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目的:采用血清药理学的方法探讨柴郁汤对CCl4造成的大鼠原代肝细胞急性损伤的保护作用。方法:建立大鼠肝细胞原代培养方法,制备大鼠药物血清。将原代肝细胞种于培养板中,37℃、5%CO2孵育箱中培养48h后,分组加入相应药物后,继续培养6h、18h、30h后,除正常血清对照组外均加入10mmol/L CCl4继续作用6h,测定MTT,分别收集这三个时间点的培养上清液以测定丙氨酸氨基转移酶(ALT)、甘胆酸(CG),并收集细胞测定细胞膜Na+-K+-ATP酶活性。结果:成功进行肝细胞分离,得率3×107个,存活率90%以上;成功制备正常大鼠血清、柴郁汤药物血清(0.2g/ml、0.6g/ml、1.8g/ml)以及熊去氧胆酸药物血清;随柴郁汤用量增加,柴郁汤药物血清使CCl4损伤大鼠肝细胞培养上清中ALT的含量下降,36 h时柴郁汤药物血清0.2g/ml组ALT的含量为6.5±2.0IU/L,柴郁汤药物血清1.8g/ml组ALT的含量为3.4±1.0IU/L。而上清中甘胆酸的含量升高,36 h柴郁汤药物血清0.2g/ml组上清中甘胆酸的含量为2.07±0.30 g/ml,柴郁汤药物血清1.8g/ml组上清中甘胆酸的含量为2.81±0.32 g/ml。且肝细胞膜Na+-K+-ATP酶活性增高,36 h柴郁汤药物血清0.2g/ml组肝细胞膜Na+-K+-ATP酶活性为0.63±0.08μmol Pi/(mg prot·h),柴郁汤药物血清1.8g/ml组Na+-K+-ATP酶活性为1.10±0.15μmol Pi/(mg prot·h),与模型组比较差异均有显著性意义。结论:柴郁汤药物血清对CCl4损伤的大鼠原代肝细胞能显著促进甘胆酸分泌。柴郁汤泌胆利胆作用机制与维持肝细胞膜系的流动性和提高肝细胞膜Na+-K+-ATP酶的活性有关。
Objective: To explore the protective effect of Chai Yu Tang on rat primary hepatocytes acute injury induced by CCl4 by serum pharmacology. Methods: The primary culture method of rat hepatocytes was established and the rat serum was prepared. Primary hepatocytes were seeded in culture plates and incubated for 48 h at 37 ° C in a 5% CO2 incubator. After the corresponding drugs were added in groups and cultured for 6h, 18h and 30h, 10mmol / L CCl4 continued to function for 6h, the MTT was measured, the culture supernatants were collected at these three time points to determine ALT and CG, and the cell membrane Na + -K + -ATPase active. Results: The successful isolation of hepatocytes was achieved with a yield of 3 × 107 and a survival rate of over 90%. The serum of normal rats, Chai Yu Tang serum (0.2g / ml, 0.6g / ml and 1.8g / ml) Ursodeoxycholic acid serum; With the increase of the amount of Chai Yu Tang, Chai Yu Tang serum CCl4 injured rat liver cell culture supernatant ALT content decreased, 36 h Chai Tang Tang serum 0.2 g / ml group ALT The content of ALT was 6.5 ± 2.0IU / L and that of Chaiyu Tang serum 1.8g / ml was 3.4 ± 1.0IU / L. While the content of glycocholate increased in the supernatant. The contents of glycocholate in the supernatant of the supernatant of 0.2% (0.2g / ml) in Chaiyu Tang for 36 h were 2.07 ± 0.30 g / ml and that of Chaiyu Tang The content of glycocholate in the supernatant was 2.81 ± 0.32 g / ml. The activity of Na + -K + -ATPase in hepatocyte membrane was increased. The activity of Na + -K + -ATPase in liver cell membrane was 0.63 ± 0.08μmol Pi / (mg prot · h) in 0.2% The activity of Na + -K + -ATPase in drug serum 1.8g / ml group was 1.10 ± 0.15μmol Pi / (mg prot · h), which was significantly different from the model group. Conclusion: Chai Yu Tang serum can significantly promote the secretion of glycocholic acid on CCl4-injured rat primary hepatocytes. Chai Yu Tang gallbladder gallbladder mechanism of action and maintain the fluidity of liver cell membrane system and improve the activity of liver cell membrane Na + -K + -ATP enzyme.