正常对照组和模型组均灌胃等量0.9%NaCl和腹腔注射等量0.9%NaCl;实验组腹腔注射等量0.9%NaCl,白细胞介素18组灌胃等量0.9%NaCl,5组大鼠每天定时给药1次,连续给药14 d。用逆转录聚合酶链反应(RT-qPCR)和蛋白质印迹法检测大鼠睾丸组织中程序性坏死关键因子受体相互作用蛋白激酶1(RIP1)、受体相互作用蛋白激酶3(RIP3)、混合谱系激酶结构域样蛋白(MLKL)和钙离子-钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达。结果正常对照组、模型组、实验组、白细胞介素18组和实验+白细胞介素18组RIP1蛋白相对表达水平分别为0.58±0.05、0.99±0.09、0.71±0.05、1.23±0.07和0.81±0.07,RIP3蛋白相对表达水平分别为0.61±0.05、1.05±0.10、0.77±0.04、1.19±0.07和0.84±0.08,MLKL蛋白相对表达水平分别为0.63±0.05、1.13±0.08、0.79±0.05、1.30±0.02和1.00±0.04,CaMKⅡ蛋白相对表达水平分别为0.54±0.04、1.12±0.07、0.77±0.05、1.36±0.04和1.00±0.07;上述指标,模型组与正常对照组比较,实验组与模型组比较,白细胞介素18组与模型组比较,实验+白细胞介素18组与白细胞介素18组比较,在统计学上差异均有统计学意义(均P<0.05)。结论RIP1/RIP3介导的程序性坏死通路在ED大鼠睾丸损伤中发挥多重调控作用,西地那非可能通过抑制上述程序性坏死信号通路,改善大鼠睾丸功能。

Objective To investigate the role of programmed necrosis pathway in testicular tissue damage in rats with erectile dysfunction(ED)and the mechanism of sildenafil intervention.Methods An ED rat model was established by high-fat chow feeding,and the rats were randomly divided into model group,experimental group,interleukin 18group,and experimental+interleukin 18 group;10 rats were randomly selected as the normal control group.The experimental group was gavaged with 20 mg·kg^(-1)sildenafil;the interleukin 18 group was injected intraperitoneally with 0.2μg·kg^(-1)interleukin 18 recombinant protein;the experimental+interleukin 18group was gavaged with an equal amount of sildenafil,and the experimental+interleukin 18 group was injected intraperitoneally with an equal amount of interleukin 18 recombinant protein;the normal control and model groups were given with an equal amount of 0.9%NaCl by gavage and intraperitoneally injected with an equal amount of saline;and the experimental group was injected with an equal amount of 0.9%NaCl.Interleukin 18 group was gavaged with an equal amount of 0.9%NaCl,and the five groups of rats were administered once a day at regular intervals for 14consecutive days.Reverse transcription polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression of receptor-interacting protein kinase 1(RIP1),receptor-interacting protein kinase 3(RIP3),mixed-spectrum kinase structural domain-like protein(MLKL),and calcium-calmodulin-dependent protein kinaseⅡ(CaMKⅡ)in rat testis tissues.Results The relative expression levels of RIP1 protein in the normal control group,model group,experimental group,interleukin 18 group and experimental+interleukin 18 group were0.58±0.05,0.99±0.09,0.71±0.05,1.23±0.07 and 0.81±0.07;the relative expression levels of RIP3 protein were 0.61±0.05,1.05±0.10,0.77±0.04,1.19±0.07 and 0.84±0.08,respectively;the relative expression levels of MLKL protein were 0.63±0.05,1.13±0.08,0.79±0.05,1.30±0.02 and 1.00±0.04,respectively;the relative expression levels of CaMKⅡprotein were 0.54±0.04,1.12±0.07,0.77±0.05,1.36±0.04 and1.00±0.07;the differences of the above indexes were statistically significant when the model group was compared with the normal control group,the experimental group was compared with the model group,the interleukin 18 group was compared with the model group,and the experimental+interleukin 18 group was compared with the interleukin 18 group(all P<0.05).Conclusion The RIP1/RIP3-mediated necroptosis pathway plays multiple regulatory roles in testicular injury in ED rats,and sildenafil may improve testicular function in rats by inhibiting the above necroptosis signaling pathway.