去甲肾上腺素和哌唑嗪对瘦素诱导肝星状细胞活化增殖和JAK2-STAT3信号通路及活性氧产生的影响

Effect of norepinephrine and prazosin on leptin-induced activation proliferation,JAK2-STAT3 signaling pathway and reactive oxygen species generation in hepatic stellate cells

目的研究去甲肾上腺素(NA)和哌唑嗪(PZ)对人肝星状细胞系(HSCLX2)活化增殖﹑JAK2-STAT_3信号通路及活性氧产生的影响。方法体外培养HSC-LX2分为对照组(单纯HSC-LX2培养,不加任何药物)、模型组(100ng·m L^(-1)瘦素)、低、中、高剂量组(1,10,100μmol·L^(-1)NA)和联合组(100μmol·L^(-1)NA+10 mmol·L^(-1)PZ)。处理24 h后,用MTT法检测细胞增殖,用Western-blot检测p-JAK2和p-STAT_3蛋白表达,用实时荧光聚合酶链式反应(RT-PCR)检测α-平滑肌肌动蛋白(α-SMA)﹑基质金属蛋白酶抑制因子-1(TIMP-1)﹑酪氨酸蛋白酶1B(PTP1B)mRNA表达,用流式细胞术检测活性氧(ROS)水平。结果对照组、模型组、低、中、高剂量组和联合组的光密度分别为(0.30±0.05),(0.48±0.06),(0.58±0.08),(0.69±0.11),(0.80±0.14),(0.60±0.10);p-JAK2蛋白表达量分别为(0.24±0.04),(0.40±0.06),(0.57±0.09),(0.66±0.11),(0.78±0.12),(0.53±0.08);p-STAT_3蛋白表达量分别为(0.30±0.05),(0.43±0.07),(0.59±0.09),(0.71±0.11),(0.82±0.12),(0.61±0.09);α-SMA mRNA表达量分别为(0.77±0.12),(0.98±0.15),(1.22±0.20),(1.40±0.23),(1.59±0.25),(1.24±0.21);TIMP-1 mRNA表达量分别为(0.61±0.10),(0.70±0.11),(0.89±0.14),(1.03±0.15),(1.32±0.22),(0.91±0.15);PTP1B mRNA表达量分别为(0.92±0.14),(0.79±0.12),(0.62±0.09),(0.50±0.08),(0.38±0.06),(0.63±0.10);ROS分别为(14.25±2.31),(23.74±3.62),(31.12±5.13),(40.69±6.02),(51.54±7.45),(32.76±5.23),低、中、高剂量组和联合组的上述指标与模型组比较差异均有统计学意义(P<0.05),且联合组的上述指标和高剂量组比较差异有统计学意义(P<0.05)。结论去甲肾上腺素主要通过α1肾上腺素受体调控瘦素诱导的HSC-LX2增殖和氧化应激,激活JAK2-STAT_3信号转导,并上调α-SMA﹑TIMP-1基因表达,下调PTP1B基因表达。

Objective To study the effect of norepinephrine（NA） and prazosin（PZ） on leptin-induced activation,proliferation,JAK2-STAT_3 signaling pathway and reactive oxygen species generation in hepatic stellate cells（HSC-LX2）.Methods Cultured HSC-LX2 were divided into 6 groups：control group（pure HSC-LX2 culture,without any drugs）,model group（100 ng·m L^（-1）leptin）,low-,middle-andhigh-dose group（1,10,100 μmol·L^（-1）NA） and combined group（100 μmol·L^（-1）NA ＋ 10 mmol·L^（-1）PZ）.After incubation for 24 h,the cell proliferation rates were determined by MTT.Phosphorylation of Janus kinase 2（JAK2）and protein expression of signal transducers and activators of transcription 3（STAT_3） were evaluated by Western-blot.The mRNA expressions of α-smooth muscle actin（α-SMA）,matrix metalloproteinase inhibition factor-1（TIMP-1） and protein tyrosine enzyme 1 B（PTP1B） were evaluated by Real time-PCR.Intracellular reactive oxygen species（ROS） level was measured by flow cytometry.Results The optical density of control group,model group,low-,middle-and high-dose groups and combined group were（0.30 ± 0.05）,（0.48 ± 0.06）,（0.58 ±0.08）,（0.69 ± 0.11）,（0.80 ± 0.14）,（0.60 ± 0.10）;the expression levels of p-JAK2 protein were（0.24 ±0.04）,（0.40 ± 0.06）,（0.57 ± 0.09）,（0.66 ± 0.11）,（0.78 ± 0.12）,（0.53 ± 0.08）;the expression levels of p-STAT_3 protein were（0.30 ± 0.05）,（0.43 ± 0.07）,（0.59 ± 0.09）,（0.71 ± 0.11）,（0.82 ± 0.12）,（0.61 ± 0.09）;the expression levels of α-SMA mRNA were（0.77 ± 0.12）,（0.98 ± 0.15）,（1.22 ± 0.20）,（1.40 ± 0.23）,（1.59 ± 0.25）,（1.24 ± 0.21）;the expression levels of TIMP-1 mRNA were（0.61 ± 0.10）,（0.70 ± 0.11）,（0.89 ± 0.14）,（1.03 ± 0.15）,（1.32 ± 0.22）,（0.91 ± 0.15）;the expression levels of PTP1 B mRNA were（0.92 ± 0.14）,（0.79 ± 0.12）,（0.62 ± 0.09）,（0.50 ± 0.08）,（0.38 ± 0.06）,（0.63 ± 0.10）;the level of ROS were（14.25 ± 2.31）,（23.74 ± 3.62）,（31.12 ± 5.13）,（40.69 ± 6.02）,（51.54 ± 7.45）,（32.76 ± 5.23）,there were statistically significant at the above indexes between low-,middle-and high-dose groups and combined group compared with model group（P 0.05）,and the difference between combined group and high-dose group were also statistically significant（P 0.05）.Conclusions NA might promote the HSC-LX2 proliferation and oxidative stress,upregulate α-SMA and TIMP-1 expression,down-regulate PTP1 B expression through promoting JAK2-STAT_3 signal transduction induced by leptin,which is mainly mediated via α1-adrenergic receptor.