Prevention of islet β-cell apoptosis by pioglitazone in NOD mice and its mechanism

Abstract

Objective To investigate the mechanism of preventing islet β-cell apoptosis in NOD mice with pioglitazone.Methods Female NOD mice at 4 weeks of age were divided into pioglitazone group ( n =21,0.02％pioglitazone was added into the feed ) and control group ( n =21,fed with regular diet).The accumulative incidence of diabetes was followed-up to 52 weeks of age in each group of NOD mice.Pancreas was removed from NOD mice at 12 weeks of age in each group ( n =15 ) to score severity of insulitis by routine H-E staining.The apoptotic β-cells in islets were observed with double-labeling technique of TUNEL in situ combined with standard sensitive avidin-biotin complex (sABC) immunohistochemical method.The spleens were taken for cell culture; IL-4 and IFN-γ levels in sera and supernatants of cultured splenocyte,the activity of PPARγ and NF-κB nuclear proteins in cultured splenocyte were measured by ELISA.Results (1)At 30 and 52 weeks of age,the respective incidences of diabetes were 57.1％ and 76.2％ in pioglitazone group,and 76.2％ and 90.5％ in control group ( all P＞0.05 ).At 15 weeks of age,the incidence became 4.8％ in pioglitazone group,and 33.3 ％ in control group ( P =0.045 ).( 2 ) At 12 weeks of age,the percentages of non infiltrated islet and peri-insulitis islet in pioglitazone group were higher than those in control group ( 14.73％ vs 5.69％,P＜0.01 ; and 26.02％ vs 15.72％,P＜0.01 ),and that of intraislet insulitis was lower than that in control group ( 59.25％ vs 78.59％,P＜0.01 ).The percentage of apoptotic β-cell in pioglitazone group was lower than that in control group( 6.17％ ±3.62％ vs 10.62％ ±4.43％,P=0.008 ).(3) In sera,IFN-γ level in pioglitazone group was lower than that in control group [( 561.05±78.61 ) vs ( 666.43 ± 28.42 ) pg/ml,P =0.045].In cultured splenocyte supernatant,the level of IFN-γ in pioglitazone group was lower than that in control group[(605.84+65.60) vs (692.20+44.98) pg/ml,P=0.041].(4) In cultured splenocyte,PPARγ nuclear protein activity in pioglitazone group was higher than that in control group ( 0.06 ± 0.01 vs 0.03 ± 0.01,P =0.013 ),and NF-κB nuclear protein activity was lower than that in control group ( 0.03 ± 0.01 vs 0.08± 0.01,P =0.001 ).Conclusions Pioglitazone activates PPARγ nuclear protein,inhibits activity of NF-κB nuclear protein,downregulates IFN-γ,diminishes differeutiation of Th cells to Th1,and subsequently prevents insulitis and β-cell apoptosis in NOD mice. Key words: Diabetes mellitus, experimeatal; Mice, inbred NOD; Apoptosis; PPARγ; NF-κB