Abstract：
OBJECTIVE: To explore whether sivelestat sodium could reduce the expression of mucin 5AC (MUC5AC) in intrahepatic bile duct epithelial cells by inhibiting neutrophil elastase (NE) and thus provide new potential therapeutic ideas for the treatment of intrahepatic bile duct stone (IBDS).
METHODS: (1) Bioinformatics analysis: differential gene analysis was performed on gallbladder stone cholecystitis sequencing data based on the gene expression omnibus (GEO) to screen for significantly different genes related to neutrophils and mucins. The search tool for the retrieval of interacting genes database (STRING) was used for protein interaction analysis to predict whether there was an interaction between NE and MUC5AC genes. (2) Animal experiment: a total of 18 male SD rats were divided into the sham-operated group, cholangitis model group and sivelestat sodium treatment group according to the random number table method, with 6 rats in each group. The cholangitis rat model was established by a one-time injection of 1.25 mg/kg lipopolysaccharide (LPS) into the right anterior lobe of the liver of rats in combination with the pre-experiment; the liver of the sham-operated group was injected with an equal volume of saline. After the modelling, 100 mg/kg of sivelestat sodium was injected into the tail vein of the cevalexin treatment group once a day for 5 days, and an equal volume of saline was injected into the tail vein of the sham-operated group and the cholangitis model group. Two weeks later, the rats were euthanized and their liver and bile duct tissues were taken. The pathological changes in the liver and bile duct tissues were observed under the light microscope. Immunohistochemical staining was used to detect the expressions of NE and MUC5AC in liver and bile duct tissues. The protein expressions of NE, MUC5AC and Toll-like receptor 4 (TLR4) were detected by Western blotting. (3) Cell experiment: primary human intrahepatic biliary epithelial cell line (HiBEpiC) was divided into blank control group, NE group (10 nmol/L NE), NE+sivelestat sodium low dose group (10 nmol/L NE+1×10-8 g/L sivelestat sodium 1 mL), NE+sivelestat sodium medium dose group (10 nmol/L NE+1×10-7 g/L sivelestat sodium 1 mL), NE+sivelestat sodium high dose group (10 nmol/L NE+1×10-6 g/L sivelestat sodium 1 mL). Cells were collected after 48 hours of culture, and EdU was performed to detect the proliferative activity of cells; enzyme linked immunosorbent assay (ELISA) and Western blotting were performed to detect the expression of MUC5AC in cells.
RESULTS: (1) Bioinformatics analysis: the NE gene (ELANE) had a reciprocal relationship with MUC5AC. (2) Animal experiment: light microscopy showed that hepatocyte edema, hepatocyte diffuse point and focal necrosis, confluent area fibrous tissue and intrahepatic bile ducts hyperplasia and inflammatory cell infiltration in the cholangitis model group; hepatic lobule structure of sivelestat sodium treatment group was clear, and the degree of peripheral inflammatory cell infiltration was reduced compared with the cholangitis model group. Immunohistochemical staining showed that the expressions of NE and MUC5AC were increased in the cholangitis model group compared with the sham-operated group, and the expressions of NE and MUC5AC were decreased in the sivelestat sodium group compared with the cholangitis model group [NE (A value): 5.23±2.02 vs. 116.67±23.06, MUC5AC (A value): 5.40±3.09 vs. 23.81±7.09, both P < 0.05]. Western blotting showed that the protein expressions of NE, MUC5AC, and TLR4 in the hepatic biliary tissues of the cholangitis model group were significantly higher than those of the sham-operated group; and the protein expressions of NE, MUC5AC, and TLR4 in the liver biliary tissues of the sivelestat sodium treatment group were significantly higher than those of the sham-operated group (NE/β-actin: 0.38±0.04 vs. 0.70±0.10, MUC5AC/β-actin: 0.37±0.03 vs. 0.61±0.05, TLR4/β-actin: 0.39±0.10 vs. 0.93±0.15, all P < 0.05). (3) Cell experiment: fluorescence microscopy showed that the proliferation of HiBEpiC cells in each group was good, and there was no significant difference in the proportion of positive cells. ELISA and Western blotting showed that the expressions of MUC5AC in cells of the NE group were significantly higher than those of the blank control group. The expressions of MUC5AC in the NE+different dose of sivelestat sodium group were significantly lower than those in the NE group, and showed a decreasing trend with the increase of sevastatin sodium concentration, especially in the highest dose group [MUC5AC (μg/L): 3.46±0.20 vs. 6.33±0.52, MUC5AC/β-actin: 0.45±0.07 vs. 1.75±0.10, both P < 0.05].
