黄连素剂量依赖性抑制脂多糖刺激下大鼠Ⅱ型肺泡上皮细胞促凝和纤溶抑制因子的表达

Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat typeⅡalveolar epithelial cells

目的探讨黄连素对脂多糖(LPS)刺激下大鼠Ⅱ型肺泡上皮细胞(AECⅡ)表达及分泌促凝和纤溶抑制相关因子的影响。方法体外培养大鼠AECⅡ细胞株RLE-6TN,取对数生长期细胞,用细胞增殖与毒性检测试剂盒(CCK-8)检测黄连素对细胞的毒性作用,根据半数抑制浓度(IC50)确定药物浓度范围。将对数生长期细胞分为5组:空白对照组使用DMEM培养基常规培养;LPS组在培养基中加入5 mg/L的LPS刺激细胞;黄连素预处理组先分别加入20、50、80μmol/L黄连素预处理1 h后,再加入5 mg/L的LPS共培养。LPS刺激24 h后收集细胞,采用蛋白质免疫印迹试验(Western blotting)及实时荧光定量反转录-聚合酶链反应(RT-qPCR)检测细胞组织因子(TF)、组织因子途径抑制物(TFPI)、纤溶酶原激活物抑制剂-1(PAI-1)的蛋白和mRNA表达;采用酶联免疫吸附试验(ELISA)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)及抗凝血酶Ⅲ(ATⅢ)的含量。结果根据抑制率曲线计算得出黄连素对RLE-6TN细胞的IC50为81.16μmol/L,故选择20、50、80μmol/L作为黄连素的干预浓度。与空白对照组相比,LPS刺激24 h后RLE-6TN细胞表达及分泌促凝和纤溶抑制相关因子异常,表现为TF、PAI-1的蛋白及mRNA表达水平均明显升高,TFPI的蛋白及mRNA表达水平明显降低;同时细胞上清液中APC、ATⅢ含量均明显降低,而PⅢP、TAT含量均明显升高。给予黄连素预处理后,LPS刺激诱导的RLE-6TN细胞表达及分泌促凝和纤溶抑制相关因子异常情况得到纠正,并呈现一定的剂量依赖性,以80μmol/L时作用更为显著;与LPS组相比,黄连素80μmol/L预处理组细胞中TF、PAI-1的蛋白及mRNA表达水平均明显降低〔TF蛋白(TF/GAPDH):0.45±0.02比0.55±0.03,TF mRNA(2^(-ΔΔCt)):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^(-ΔΔCt)):1.14±0.29比4.18±0.44,均P<0.01〕,TFPI蛋白及mRNA表达水平明显升高〔TFPI蛋白(TFPI/GAPDH):0.53±0.02比0.45±0.02,TFPI mRNA(2^(-ΔΔCt)):0.94±0.08比0.40±0.05,均P<0.01〕;同时细胞上清液中APC、ATⅢ含量明显升高〔APC(μg/L):1358.5±26.0比994.2±23.1,ATⅢ(μg/L):118.0±7.4比84.4±2.7,均P<0.01〕,而PⅢP和TAT含量则均明显降低〔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〕。结论黄连素可以剂量依赖性抑制LPS刺激下大鼠AECⅡ细胞促凝及纤溶抑制相关因子表达和分泌,促进抗凝因子表达和分泌,有望成为有效防治急性呼吸窘迫综合征(ARDS)肺泡过度促凝和纤溶抑制的新靶点。

Objective To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat typeⅡalveolar epithelial cell(AECⅡ)induced by lipopolysaccharide(LPS).Methods AECⅡcells(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(IC50).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 80μmol/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),precollagenⅢpeptide(PⅢP),thrombin-antithrombin complex(TAT)and antithrombinⅢ(ATⅢ)in the cell supernatant were measured by enzyme linked immunosorbent assay(ELISA).Results According to the inhibition rate curve,the IC50 of berberine on RLE-6TN cells was 81.16μmol/L.Therefore,20,50 and 80μmol/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 ATⅢin 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 80μmol/L.Compared with the LPS group,the protein and mRNA expression levels of TF and PAI-1 in the berberine 80μmol/L group were significantly decreased[TF protein(TF/GAPDH):0.45±0.02 vs.0.55±0.03,TF mRNA(2^(-ΔΔCt)):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^(-ΔΔCt)):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^(-ΔΔCt)):0.94±0.08 vs.0.40±0.05,both P<0.01].Meanwhile,the levels of APC and ATⅢin the cell supernatant were significantly increased[APC(μg/L):1358.5±26.0 vs.994.2±23.1,ATⅢ(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].Conclusions Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AECⅡcells 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).