青藤碱对PKC⁃α/NF⁃κB⁃p65通路的抑制作用及机制
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国家自然科学基金(81603358,31470853,31770934,81971468)


Inhibitory effects and mechanisms of snomenine on PKC⁃α/NF⁃κB⁃p65 pathway
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    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。

    Abstract:

    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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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(自然科学版),2020,(11):1583-1589

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  • 收稿日期:2020-04-27
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  • 在线发布日期: 2020-12-04
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