CONCLUSIONS: LPS can upregulate the expression of NE and MUC5AC in rats with cholangitis, while sodium sivelestat can reduce the expression of MUC5AC in in intrahepatic biliary epithelial cells by inhibiting NE, providing a new direction for the treatment of IBDS.
METHODS: (1) Bioinformatics analysis: differential gene analysis was performed on gallbladder stone cholecystitis sequencing data based on the gene expression omnibus (GEO) to screen for significantly different genes related to neutrophils and mucins. The search tool for the retrieval of interacting genes database (STRING) was used for protein interaction analysis to predict whether there was an interaction between NE and MUC5AC genes. (2) Animal experiment: a total of 18 male SD rats were divided into the sham-operated group, cholangitis model group and sivelestat sodium treatment group according to the random number table method, with 6 rats in each group. The cholangitis rat model was established by a one-time injection of 1.25 mg/kg lipopolysaccharide (LPS) into the right anterior lobe of the liver of rats in combination with the pre-experiment; the liver of the sham-operated group was injected with an equal volume of saline. After the modelling, 100 mg/kg of sivelestat sodium was injected into the tail vein of the cevalexin treatment group once a day for 5 days, and an equal volume of saline was injected into the tail vein of the sham-operated group and the cholangitis model group. Two weeks later, the rats were euthanized and their liver and bile duct tissues were taken. The pathological changes in the liver and bile duct tissues were observed under the light microscope. Immunohistochemical staining was used to detect the expressions of NE and MUC5AC in liver and bile duct tissues. The protein expressions of NE, MUC5AC and Toll-like receptor 4 (TLR4) were detected by Western blotting. (3) Cell experiment: primary human intrahepatic biliary epithelial cell line (HiBEpiC) was divided into blank control group, NE group (10 nmol/L NE), NE+sivelestat sodium low dose group (10 nmol/L NE+1×10-8 g/L sivelestat sodium 1 mL), NE+sivelestat sodium medium dose group (10 nmol/L NE+1×10-7 g/L sivelestat sodium 1 mL), NE+sivelestat sodium high dose group (10 nmol/L NE+1×10-6 g/L sivelestat sodium 1 mL). Cells were collected after 48 hours of culture, and EdU was performed to detect the proliferative activity of cells; enzyme linked immunosorbent assay (ELISA) and Western blotting were performed to detect the expression of MUC5AC in cells.
RESULTS: (1) Bioinformatics analysis: the NE gene (ELANE) had a reciprocal relationship with MUC5AC. (2) Animal experiment: light microscopy showed that hepatocyte edema, hepatocyte diffuse point and focal necrosis, confluent area fibrous tissue and intrahepatic bile ducts hyperplasia and inflammatory cell infiltration in the cholangitis model group; hepatic lobule structure of sivelestat sodium treatment group was clear, and the degree of peripheral inflammatory cell infiltration was reduced compared with the cholangitis model group. Immunohistochemical staining showed that the expressions of NE and MUC5AC were increased in the cholangitis model group compared with the sham-operated group, and the expressions of NE and MUC5AC were decreased in the sivelestat sodium group compared with the cholangitis model group [NE (A value): 5.23±2.02 vs. 116.67±23.06, MUC5AC (A value): 5.40±3.09 vs. 23.81±7.09, both P < 0.05]. Western blotting showed that the protein expressions of NE, MUC5AC, and TLR4 in the hepatic biliary tissues of the cholangitis model group were significantly higher than those of the sham-operated group; and the protein expressions of NE, MUC5AC, and TLR4 in the liver biliary tissues of the sivelestat sodium treatment group were significantly higher than those of the sham-operated group (NE/β-actin: 0.38±0.04 vs. 0.70±0.10, MUC5AC/β-actin: 0.37±0.03 vs. 0.61±0.05, TLR4/β-actin: 0.39±0.10 vs. 0.93±0.15, all P < 0.05). (3) Cell experiment: fluorescence microscopy showed that the proliferation of HiBEpiC cells in each group was good, and there was no significant difference in the proportion of positive cells. ELISA and Western blotting showed that the expressions of MUC5AC in cells of the NE group were significantly higher than those of the blank control group. The expressions of MUC5AC in the NE+different dose of sivelestat sodium group were significantly lower than those in the NE group, and showed a decreasing trend with the increase of sevastatin sodium concentration, especially in the highest dose group [MUC5AC (μg/L): 3.46±0.20 vs. 6.33±0.52, MUC5AC/β-actin: 0.45±0.07 vs. 1.75±0.10, both P < 0.05].
