灰兜巴中过氧化物酶体增殖物激活受体γ活性部位的筛选

Screening of active site of peroxisome proliferator-activated receptorγin Huidouba

目的通过构建过氧化物酶体增殖物激活受体γ(PPARγ)激动剂的细胞筛选模型,检测灰兜巴的不同极性提取物对PPARγ的激活效率。为开发新型、高效、安全的天然PPARγ激动剂提供可靠的实验依据。方法采用Lipofectamine3000转染试剂将重组质粒pBIND-PPARγ-LBD与报告质粒pGL4.35共同转入293T细胞中,同时在293T细胞中只转染pGL4.35报告质粒作为阴性对照,以PPARγ激动剂人工合成配体吡格列酮作为阳性对照药,通过检测荧光素酶活性来计算灰兜巴的不同极性提取物对PPARγ的激活作用。所有结果均使用Origin2021软件分析计算,样品间差异有统计学意义(P<0.05)。结果阳性对照组加入0.75μmol/L的吡格列酮后荧光强度较空白对照组荧光强度增加1.27倍,差异有统计学意义(P<0.05),阴性对照组加入吡格列酮后未见荧光素酶被激活;共转染细胞组加入不同浓度(400、200μg/mL和100μg/mL)灰兜巴的不同极性提取物后,荧光素酶激活程度各不相同,其中400μg/mL的正己烷、正丁醇、水提醇沉上清、水提醇沉部分的激活能力相当于空白对照组的145%、128%、147%、157%,差异有统计学意义(P<0.01)。结论将PPARγ重组质粒和报告质粒pGL4.35共转染入293T细胞中可作为PPARγ激活能力的细胞筛选模型,灰兜巴正己烷、正丁醇、水提醇沉上清、水提醇沉提取物可作为PPARγ的天然激动剂。

Objective To establish a cell screening model of peroxisome proliferator-activated receptorγ(PPARγ)agonist,in order to detect the activation efficiency of different polar extracts of Huidouba on PPARγ.And to provide a reliable experimental basis for the development of new,efficient and safe natural PPARγagonists.Methods The recombinant plasmid bind-PPARγ-LBD and the reporter plasmid pGL4.35 were co-transfected into 293T cells with Lipofectamine3000 transfection reagent.Meanwhile,the reporter plasmid pGL4.35 transfected in 293T cells only was used as a negative control.The PPARγagonist synthetic ligand pioglitazone was used as the positive control drug.The activation effect by different polar extracts in Huidouba on PPARγwas calculated by detecting luciferase activity.All results were calculated using Origin 2021 analysis with statistically significant differences between samples(P<0.05).Results After adding 0.75μmol/L of pioglitazone in the positive control group,the fluorescence intensity was 1.27 times higher than that of the blank control group,and the difference was statistically significant(P<0.05).No luciferase was activated after adding pioglitazone to the negative control group.After different concentrations(400μg/mL,200μg/mL and 100μg/mL)of different polar extracts were added to the co-transfected cell group,the activation degree of firefly luciferase was different.The activation capacity of 400μg/mL n-hexane,n-butanol,water-extracted alcohol precipitation supernatant and water-extracted alcohol precipitation extract was equivalent to 145%,128%,147%and 157%of the blank control group(P<0.01).Conclusion PPARγrecombinant plasmid and reporter plasmid pGL4.35 are co-transfected into 293T cells,which can be used as the cell screening model of PPARγactivation.n-hexane,n-butanol,water-extracted alcohol precipitation supernatant and water-extracted alcohol precipitation extract can be used as the natural agonist of PPARγ.