肝纤维化小鼠模型中弹性蛋白及其调控因子在肝纤维化形成和逆转中的分子机制

Molecular mechanism of elastin and its regulatory factors on the formation and reversal of liver fibrosis in a mouse model of liver fibrosis

目的明确弹性蛋白在四氯化碳(CCl4)小鼠肝纤维化模型中的表达特点,初步探索弹性蛋白沉积的相关分子机制。方法建立CCl4诱导的小鼠肝纤维化形成模型,通过Western Blot、可溶性蛋白及维多利亚蓝染色检测纤维化早期(CCl4注射4周)和进展期(CCl4注射8周)弹性蛋白的变化,同时以橄榄油做为健康对照组;其次,建立纤维化自发逆转模型,通过LC-MS/MS检测不同逆转时间(逆转0周、逆转4周、逆转8周、逆转12周)血清蛋白组学的变化,寻找潜在的与弹性蛋白沉积相关分子,并通过q PCR进行验证。计量资料多组间比较采用单因素方差分析,进一步两两比较采用Tukey检验。结果弹性蛋白的相对表达量在早期纤维化组和进展期纤维化组分别为0. 82±0. 05、1. 59±0. 58,与健康对照组(1. 00±0. 11)相比,进展期纤维化组显著上升(P <0. 05)。对不可溶性弹性蛋白的检测发现,与健康对照组(207. 9±34. 7μg/10 mg)相比,早期纤维化组(213. 5±26. 7μg/10 mg)无明显变化(P>0. 05),而进展期组显著升高[(352. 0±57. 0)μg/10 mg,P <0. 05]。肝组织弹性蛋白染色发现,与健康对照组(1. 03±0. 14)相比,弹性蛋白阳性面积早期纤维化组(2. 08±0. 16)和进展期纤维化组(4. 39±0. 51)均显著增加(P值均<0. 05)。进一步检测了弹性蛋白酶12的基因表达,与健康对照组(1. 30±0. 10)相比,弹性蛋白酶12的基因水平随纤维化进展表达显著升高(早期纤维化:15. 50±2. 90;进展期纤维化:22. 70±4. 10,P值均<0. 05)。利用LC-MS/MS分离并鉴定逆转过程中与肝纤维化进展和逆转相关的蛋白45种,其中与弹性蛋白沉积相关的蛋白包括赖氨酸氧化酶-1(LOXL-1)和Fibulin-1(FBLN-1)。通过q PCR对LOXL-1和FBLN-1基因验证证实,LOXL-1和FBLN-1的基因水平在CCl4注射8周(逆转0周)达到高峰,且随着逆转时间的延长表达逐渐下降。结论弹性蛋白沉积是进展期小鼠肝纤维化模型的重要特征之一,LOXL-1和FBLN-1介导的弹性蛋白沉积有可能成为肝纤维化特别是肝硬化和终末期肝病的治疗靶点。

Objective To investigate the expression of elastin in a mouse model of carbon tetrachloride(CCl 4)-induced liver fibrosis and the molecular mechanism of elastin deposition. Methods A mouse model of CCl 4-induced liver fibrosis was established.Western blot,soluble protein,and Victoria blue staining were used to measure the change in elastin in the early stage of liver fibrosis(after 4 weeks of CCl 4 injection)and the progressive stage of liver fibrosis(after 8 weeks of CCl 4 injection).The mice treated with olive oil were enrolled as healthy control group.A model of spontaneous reversal of fibrosis was established;liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to measure the change in serum proteomics at different time points of reversal(at weeks 0,4,8,and 12 of reversal)and identify the potential molecules associated with elastin deposition,and qPCR was used for validation.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least signficant difference Tukey test was used for further comparison between two groups. Results The relative expression of elastin was 0.82±0.05 in the early fibrosis group and 1.59±0.58 in the progressive fibrosis group,and compared with the healthy control group,the progressive fibrosis group had a significant increase in the expression of elastin(1.59±0.58 vs 1.00±0.11,P<0.05).The measurement of insoluble elastin showed that compared with the healthy control group(207.9±34.7μg/10 mg),the early fibrosis group had no significant change in insoluble elastin(213.5±26.7μg/10 mg,P>0.05),and the progressive fibrosis group had a significant increase in insoluble elastin(352.0±57.0μg/10 mg,P<0.05).Elastin staining of liver tissue showed that compared with the healthy control group,the early fibrosis group and the progressive fibrosis group had a significant increase in the area of liver tissue with positive elastin expression(2.08±0.16/4.39±0.51 vs 1.03±0.14,both P<0.05).The mRNA expression of elastase 12 was measured,and the results showed that compared with the healthy control group,the early fibrosis group and the progressive fibrosis group had a significant increase in the mRNA expression of elastase 12(15.50±2.90/22.70±4.10 vs 1.30±0.10,both P<0.05),suggesting that the mRNA expression of elastase 12 increased with the progression of liver fibrosis.During the reversal of liver fibrosis,45 proteins associated with the progression and reversal of liver fibrosis were isolated and identified by LC-MS/MS,among which lysyl oxidase-like protein-1(LOXL-1)and fibulin-1(FBLN-1)were associated with elastin deposition.Validation of LOXL-1 and FBLN-1 by qPCR showed that the mRNA expression of LOXL-1 and FBLN-1 reached the peak after 8 weeks of CCl 4 injection(week 0 of reversal)and then gradually decreased over the time of reversal. Conclusion Elastin deposition is one of the important features in a mouse model of progressive liver fibrosis,and elastin deposition mediated by LOXL-1 and FBLN-1 may become a treatment target for liver fibrosis,liver cirrhosis,and end-stage liver disease.