他克林对脂多糖诱导小鼠睾丸支持细胞炎性损伤的保护作用  

Protective effect of tacrine on inflammatory injury of LPS-induced TM4 cells

作　　者：蔡金霞 黄明光 胡传活[1] 张鑫 梁莹莹 纪伟霞 冯妮 葛晨玲 陈志英 李珣[1] 王晓晔[1] CAI Jin-xia;HUANG Ming-guang;HU Chuan-huo;ZHANG Xin;LIANG Ying-ying;JI Wei-xia;FENG Ni;GE Chen-ling;CHEN Zhi-ying;LI Xun;WANG Xiao-ye(College of Animal Science and Technology,Guangxi University,Nanning 530004,China;Guangxi Institute of Animal Sciences,Nanning 530001,China)

机构地区：[1]广西大学动物科学技术学院,南宁530004 [2]广西畜牧研究所,南宁530001

出　　处：《西南农业学报》2022年第3期700-706,共7页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31660700);广西自然科学基金项目(2016GXNSFAA380024)。

摘　　要：【目的】分析他克林对脂多糖(LPS)诱导小鼠睾丸支持细胞(TM4)炎性损伤的保护作用,从炎症角度探索他克林的功能,为动物生产过程中雄性动物生殖疾病相关抗炎药物的利用及精子质量提升提供更多药物选择。【方法】采用CCK8法检测0.01、0.10、1.00、10.00和100.00μg/mL LPS分别诱导3.0、6.0、12.0和24.0 h对TM4的适宜损伤条件;同时分析0.01、0.10、1.00、10.00、100.00、1000.00、10000.00和100000.00μg/mL他克林在加入LPS前预处理0.5、1.0和2.0 h或加入LPS后处理0.5、1.5、3.0、6.0、12.0和24.0 h对TM4炎性损伤的适宜保护条件。以荧光定量PCR(qRT-PCR)法检测TNF-α和IL-6基因mRNA表达水平,以ELISA法进一步评价细胞上清TNF-α和IL-6蛋白表达量,以免疫蛋白印迹(WB)法分析NF-κB蛋白表达量。【结果】不同浓度不同处理时间下,LPS对TM4增殖的影响情况存在差异,其中1.00μg/mL LPS处理12 h TM4的增殖率极显著低于空白组(P<0.01,下同)。他克林对TM4炎性损伤的保护作用与处理时间和剂量有关,其中,10000.00μg/mL他克林预处理2.0 h的TM4增殖率显著低于空白组(P<0.05,下同),10000.00、100000.00μg/mL他克林预处理0.5和1.0 h或后处理0.5、1.5、3.0、6.0、12.0和24.0 h的TM4增殖率均极显著低于空白组,而0.01μg/mL他克林后处理6.0 h的保护作用最佳,其TM4增殖率显著高于空白组。与空白组相比,LPS可显著上调TM4的IL-6和TNF-α基因mRNA及蛋白表达量,而与LPS组相比,0.01μg/mL他克林显著下调其表达量。与空白组相比,LPS可显著上调TM4的NF-κB蛋白表达量;1.00、100.00和10000.00μg/mL他克林可极显著上调NF-κB蛋白表达量,而与LPS相比,0.01μg/mL他克林降低NF-κB蛋白表达量,但差异不显著(P>0.05)。【结论】0.01μg/mL他克林后处理6.0 h对TM4的保护作用最佳,其机制可能与IL-6和TNF-α基因mRNA及蛋白表达量下调有关。【Objective】To analyze the protective effect of tacrine on lipopolysaccharide(LPS)-induced inflammatory damage in mouse testicular sertoli cells(TM4),and to further explore the function of tacrine from an inflammatory perspective,in order to provide more drug options for the use of anti-inflammatory drugs related to reproductive diseases and sperm quality enhancement in male animals during animal production.【Method】The CCK8 method was used to detect the appropriate damage conditions for TM4 induced by 0.01,0.10,1.00,10.00 and 100.00μg/mL LPS for 3.0,6.0,12.0 and 24.0 hours,respectively;And to analyze the appropriate protection conditions for TM4 pretreated by 0.01,0.10,1.00,10.00,100.00,1000.00,10000.00 and 100000.00μg/mL tacrine for 0.5,1.0 and 2.0 hours prior to the addition of LPS or treated for 0.5,1.5,3.0,6.0,12.0 and 24.0 hours after the addition of LPS.The mRNA expression levels of TNF-αand IL-6 genes were measured by fluorescent quantitative PCR(qRT-PCR).The expression levels of TNF-αand IL-6 proteins in cell supernatants were further evaluated by ELISA,and the expression of NF-κB protein were analyzed by immunoprotein blotting(WB).【Result】There were differences in the effects of LPS on the proliferation of TM4 under different concentrations and different treatment times,and the inflammatory damage induced by 1.00μg/mL LPS treatment of TM4 for 12 hours was significantly lower than that in the blank group(P<0.01,the same as below).The protective effect of tacrine on TM4 was time and dose-dependent,with the TM4 proliferation rate of 10000.00μg/mL tacrine pretreatment for 2.0 hours being significantly(P<0.05,the same as below)lower than that of the blank group,and 100000.00μg/mL tacrine pretreatment for 0.5 and 1.0 hours or post-treatment for 0.5,1.5,3.0,6.0,12.0 and the TM4 proliferation rate at 24.0 hours was highly significantly lower than that of the blank group,while the best protection was achieved by 0.01μg/mL tacrine post-treatment for 6.0 hours,which had a significantly higher TM

关 键 词：他克林 脂多糖 小鼠睾丸支持细胞 炎性损伤 

分 类 号：S859.1[农业科学—临床兽医学]