Li Xiang, Yang Guixia, Shen Feng, Zheng Xinghao, He Tianhui, Li Shuwen, Cheng Yumei, Li Qing, Li Wei, Qin Jincheng
Department of Critical Care Medicine, Guizhou Medical University Affiliated Hospital, Guiyang 550004, Guizhou, China. Corresponding author: Shen Feng, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Jan;33(1):53-58. doi: 10.3760/cma.j.cn121430-20200917-00636.
To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS).
AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 μmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA).
According to the inhibition rate curve, the IC of berberine on RLE-6TN cells was 81.16 μmol/L. Therefore, 20, 50 and 80 μmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 μmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 μmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (μg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (μg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (μg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01].
Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).
观察黄连素对脂多糖（LPS）诱导的大鼠Ⅱ型肺泡上皮细胞（AEC II）产生的促凝和纤溶抑制因子的影响。
体外培养AEC II细胞（RLE-6TN细胞），收集对数生长期细胞。采用细胞计数试剂盒-8（CCK-8）检测黄连素的细胞毒性，根据半数细胞抑制浓度（IC）确定药物浓度范围。将RLE-6TN细胞分为五组，空白对照组细胞用DMEM培养；LPS组细胞用5 mg/L LPS刺激；黄连素预处理组细胞分别用20、50和80 μmol/L黄连素预处理1小时，然后与5 mg/L LPS共培养。LPS诱导24小时后收集细胞。采用蛋白质印迹法和实时荧光定量逆转录-聚合酶链反应（RT-qPCR）检测细胞中组织因子（TF）、组织因子途径抑制物（TFPI）和纤溶酶原激活物抑制因子-1（PAI-1）的蛋白质和mRNA表达水平。采用酶联免疫吸附测定（ELISA）法检测细胞上清液中活化蛋白C（APC）、Ⅲ型前胶原肽（PIIIP）、凝血酶-抗凝血酶复合物（TAT）和抗凝血酶Ⅲ（AT III）的水平。
根据抑制率曲线，黄连素对RLE-6TN细胞的IC为81.16 μmol/L。因此，选择20、50和80 μmol/L作为黄连素的干预浓度。与空白对照组相比，LPS诱导24小时后RLE-6TN细胞促凝和纤溶抑制因子的表达及分泌异常。LPS组TF和PAI-1的蛋白质和mRNA表达水平显著升高，但TFPI的蛋白质和mRNA表达水平显著降低。同时，细胞上清液中APC和AT III水平显著降低，而PIIIP和TAT水平显著升高。黄连素预处理后，LPS诱导的促凝和纤溶抑制因子的异常表达及分泌呈剂量依赖性得到纠正，尤其是80 μmol/L组。与LPS组相比，黄连素80 μmol/L组TF和PAI-1的蛋白质和mRNA表达水平显著降低[TF蛋白（TF/GAPDH）：0.45±0.02比0.55±0.03，TF mRNA（2）：0.39±0.08比1.48±0.11，PAI-1蛋白（PAI-1/GAPDH）：0.37±0.02比0.64±0.04，PAI-1 mRNA（2）：1.14±0.29比4.18±0.44，均P<0.01]，TFPI的蛋白质和mRNA表达水平显著升高[TFPI蛋白（TFPI/GAPDH）：0.53±0.02比0.45±0.02，TFPI mRNA（2）：0.94±0.08比0.40±0.05，均P<0.01]。同时，细胞上清液中APC和AT III水平显著升高[APC（μg/L）：1 358.5±26.0比994.2±23.1，AT III（μg/L）：118.0±7.4比84.4±2.7，均P<0.01]，而PIIIP和TAT水平显著降低[PIIIP（μg/L）：11.2±0.4比18.6±0.9，TAT（ng/L）：222.1±2.8比287.6±7.0，均P<0.01]。
黄连素可抑制LPS诱导的大鼠AEC II细胞促凝和纤溶抑制因子的表达，并呈剂量依赖性促进抗凝因子的表达。黄连素可能是急性呼吸窘迫综合征（ARDS）肺泡高凝和纤溶抑制的新治疗靶点。
