Objective: To investigate the regulation effects on LPS-mediated cytokine secretion and dexamethasone- induced apoptosis in macrophages by transient overexpression of hDaxx. Methods: An eukaryotic expression vector pE...Objective: To investigate the regulation effects on LPS-mediated cytokine secretion and dexamethasone- induced apoptosis in macrophages by transient overexpression of hDaxx. Methods: An eukaryotic expression vector pEGFP/hDaxx, which could express a fusion protein GFP-Daxx, was transfected into macrophages. The expression and localization of GFP-hDaxx fusion protein was analyzed by fluorescent microscope and western blot. The effects of transient overexpression of GFP-hDaxx fusion protein on the lipopolysaccharide(LPS)-mediated secretion of TNF-α and IL-1β were determined by ELISA. Moreover, the dexamethasone-induced apoptosis was determined morphologically by Giemsa stain. Results: The results observed showed that GFP-hDaxx fusion protein overexpressed in macrophages and localized in nuclei but GFP in cytoplasm under fluorescent microscope. The overexpression of GFP-hDaxx fusion protein could be detected by Western blot with an antibody against C-terminal of hDaxx. In the group with overexpressed GFP-hDaxx fusion protein, the LPS-mediated cytokine secretion decreased remarkably at 1 h, 3 h, 6 h respectively after LPS stimulation, and the dexamethasone- induced apoptosis reduced notably at 6 h, 12 h and 24 h respectively after addition of dexamethasone. There were remarkable difference between pEGFP/hDaxx group and control group (P<0.01) at different time. Conclusion: Transient overexpression of hDaxx down-regulates LPS-mediated cytokine secretion in macrophages and inhibits dexamethasone-induced macrophages apoptosis.展开更多
Crassostrea sikamea(C.sikamea)is an important edible and medicinal seafood in China.In the present study,a compound named flazin was separated and identified from the ethyl acetate extract of C.sikamea(EAECs)for the f...Crassostrea sikamea(C.sikamea)is an important edible and medicinal seafood in China.In the present study,a compound named flazin was separated and identified from the ethyl acetate extract of C.sikamea(EAECs)for the first time.In addition,the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium(MTS)assay revealed that EAECs and flazin inhibited the transformation of splenic lymphocytes in vitro.Moreover,flazin(20μg·mL^(−1))altered the populations of splenic lymphocyte subtypes.Real-time quantitative PCR(RT-qPCR)analysis and enzyme-linked immunosorbent assay(ELISA)showed that flazin suppressed the mRNA expression and secretion of TNF-αand IL-2,and reversed Concanavalin A(ConA)-induced mRNA up-regulation and protein secretion of TNF-αand IL-2.Western blot results showed that flazin reversed ConA-induced increases in p-ERK1/2 and p-p38 in splenocytes.In conclusion,flazin exhibits effective immunomodulatory function and may be useful for treating immune-related disorders,which indicates the application potential of C.sikamea as a functional food or immunomodulator.展开更多
基金This work was supported by a grant from theEducation Committee of Hunan Province(No.02C391)
文摘Objective: To investigate the regulation effects on LPS-mediated cytokine secretion and dexamethasone- induced apoptosis in macrophages by transient overexpression of hDaxx. Methods: An eukaryotic expression vector pEGFP/hDaxx, which could express a fusion protein GFP-Daxx, was transfected into macrophages. The expression and localization of GFP-hDaxx fusion protein was analyzed by fluorescent microscope and western blot. The effects of transient overexpression of GFP-hDaxx fusion protein on the lipopolysaccharide(LPS)-mediated secretion of TNF-α and IL-1β were determined by ELISA. Moreover, the dexamethasone-induced apoptosis was determined morphologically by Giemsa stain. Results: The results observed showed that GFP-hDaxx fusion protein overexpressed in macrophages and localized in nuclei but GFP in cytoplasm under fluorescent microscope. The overexpression of GFP-hDaxx fusion protein could be detected by Western blot with an antibody against C-terminal of hDaxx. In the group with overexpressed GFP-hDaxx fusion protein, the LPS-mediated cytokine secretion decreased remarkably at 1 h, 3 h, 6 h respectively after LPS stimulation, and the dexamethasone- induced apoptosis reduced notably at 6 h, 12 h and 24 h respectively after addition of dexamethasone. There were remarkable difference between pEGFP/hDaxx group and control group (P<0.01) at different time. Conclusion: Transient overexpression of hDaxx down-regulates LPS-mediated cytokine secretion in macrophages and inhibits dexamethasone-induced macrophages apoptosis.
基金Education Department of Jilin Province(No.JJKH20201177KJ)the Foundation of Department of Human Resources and Social Security of Jilin Province(No.2020009)the Foundation of Jilin Province Development and Reform Commission(No.2020C015).
文摘Crassostrea sikamea(C.sikamea)is an important edible and medicinal seafood in China.In the present study,a compound named flazin was separated and identified from the ethyl acetate extract of C.sikamea(EAECs)for the first time.In addition,the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium(MTS)assay revealed that EAECs and flazin inhibited the transformation of splenic lymphocytes in vitro.Moreover,flazin(20μg·mL^(−1))altered the populations of splenic lymphocyte subtypes.Real-time quantitative PCR(RT-qPCR)analysis and enzyme-linked immunosorbent assay(ELISA)showed that flazin suppressed the mRNA expression and secretion of TNF-αand IL-2,and reversed Concanavalin A(ConA)-induced mRNA up-regulation and protein secretion of TNF-αand IL-2.Western blot results showed that flazin reversed ConA-induced increases in p-ERK1/2 and p-p38 in splenocytes.In conclusion,flazin exhibits effective immunomodulatory function and may be useful for treating immune-related disorders,which indicates the application potential of C.sikamea as a functional food or immunomodulator.