CONCLUSIONS: LPS can upregulate the expression of NE and MUC5AC in rats with cholangitis, while sodium sivelestat can reduce the expression of MUC5AC in in intrahepatic biliary epithelial cells by inhibiting NE, providing a new direction for the treatment of IBDS.
摘要：
目的：探讨西维来司钠是否通过抑制中性粒细胞弹性蛋白酶(NE)降低肝内胆管上皮细胞黏蛋白5AC(MUC5AC)的表达，为肝内胆管结石(IBDS)的治疗提供新的潜在治疗思路。
方法：(1)生物信息学分析：根据基因表达综合(GEO)对胆囊结石性胆囊炎测序数据进行差异基因分析，筛选与中性粒细胞和黏蛋白相关的显著不同基因。用于检索相互作用基因数据库（STRING）的搜索工具用于蛋白质相互作用分析，以预测NE和MUC5AC基因之间是否存在相互作用。(2)动物实验:18只雄性SD大鼠分为假手术组,胆管炎模型组和西维司他钠治疗组,按照随机数字表法,每组6只大鼠。采用大鼠肝右前叶一次性注射脂多糖(LPS)1.25mg/kg,结合预实验建立胆管炎大鼠模型;假手术组肝脏注射等体积生理盐水。建模之后,西维来司钠100mg/kg，每天1次，连续5天，假手术组和胆管炎模型组尾静脉注射等体积生理盐水。两周后,对大鼠实施安乐死,取其肝脏和胆管组织。光镜下观察肝脏和胆管组织的病理变化。免疫组化染色检测NE和MUC5AC在肝脏和胆管组织中的表达。NE的蛋白质表达，通过蛋白质印迹法检测MUC5AC和Toll样受体4(TLR4)。(3)细胞实验:将原代人肝内胆管上皮细胞系(HibepiC)分为空白对照组,NE组（10nmol/LNE），NE+西维来司钠低剂量组（10nmol/LNE+1×10-8g/L西维来司钠1mL），NE+西维来司钠中剂量组（10nmol/LNE+1×10-7g/L西维来司钠1mL），NE+西维来司钠高剂量组(10nmol/LNE+1×10-6g/L西维来司钠1mL)。培养48小时后收集细胞，并进行EdU检测细胞的增殖活性；进行酶联免疫吸附试验（ELISA）和Westernblotting检测细胞中MUC5AC的表达。
结果：（1）生物信息学分析：NE基因（ELANE）与MUC5AC具有相互关系。(2)动物实验:光镜显示肝细胞水肿,肝细胞弥漫性点和局灶性坏死，胆管炎模型组融合区纤维组织和肝内胆管增生及炎性细胞浸润;西维来司钠治疗组肝小叶结构清晰,与胆管炎模型组相比，外周炎性细胞浸润程度减轻。免疫组化染色显示,与假手术组相比,胆管炎模型组NE和MUC5AC的表达增加,与胆管炎模型组相比，西维来司钠组NE和MUC5AC的表达降低[NE（A值）：5.23±2.02。116.67±23.06,MUC5AC(A值):5.40±3.09vs.23.81±7.09,均P<0.05。Westernblotting显示NE的蛋白表达,MUC5AC,胆管炎模型组肝胆管组织TLR4明显高于假手术组,MUC5AC,西维来司钠治疗组肝胆管组织TLR4明显高于假手术组（NE/β-肌动蛋白：0.38±0.04vs.0.70±0.10，MUC5AC/β-肌动蛋白：0.37±0.03vs.0.61±0.05，TLR4/β-肌动蛋白：0.39±0.10vs.0.93±0.15,均P<0.05)。(3)细胞实验:荧光显微镜显示各组HibepiC细胞增殖良好,阳性细胞比例差异无统计学意义。ELISA和Westernblotting结果显示，NE组细胞中MUC5AC的表达明显高于空白对照组。NE+不同剂量西维来司钠组MUC5AC的表达明显低于NE组,随着舒伐他汀钠浓度的增加呈下降趋势,特别是在最高剂量组[MUC5AC（μg/L）：3.46±0.20vs.6.33±0.52，MUC5AC/β-肌动蛋白：0.45±0.07vs.1.75±0.10,均P<0.05]。
