细胞保护蛋白PTD-FNK对脂多糖诱导的小鼠睾丸炎性损伤的保护作用

The effect of cytoprotective protein PTD-FNK on lipopolysaccharideinduced inflammatory testicular injury in mice

本试验旨在探索细胞保护蛋白PTD-FNK对脂多糖诱导的小鼠睾丸炎性损伤的作用机制。将30只C57BL/6J雄性小鼠随机分为空白组、LPS组(20 mg/kg)、LPS+PTD-FNK(0.1、0.3、0.5 mg/kg)组。空白组腹腔注射0.9%生理盐水,LPS组腹腔注射含LPS(20 mg/kg)的生理盐水,LPS+PTD-FNK组先腹腔注射含有不同浓度PTD-FNK的生理盐水预保护1 h,再以20 mg/kg的LPS腹腔注射诱导其睾丸炎症6 h,随后颈椎脱位处死,分别取各组小鼠睾丸用于后续试验。HE染色观察小鼠睾丸组织形态结构变化,Western-blot检测睾丸细胞焦亡相关蛋白NLRP3、Caspase-1、GSDMD、IL-1β的表达,酶联免疫吸附试验(ELISA)检测睾酮分泌水平,实时荧光定量PCR(RT-qPCR)检测小鼠睾酮合成相关基因(STAR、CYP11A1、17β-HSD、3β-HSD)、炎症因子相关基因(IL-6、IL-1β、TNF-α)及血睾屏障(BTB)相关基因(CX-43、ZO-1、Occludin)mRNA的表达。结果显示,与LPS组相比,LPS+PTD-FNK组小鼠睾丸病理损伤得到明显改善;IL-6、TNF-α、IL-1βmRNA的表达显著降低,CYP11A1、17β-HSD、3β-HSD、CX-43、ZO-1、Occludin mRNA的表达显著升高;小鼠睾酮分泌水平显著提高,焦亡蛋白NLRP3、Caspase-1、GSDMD、IL-1β的表达显著下降。结果表明,0.3 mg/kg的PTD-FNK可在一定程度上缓解小鼠睾丸炎性损伤,降低睾丸细胞焦亡,增强血睾屏障功能以及提高小鼠睾酮合成与分泌能力。

The objective of this study was to explore the mechanism of cytoprotective protein PTD-FNK on lipopolysaccharids-induced inflammatory testicular injury in mice.30 C57BL/6J male mice were randomly divided into blank group,LPS group(20 mg/kg),LPS+PTD-FNK group(0.1 mg/kg,0.3 mg/kg,and 0.5 mg/kg).The blank group mice were intraperitoneally injected with 0.9%normal saline,and the LPS+PTD-FNK group mice were intraperitoneally injected with normal saline containing different concentrations of PTD-FNK for pre-protection for 1 h,and then the mice were intraperitoneally injected with 20 mg/kg LPS to induce testicular inflammation for 6 h,and then the mice were killed by cervical dislocation.Testis of mice in each group were taken for subsequent experiments.The morphological and structural changes of testicular tissue were observed after HE staining.The protein expressions of NLRP3,Caspase-1,GSDMD and IL-1βin testicular cells were detected using Western-blot,and the levels of testosterone secretion were detected using enzyme linked immunosorbent assay(ELISA).Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the mRNA expression of testosterone synthesis-related genes(STAR,CYP11A1,17β-HSD,and 3β-HSD),inflammatory factor related genes(IL-6,IL-1β,and TNF-α)and blood-testis barrier(BTB)related genes(CX-43,ZO-1,and Occludin)in mice.Compared with LPS group,the pathological injury of testis in LPS+PTD-FNK group was significantly improved.The mRNA expression of IL-6,TNF-αand IL-1βsignificantly decreased,while the mRNA expression of CYP11A1,17β-HSD,3β-HSD,CX-43,ZO-1 and Occludin significantly increased.The levels of testosterone secretion in mice were significantly increased,and the pyroptosis related proteins expressions of NLRP3,Caspase-1,GSDMD and IL-βwere significantly decreased.In conclusion,the study indicated 0.3 mg/kg PTD-FNK could alleviate testicular inflammatory injury and reduce testicular cell pyrodeath.0.3 mg/kg PTD-FNK could enhance the function of blood-testis barrier and improve the synthesis and secretion of testosterone in mice to a certain extent.