Convergence role of transcriptional coactivator p300 and apparent modification on hmcs metabolic memory induced by high glucose / 解放军医学杂志

ABSTRACT

Objective To investigate the protein expression of transcriptional coactivator p300, acetylated histone H3 (Ac-H3) and Ac-H4 in human renal mesangial cell (HMCs) as imitative "metabolic memory" in vitro, and explore the potential role of convergence point of p300. Methods The HMCs were divided into the following groups {circled digit 1} High glucose metabolic memory model normal glucose group (NG, S.Smmol/L D-glucose × 2d), high glucose group (HG, 2Smmol/L D-glucose × 2d), memory groups (Ml, M2, M3, 2Smmol/L D-glucose × 2days + S.Smmol/L D-glucose × 3d, 6d or 9d), persisting normal glucose group (NG, S.Smmol/L D-glucose × 9d). {circled digit 2} Advanced glycation end products memory model normal glucose group (NG, S.Smmol/ L D-glucose × 2d), NG+AGEs group (AGEs, S.Smmol/L D-glucose+2S0μg/ml AGEs × 2d); AGEs memory group (AGEs-M, S.Smmol/L D-glucose + 2S0μg/ml AGEs × 2d + S.Smmol/L D-glucose × 3d); BSA control group (NG+BSA, S.Smmol/L D-glucose + 2S0μg/ml BSA × 2d). {circled digit 3} H202 was used to simulate oxidative stress memory model normal glucose group (NG, S.Smmol/L D-glucose × 2d), NG+H202 group (H202, S.Smmol/L D-glucose +100μmol/L H202 × 30min); H202 memory group [(S.Smmol/ L D-glucose + 100μmol/L H202 × 30min) + S.Smmol/L D-glucose × 3d]; normal glucose control group (NG3, S.Smmol/L D-glucose X 3d). {circled digit 4} Transfection with PKCβ2 memory model normal glucose group (NG, S.Smmol/L D-glucose × 2d); high glucose group (HG, 2Smmol/L D-glucose × 2d); memory group (M, 2Smmol/L D-glucose × 2d + S.Smmol/L D-glucose × 3d); AdS-null memory group (HN, 2Smmol/L D-glucose + AdS-null × 2d + S.Smmol/L D-glucose × 3d); PKCβ2 memory group (PO, 2Smmol/L D-glucose + AdS-PKCβ2 × 2d + S.Smmol/L D-glucose × 3d); inhibitor of PKCβ2 memory group (PI, 2Smmol/L D-glucose × 2d + 10μmol/L CGPS33S3 + S.Smmol/L D-glucose × 3d). The expression of intracellular reactive oxygen species (ROS) was detected by fluorescence microscope and fluorescence microplate reader. The expression levels of p300, Ac-H3, Ac-H4 and PKCβ2 proteins were determined by Western blotting. Results The expression levels of p300, Ac-H3 and Ac-H4 protein in HG group increased, being 2.15, 1.93 and 1.87 fold of those in group NG (P<0.05), accompanying with the up-regulation of PKCβ2 protein and ROS levels in HG group. The p300, Ac-H3, Ac-H4, PKCβ2 protein expression and ROS levels in Ml, M2, M3 group were higher than those in NG group, and was 1.75, 1.49, 1.47, 1.98 and 1.48 fold higher in M3 group than in NG group. The protein expressions of p300, Ac-H3 and Ac-H4 in AGEs group were increased by 1.73, 1.08 and 1.05 folds, and in AGE-M group increased by 1.47, 0.95 and 1.03 folds of that in control group (P<0.05). The protein expression levels of p300, Ac-H3 and Ac-H4 in H202 group increased by 1.03, 0.85 and 0.79 folds of those in control group (P<0.05). However, no significantly difference in these indices was found between H202-M and control groups. The protein expression levels of p300, Ac-H3 and Ac-H4 in PO group increased more obviously by 1.25, 1.06 and 1.10 folds of those in M group (P<0.05). However, the elective PKCβ2 inhibitor CGP533S3 could lower those indices significantly. Conclusion Persistent activation of transcriptional coactivator p300 and apparent modification may be normalized in HMCs. p300 may be the convergent point of glucose-induced metabolic "memory" stimulations.