结论：LPS可上调胆管炎大鼠NE和MUC5AC的表达，西维来司钠通过抑制NE降低肝内胆管上皮细胞MUC5AC的表达,为IBDS的治疗提供了新的方向。
方法：(1)生物信息学分析：根据基因表达综合(GEO)对胆囊结石性胆囊炎测序数据进行差异基因分析，筛选与中性粒细胞和黏蛋白相关的显著不同基因。用于检索相互作用基因数据库（STRING）的搜索工具用于蛋白质相互作用分析，以预测NE和MUC5AC基因之间是否存在相互作用。(2)动物实验:18只雄性SD大鼠分为假手术组,胆管炎模型组和西维司他钠治疗组,按照随机数字表法,每组6只大鼠。采用大鼠肝右前叶一次性注射脂多糖(LPS)1.25mg/kg,结合预实验建立胆管炎大鼠模型;假手术组肝脏注射等体积生理盐水。建模之后,西维来司钠100mg/kg，每天1次，连续5天，假手术组和胆管炎模型组尾静脉注射等体积生理盐水。两周后,对大鼠实施安乐死,取其肝脏和胆管组织。光镜下观察肝脏和胆管组织的病理变化。免疫组化染色检测NE和MUC5AC在肝脏和胆管组织中的表达。NE的蛋白质表达，通过蛋白质印迹法检测MUC5AC和Toll样受体4(TLR4)。(3)细胞实验:将原代人肝内胆管上皮细胞系(HibepiC)分为空白对照组,NE组（10nmol/LNE），NE+西维来司钠低剂量组（10nmol/LNE+1×10-8g/L西维来司钠1mL），NE+西维来司钠中剂量组（10nmol/LNE+1×10-7g/L西维来司钠1mL），NE+西维来司钠高剂量组(10nmol/LNE+1×10-6g/L西维来司钠1mL)。培养48小时后收集细胞，并进行EdU检测细胞的增殖活性；进行酶联免疫吸附试验（ELISA）和Westernblotting检测细胞中MUC5AC的表达。
结果：（1）生物信息学分析：NE基因（ELANE）与MUC5AC具有相互关系。(2)动物实验:光镜显示肝细胞水肿,肝细胞弥漫性点和局灶性坏死，胆管炎模型组融合区纤维组织和肝内胆管增生及炎性细胞浸润;西维来司钠治疗组肝小叶结构清晰,与胆管炎模型组相比，外周炎性细胞浸润程度减轻。免疫组化染色显示,与假手术组相比,胆管炎模型组NE和MUC5AC的表达增加,与胆管炎模型组相比，西维来司钠组NE和MUC5AC的表达降低[NE（A值）：5.23±2.02。116.67±23.06,MUC5AC(A值):5.40±3.09vs.23.81±7.09,均P<0.05。Westernblotting显示NE的蛋白表达,MUC5AC,胆管炎模型组肝胆管组织TLR4明显高于假手术组,MUC5AC,西维来司钠治疗组肝胆管组织TLR4明显高于假手术组（NE/β-肌动蛋白：0.38±0.04vs.0.70±0.10，MUC5AC/β-肌动蛋白：0.37±0.03vs.0.61±0.05，TLR4/β-肌动蛋白：0.39±0.10vs.0.93±0.15,均P<0.05)。(3)细胞实验:荧光显微镜显示各组HibepiC细胞增殖良好,阳性细胞比例差异无统计学意义。ELISA和Westernblotting结果显示，NE组细胞中MUC5AC的表达明显高于空白对照组。NE+不同剂量西维来司钠组MUC5AC的表达明显低于NE组,随着舒伐他汀钠浓度的增加呈下降趋势,特别是在最高剂量组[MUC5AC（μg/L）：3.46±0.20vs.6.33±0.52，MUC5AC/β-肌动蛋白：0.45±0.07vs.1.75±0.10,均P<0.05]。
结论：LPS可上调胆管炎大鼠NE和MUC5AC的表达，西维来司钠通过抑制NE降低肝内胆管上皮细胞MUC5AC的表达,为IBDS的治疗提供了新的方向。
