内毒素对原代大鼠肝星状细胞表达结缔组织生长因子影响机制的研究

Study of the mechanism of lipopolysaccharide on the expression of connective tissue growth factor in primary hepatic stellate cells of rats

目的研究内毒素(LPS)与其刺激的Kupffer细胞(KCs)培养上清(KCCM)及NF-κB通路抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)对大鼠原代肝星状细胞(HSCs)表达结缔组织生长因子(CTGF)的影响,探讨LPS刺激HSCs表达CTGF的机制。方法分离培养原代大鼠HSCs、KCs。培养72 h的KCs经1 mg/L LPS刺激48 h后收集上清标记为KCCM1,未经LPS刺激的上清标记为KCCM0。将培养72 h的HSCs随机分为空白对照组、KC-CM0组、LPS组、KCCM1组、PDTC组、(PDTC+LPS)组、(PDTC+KCCM1)组。各组HSCs经相应的处理48 h后,Western blot检测各组HSCs表达CTGF水平,ELISA检测培养上清中Ⅰ型胶原含量。结果各组HSCs中CTGF蛋白表达水平为:空白对照组(1.95±0.67)、KCCM0组(2.15±1.10)、LPS组(4.56±4.16)、KCCM1组(5.21±3.23)、PDTC组(0.06±0.02)、(PDTC+LPS)组(0.10±0.08)、(PDTC+KCCM1)组(2.77±4.08)。LPS与KCCM1作用于HSCs后CTGF表达增加(P<0.05);PDTC几乎可以完全阻断正常及LPS剌激的HSCs表达CTGF(P<0.01),但只能降低KCCM1组CTGF的表达(P<0.05);KCCM1组HSCs上清中Ⅰ型胶原的含量增加(P<0.05)。结论 LPS可直接通过HSCs内的NF-κB通路使其表达CTGF水平上调,LPS刺激的KCs还可能通过其他途经使HSCs表达CTGF水平上调。

Objective To study the effects of lipopolysacchoride （LPS） and the supernatant of Kupffer cells （KCs） stimulated by LPS （KCCM） and Pyrrolidinedithiocarbamic acid （PDTC, inhibitor of NF-κB） on the expression of con- nective tissue growth factor （CTGF）in primary cultured hepatic stellate cells （HSCs） of rat, explore the mechanism of CTGF expression stimulated by LPS in HSCs. Methods HSCs and KCs were isolated and cultured from the liver of rats. After 72 h KCs were stimulated by 1 mg/L LPS and collected the supernatant after 48 h, marked it for KCCM1, without LPS stimulated for KCCM0. After 3 days of cultivation, HSCs were randomized into KCCMO group, LPS group, KCCMlgroup, PDTC group, （PDTC＋LPS） group, （PDTC＋KCCM1） group and control group. After co-cultivation for 48 h, the expression of CTGF in HSCs was detected by western blot, the content of type I collagen was measured by ELISA. Results The expression of CTGF in HSCs： control group （1.95±0.67）, KCCMO group （2115＋1.10）, LPS group （4.56＋4.16）, KC- CM1 group （5.21±3.23）, PDTC group （0.06±0.02）, （PDTC＋LPS） group （0.10±0.08） group, （PDTC＋KCCM1） group （2.77± 4.08）. LPS and KCCM1 had significantly enhanced the CTGF expression in HSCs （P 〈 0.05）; PDTC almost completely blocked the CTGF expression in the control and LPS groups （P 〈 0.01）, but only reduced it in KCCM1 stimulated HSCs （P 〈 0.05）; （KCCM＋LPS） enhanced type I collagen expressions in HSCs （P 〈 0.05）. Conclusion LPS can directly en- hance the expression of CTGF in HSCs through NF-κB pathway, while Kupffer cells stimulated by LPS may enhance the expression of CTGF in HSCs through some pathways else.