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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 investigate the effects of norepinephrine (NA) and prazosin (PZ) on the activation and proliferation of human hepatic stellate cell line (HSCLX2), the JAK2-STAT3 signaling pathway and the production of reactive oxygen species. Methods HSC-LX2 was divided into control group (HSC-LX2 cultured without any drug), model group (100ng · m L -1) leptin, low, medium and high dose group μmol·L -1 NA) and combination group (100μmol·L -1 NA + 10 mmol·L -1 PZ). The cell proliferation was detected by MTT assay after 24 h of treatment. The expression of p-JAK2 and p-STAT3 protein was detected by Western-blot and the expression of α-smooth muscle actin (α-SMA) was detected by real-time fluorescence polymerase chain reaction (SMA), TIMP-1 and PTP1B mRNA were detected by flow cytometry. The level of reactive oxygen species (ROS) was detected by flow cytometry. Results The optical density of the control group, the model group, the low, middle and high dose groups and the combination 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), respectively. The expression 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, ); The expression of p-STAT3 was (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 TIMP-1 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) ± 0.11, 0.89 ± 0.14, 1.03 ± 0.15, 1.91 ± 0.22, 0.91 ± 0.15, respectively. The expressions of PTP1B 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), respectively; ROS were (14.25 ± 2.31), (23.74 ± 3.62), (31.12 ± 5.13) 6.02), (51.54 ± 7.45) and (32.76 ± 5.23) respectively. There was significant difference between the above indexes in the low, medium and high dose groups and the combination group with the model group (P <0.05) Compared with high dose group, the difference was statistically significant (P <0.05). Conclusion Norepinephrine regulates leptin-induced HSC-LX2 proliferation and oxidative stress through α1 adrenoceptor, activates JAK2-STAT3 signaling, upregulates the expression of α-SMA and TIMP-1, and down-regulates the expression of PTP1B .