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目的观察七肽LPAAQKT在布鲁氏菌侵染人单核细胞系THP-1过程中对MAPK通路的影响。方法通过MTT法检测合成七肽对THP-1的毒性;采用AnnexinV-FITC/PI双染法检测七肽对感染细胞凋亡的影响;通过CFU计数分析七肽对感染细胞内细菌数量的影响;采用qRT-PCR和Western blot检测牛种布鲁氏菌RB51感染THP-1不同阶段MAPK通路中ERK1/2、JNK1/2、P38的mRNA和蛋白活化及表达情况。结果合成七肽对THP-1无毒性作用;流式细胞术检测七肽对THP-1细胞凋亡作用的影响为:感染复数50∶1组4h时对细胞凋亡有促进作用,48h时对细胞凋亡有抑制作用;CFU计数显示在感染前预先加入七肽导致THP-1细胞内布鲁氏菌数少于未加七肽组;qRTPCR和磷酸化Western blot显示,感染4h时,七肽降低感染组中erk1的转录与蛋白活化,感染48h时七肽降低jnk1/2的转录与蛋白活化。结论环化七肽CLPAAQKTC参与感染细胞的凋亡过程,因此有望用于布鲁氏菌病治疗药物的研发。
Objective To investigate the effect of heptapeptide LPAAQKT on the MAPK pathway in Brucella infection of human monocytic cell line THP-1. Methods The cytotoxicity of heptapeptide to THP-1 was detected by MTT assay. The effect of heptapeptide on the apoptosis of infected cells was detected by Annexin V-FITC / PI double staining. The influence of heptapeptide on the number of bacteria in infected cells was analyzed by CFU counting. The mRNA and protein expressions of ERK1 / 2, JNK1 / 2 and P38 in MAPK pathway during different stages of THP-1 infection by Brucella RB51 were detected by qRT-PCR and Western blot. Results The heptapeptide had no toxic effect on THP-1. The effect of heptapeptide on the apoptosis of THP-1 cells by flow cytometry was as follows: CFU count showed that pre-addition of heptapeptide before infection resulted in less number of brucella in THP-1 cells than in heptapeptide group. QRTPCR and phosphorylation Western blot showed that heparin Reduce erk1 transcription and protein activation in infected group, heptapeptide reduced Jnk1 / 2 transcription and protein activation 48h infection. Conclusion CLPAAQKTC, a cyclized heptapeptide, is involved in the process of apoptosis in infected cells. Therefore, CLPAAQKTC is expected to be used in the research and development of drugs for the treatment of brucellosis.