PPARγ、NF-κB的表达与砷暴露致大鼠肝纤维化的相关性

Correlation of PPARγ and NF-κB expression with arsenicinduced hepatic fibrosis in rats

目的:初步探讨过氧化物酶体增殖物激活受体γ(PPARγ)、核转录因子B(NF-κB)的表达与水砷暴露致大鼠肝纤维化的相关性.方法:110只SD大鼠随机分成对照组(自来水)、模型组(浓度100mg/L亚砷酸钠溶液)、自然恢复组(浓度100mg/L亚砷酸钠溶液+自来水).对照组和模型组分别于第l、2、3、4月末各处死10只,自然恢复组先给予砷溶液,分别在第l、2、3月末取出10只改给予1mo自来水饮用后处死.肝组织病理学检查以观察肝纤维化的动态变化,实时荧光定量RT-PCR法和Western blot法检测PPARγ、NF-κB的mRNA及蛋白表达水平.结果:(1)病理结果:HE染和Masson染色可见,随砷暴露时间的延长,肝细胞变性、坏死增多,汇管区炎症细胞浸润加重,纤维组织增生增多,肝纤维化趋势明显.砷暴露1mo脱离自然恢复1mo后较同月模型组肝细胞变性、坏死及炎细胞浸润程度明显减轻,胶原生成减少.砷暴露2、3mo脱离自然恢复1mo后较同月模型组病理结果差异不明显;(2)mRNA水平:模型组PPARγ mRNA含量逐渐降低,与对照组比较差异均有统计学意义(174.99±41.48,114.55±21.30,64.67±9.83,19.20±16.10 vs 218.40±47.85,P<0.05),砷暴露1、2、3mo后分别自然恢复1mo大鼠肝组织中PPARγ mRNA表达均低于同月造模组,仅砷暴露1mo自然恢复组1mo组PPARγ mRNA降低有统计学意义(174.99±41.48 vs 215.97±45.96,P<0.05);模型组NF-κB mRNA含量逐渐升高,与对照组比较差异均有统计学意义(65.58±13.17,90.23±15.68,117.95±18.19,172.86±32.92 vs 30.84±15.24,P<0.05),砷暴露1、2、3mo后分别自然恢复1mo大鼠肝组织中NF-κB mRNA表达均高于同月造模组,仅砷暴露1mo自然恢复组1mo组NF-κB mRNA升高有统计学意义(65.58±13.17 vs 40.45±19.56,P<0.05);(3)蛋白水平:模型组大鼠肝组织中PPARγ的蛋白含量表达均低于对照组,造模3、4mo组与造模1mo组比较差异有统计学意义(0.63±0.06,0.55±0.11 vs 0.85±0.08,P<0.05);模型组大鼠肝组织中NF-κB的蛋白含量均高于对照组,造模3、4mo组与造模1mo组比较差异有统计学意义(3.25±0.89,4.27±1.26 vs 1.6±0.57,P<0.05);(4)PPARγ和NF-κB的相关性:两者mRNA的表达呈负相关(r=0.847,P<0.01),两者蛋白表达也呈负相关(r=0.529,P<0.05).结论:肝纤维化程度随砷暴露时间延长而加重,越早脱离砷环境,肝损伤自然恢复越快;砷暴露时间越长,PPARγ mRNA及蛋白表达越低,NF-κB mRNA及蛋白表达越高,二者存在一反馈抑制通路;PPARγ-NF-κB信号传导通路参与砷暴露致肝纤维化形成机制.

AIM：To investigate the significance of peroxisome proliferator activated receptor γ（PPARγ） and nuclear transcription factor kappa B（NF-κB） expression in arsenic-induced hepatic f ibrosis in rats.METHODS：One hundred and ten Sprague-Dawley rats were randomly divided into control group（tap water）,model group（100 mg/L NaASO2） and spontaneous recovery group（100 mg/L NaASO2 ＋ tap water）.Ten rats of each group were sacrificed on days 30,60,90 and 120.Hepatic histological changes and liver fibrosis were evaluated by hematoxylin and eosin staining and Masson staining.The mRNA and protein expression of PPARγ and NF-κB was detected by real-time PCR and Western blot,respectively.RESULTS：（1） Pathology：With the prolongation of arsenic exposure,hepatic injury became more serious.Compared with the model group,liver injury was signif icantly relieved on day 30 after one-month arsenic-exposure in the spontaneous recovery,but showed no signif icant changes on day 30 after two-or three-month arsenic-exposure;（2） mRNA：Compared with the control group,the expression levels of PPARγ mRNA were significantly reduced at different time points in the model group（174.99 ± 41.48 vs 218.40 ± 47.85,114.55 ± 21.30 vs 218.40 ± 47.85,64.67 ± 9.83 vs 218.40 ± 47.85,19.20 ± 16.10 vs 218.40 ± 47.85,all P〈 0.05）.The expression level of PPARγ mRNA on day 30 after one-month arsenic-exposure in the spontaneous recovery group was significantly higher than that in the corresponding model group（215.97 ± 45.96 vs 174.99 ± 41.48,P 〈0.05）.Compared with the control group,the levels of NF-κB mRNA were signif icantly elevated at different time points in the model group（65.58 ± 13.17 vs 30.84 ± 15.24,90.23 ± 15.68 vs 30.84 ± 15.24,117.95 ± 18.19 vs 30.84 ± 15.24,172.86 ± 32.92 vs 30.84 ± 15.24,all P 〈0.05）.The expression of NF-κB mRNA on day 30 after one-month arsenic-exposure in the spontaneous recovery group was significantly higher than that in the corresponding model group（40.45 ± 19.56 vs 65.58 ± 13.17,P〈 0.05）;（3） Protein：Compared with the control group,the level of PPARγ protein was reduced in the model group.Compared with the control group,the level of NF-κB protein was elevated in the model group;（4） Correlation：There is a negative correlation between PPARγ and NF-κB mRNA expression（r = 0.847,P〈 0.05） as well as between PPARγ and NF-κB protein expression（r = 0.529,P 〈0.05）.CONCLUSION：Liver tissue injury is aggravated gradually with the prolongation of arsenic exposure.Earlier avoidance of arsenic exposure is associated with a quicker recovery from liver injury.In arsenic-induced hepatic f ibrosis,PPARγ mRNA and protein expression is reduced,while that of NF-κB is elevated.There is a negative correlation between PPARγ and NF-κB expression in arsenic-induced hepatic f ibrosis.