慢性氟中毒大鼠肝组织Col-Ⅰ、Col-Ⅲ及α-SMA的表达

Expressions of Col-Ⅰ,Col-Ⅲ andα-SMA in liver of rats with chronic fluorosis

目的观察慢性氟中毒大鼠肝组织胶原蛋白-Ⅰ型(Col-Ⅰ)、胶原蛋白-Ⅲ型(Col-Ⅲ)及α-平滑肌动蛋白(α-SMA)表达情况,探讨慢性氟中毒是否有导致大鼠肝纤维化的作用。方法将48只SD大鼠按体重(90~100 g)采用随机数字表法分为对照组(饮用水氟离子浓度<0.5 mg/L),低、中、高浓度氟组(饮用水氟离子浓度分别为5.0、50.0、100.0 mg/L),每组12只(雌、雄各半),饲养6个月。采用氟离子选择电极法检测骨氟、尿氟含量;苏木精-伊红(HE)染色及马松染色法光镜下观察肝组织病理形态学改变及胶原纤维沉积情况;实时荧光定量PCR及免疫组织化学染色法观察肝组织Col-Ⅰ、Col-Ⅲ、α-SMA mRNA及蛋白表达情况。结果4组大鼠骨氟、尿氟[骨氟:(92.52±5.64)、(112.21±11.86)、(142.99±7.87)、(235.63±11.55)mg/kg,尿氟:(5.47±0.88)、(17.78±1.48)、(54.16±5.96)、(121.11±6.32)mg/L]比较,差异均有统计学意义(P均<0.001)。光镜下,随着染氟浓度增加,肝细胞水肿程度加重,中央静脉周围及汇管区胶原纤维沉积显著增多。低、中、高浓度氟组大鼠Col-ⅠmRNA表达水平(1.20±0.09、1.80±0.08、1.58±0.06)均高于对照组(1.00±0.00,P均<0.05);中、高浓度氟组大鼠Col-Ⅲ、α-SMA mRNA表达水平(Col-Ⅲ:1.15±0.14、1.64±0.24,α-SMA:1.69±0.02、2.34±0.06)均高于低浓度氟组(Col-Ⅲ:0.59±0.17,α-SMA:0.80±0.13,P均<0.05)。随着染氟浓度增加,大鼠肝组织Col-Ⅰ(0.00±0.00、0.03±0.01、0.08±0.01、0.13±0.02)、Col-Ⅲ(17803.05±3221.16、47523.15±3490.10、127786.35±13008.86、237233.03±47614.63)、α-SMA(516.83±181.18、2885.03±864.92、11186.94±2394.08、37182.43±12390.59)蛋白表达水平均增加(P均<0.05)。结论长期摄入过量氟可能引起大鼠肝脏中央静脉周围及汇管区胶原纤维产生增多,进而引起肝纤维化形成。

Objective To explore whether chronic fluorosis can cause liver fibrosis in rats by observing expression changes in type Ⅰ collagen(Col-I),type Ⅲ collagen(Col-Ⅲ)and alpha smooth actin(α-SMA)in the liver tissue of chronic fluorosis rats.MethodsAccording to body weight(90-100 g),forty-eight SD rats were randomly divided into control group(drinking water fluoride ion concentration<O.5 mg/L),low,medium and high concentration fluoride groups(drinking water fluoride ion concentration of 5.0,50.0 and 100.0 mg/L),with 12 rats in each group(half male and half female),and fed for 6 months.Fluoride ion selective electrode method was used to detect bone fluoride and urinary fluoride levels;hematoxylin-eosin staining(HE staining)and Masson staining were used to observe the pathological and morphological changes and the collagen deposition of liver tissue;quantitative real-time polymerase chain reaction and immunohistochemical staining were used to observe Col-I,Col-II andα-SMA mRNA and protein expressions.Results There was significant dfference in bone fluoride and urine fluoride between the 4 groups[bone fluoride:(92.52±5.64),(112.21±11.86),(142.99±7.87),(235.63±11.55)mg/kg;urinary fluoride:(5.47±0.88),(17.78±1.48),(54.16±5.96),(121.11±6.32)mg/L,P<0.001).Under light microscope,with the increase of fluoride concentration,the degree of hepatic cell edema was aggravated,and the deposition of collagen fiber around the central vein and the portal area increased significantly.The mRNA expression level of Col-I in low,medium and high concentration fluoride groups(1.20±0.09,1.80±0.08,1.58±0.06)was significantly higher than that in control group(1.00±0.00,P<0.05);Col-Ⅲ andα-SMA mRNA expression levels in medium and high concentration fluoride groups(Col-II:1.15±0.14,1.64±0.24;α-SMA:1.69±0.02,2.34±0.06)were significantly higher than those of low concentration fluoride group(Col-II:0.59±0.17;α-SMA:0.80±0.13,P<0.05).With the increase of fluoride concentration,the liver tissue Col-I(0.00±0.00,0.03±0.01,0.08±0.01,0.13±0.02),ColⅢ(17803.05±3221.16,47523.15±3490.10,127786.35±13008.86,237233.03±47614.63)andαSMA(516.83±181.18,2885.03±864.92,11186.94±2394.08,37182.43±12390.59)protein levels were also increased significantly(P<0.05).Conclusion Long-term excessive intake of fluorine may cause the production of collagen fibers around the central vein and the portal area of the liver in rats to increase,and then lead to the formation of liver fibrosis.