文章摘要
王文博,罗 灿,吴志皎,彭明玉,何庆玲,王迎伟,季明德,邱 文.青藤碱对PKC⁃α/NF⁃κB⁃p65通路的抑制作用及机制[J].南京医科大学学报,2020,(11):1583~1589
青藤碱对PKC⁃α/NF⁃κB⁃p65通路的抑制作用及机制
Inhibitory effects and mechanisms of snomenine on PKC⁃α/NF⁃κB⁃p65 pathway
投稿时间:2020-04-27  
DOI:10.7655/NYDXBNS20201103
中文关键词: 蛋白激酶C⁃α  核因子⁃κB⁃p65  青藤碱
英文关键词: protein kinase C⁃α  nuclear factor⁃κB⁃p65  sinomenine
基金项目:国家自然科学基金(81603358,31470853,31770934,81971468)
作者单位
王文博 南京医科大学免疫学系江苏 南京 211166 
罗 灿 南京医科大学免疫学系江苏 南京 211166 
吴志皎 南京医科大学免疫学系江苏 南京 211166 
彭明玉 南京医科大学免疫学系江苏 南京 211166 
何庆玲 南京医科大学免疫学系江苏 南京 211166 
王迎伟 南京医科大学免疫学系江苏 南京 211166 
季明德 江苏省中医院检验科江苏 南京 210006 
邱 文 南京医科大学免疫学系江苏 南京 211166 
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中文摘要:
      目的:探讨过表达蛋白激酶C?α(protein kinase C?α,PKC?α)对HEK?293T细胞中核因子?κB?p65(nuclear factor?κB?p65,NF?κB?p65)磷酸化水平的影响,并检查青藤碱(sinomenine,SIN)对PKC?α/NF?κB?p65信号通路的抑制作用。方法:采用PCR技术扩增大鼠PKC?α基因蛋白质编码区(complete sequence coding,CDS)序列,将其插入到pIRES2?EGFP空载质粒中,构建野生型(wide type,WT)PKC?α过表达质粒(pIRES2?PKC?α?WT,PKC?αWT);在PKC?αWT质粒的基础上,将PKC?α第25位丙氨酸(A)与368位赖氨酸(K)分别突变为谷氨酸(E)和精氨酸(R),即构建持续活化(constitutively active,CA)突变型PKC?α过表达质粒(pIRES2?PKC?α?A25E,PKC?αCA)和显性负性(dominant negative,DN)突变型PKC?α过表达质粒(pIRES2?PKC?α?K368R,PKC?αDN)。本课题组前期构建的大鼠野生型NF?κB?p65过表达质粒(pIRES2?NF?κB?p65,p65WT)分别与上述各PKC?α过表达质粒共同转染HEK?293T细胞,免疫印迹法检测PKC?α、NF?κB?p65的表达及磷酸化水平。再将上述不同质粒转染HEK?293T细胞,46 h后予50 ng/mL SIN处理细胞2 h,再行免疫印迹实验分析SIN对PKC?α、NF?κB?p65磷酸化的影响。结果:菌液PCR及测序证实,上述PKC?α过表达质粒构建成功。在HEK?293T中过表达PKC?αWT或PKC?αCA可促进NF?κB?p65的磷酸化,且过表达PKC?αCA时尤为显著,而过表达PKC?αDN后NF?κB?p65的磷酸化无明显变化。SIN处理可抑制PKC?αWT、PKC?αCA和PKC?αDN转染组HEK?293T细胞中PKC?α的磷酸化,同时也能抑制过表达PKC?αWT上调的NF?κB?p65磷酸化修饰,但不影响过表达PKC?αCA和PKC?αDN对NF?κB?p65的磷酸化作用。结论:过表达PKC?α可促进HEK?293T细胞中NF?κB?p65的磷酸化,SIN处理HEK?293T细胞后可通过抑制PKC?α的磷酸化下调NF?κB?p65的磷酸化修饰。
英文摘要:
      Objective:This study aims to investigate the effects of protein kinase C?α(PKC?α)over?expression on the phosphorylation level of nuclear factor?κB?p65(NF?κB?p65)in human embryonic kidney 293T(HEK?293T)cells,and examine the inhibitory effect of sinomenine(SIN)on PKC?α/NF?κB?p65 signaling pathway. Methods:To construct the rat wild type(WT)PKC?α over?expression plasmid(pIRES2?PKC?α WT,PKC?αWT),rat PKC?α complete sequence coding(CDS)was amplified by PCR and cloned into pIRES2?EGFP. Then,alanine(A) at the site of 25 and lysine(K) at the site of 368 were mutated to glutamic(E) and arginine(R),respectively based on PKC?αWT to construct PKC?α constitutively active(CA) mutant(pIRES2?PKC?α A25E,PKC?αCA)and PKC?α dominant negative(DN) mutant(pIRES2?PKC?αK368R,PKC?αDN). The rat wild?type NF?κB?p65 over?expression plasmid,namely pIRES2?NF?κB?p65(p65WT),was constructed by our research group previously. The plasmid of p65WT was transfected into HEK?293T cells together with the above?mentioned different PKC?α over?expression plasmids respectively. The expression and phosphorylation levels of PKC?α and NF?κB?p65 were detected by Western blot. Next,HEK?293T cells were co?transfected with the above?mentioned plasmids in different groups,and after 46 h cells were continuously treated with SIN at the dose of 50 ng/mL for 2 h. Then,the effects of SIN on phosphorylation of PKC?α and NF?κB?p65 were determined by Western blot. Results:PCR analysis and nucleotide sequencing verified that the above?mentioned PKC?α over?expression plasmids were constructed successfully. The phosphorylation of NF?κB?p65 in HEK?293T cells was increased in response to PKC?αWT and PKC?αCA over?expression,especially PKC?αCA over?expression. However,there was no significant change of NF?κB?p65 phosphorylation after PKC?αDN over?expression. SIN treatment could inhibit the phosphorylation of PKC?α in HEK?293T cells transfected with PKC?αWT,PKC?αCA and PKC?αDN. SIN could also inhibit the phosphorylation of NF?κB?p65 in HEK?293T cells caused by PKC?αWT over?expression,but not by PKC?αCA or PKC?αDN over?expression. Conclusion:Over?expression of PKC?α can promote the phosphorylation of NF?κB?p65 in HEK?293T cells. SIN treatment can down?regulate NF?κB?p65 phosphorylation through inhibition of PKC?α phosphorylation in HEK?293T cells.
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