[Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro]
- PMID: 36642896
- DOI: 10.16250/j.32.1374.2022163
[Recombinant Schistosoma japonicum egg ribonuclease SjCP1412 inhibits the activation of LX-2 hepatic stellate cells in vitro]
Abstract
Objective: To investigate the effect of recombinant Schistosoma japonicum egg ribonuclease SjCP1412 (rSjCP1412) on proliferation, cell cycle, apoptosis and activation of human hepatic stellate cells LX-2 in vitro, and explore the underlying mechanisms.
Methods: The rSjCP1412 protein was expressed in Escherichia coli BL21 by prokaryotic expression, and the highly purified soluble rSjCP1412 protein was prepared by Ni NTA affinity chromatography and urea gradient refolding dialysis. Yeast RNA was digested using 12.5, 25.0, 50.0 µg rSjCP1412 proteins at 37 °C for 2, 3, 4 h, and the enzymatic products were electrophoresed on 1.5% agarose gel to observe the RNAase activity of rSjCP1412 protein. The proliferation of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was measured using CCK-8 assay, and the apoptosis of LX-2 cells stimulated by different doses of rSjCP1412 protein for 48 hours was detected using the Annexin V-FITC/PI double staining, while the percentage of LX-2 cells at G0/G1, S and G2/M phases of cell cycle following stimulation with different doses of rSjCP1412 protein for 48 h was detected by DAPI staining. The type I collagen, type III collagen and α-smooth muscle actin (α-SMA) mRNA expression was quantified using quantitative florescent real-time PCR (qPCR) assay and Western blotting at transcriptional and translational levels in LX-2 cells following stimulation with different doses of rSjCP1412 protein for 48 h, while soluble egg antigen (SEA) served a positive control and PBS without rSjCP1412 protein as a normal control in the above experiments. The expression of collagen I, α-SMA and Smad4 protein was determined using Western blotting in LX-2 cells following stimulation with rSjCP1412 protein, transforming growth factor-β1 (TGF-β1) alone or in combination, to examine the signaling for the effect of rSjCP1412 protein on LX-2 cells.
Results: The rSjCP1412 protein was successfully expressed and the highly purified soluble rSjCP1412 protein was prepared, which had a RNase activity. Compared with the normal group, the survival rates of LX-2 cells significantly decreased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein and SEA for 48 h (F = 22.417 and 20.448, both P values < 0.05). The apoptotic rates of LX-2 cells significantly increased post-treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h (F = 11.350, P < 0.05), and treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h resulted in arrest of LX-2 cells in G0/G1 phase (F = 20.710, P < 0.05). Treatment with 12.5, 25.0, 50.0 µg/mL rSjCP1412 protein for 48 h caused a significant reduction in relative expression levels of collagen I (F = 11.340, P < 0.05), collagen III (F = 456.600, P < 0.05) and α-SMA mRNA (F = 23.100, P < 0.05) in LX-2 cells, and both rSjCP1412 protein and SEA treatment caused a significant reduction in collagen I (F = 1 302.000, P < 0.05), α-SMA (F = 49.750, P < 0.05) and Smad4 protein expression (F = 52.420, P < 0.05) in LX-2 cells. In addition, rSjCP1412 protein treatment inhibited collagen I (F = 66.290, P < 0.05), α-SMA (F = 31.300, P < 0.05) and Smad4 protein expression (F = 27.010, P < 0.05) in LX-2 cells activated by TGF-β1.
Conclusions: rSjCP1412 protein may induce apoptosis of LX-2 cells and inhibit proliferation, cell cycle and activation of LX-2 cells through down-regulating Smad4 signaling molecules.
［摘要］ 目的观察重组日本血吸虫虫卵核糖核酸酶SjCP1412(rSjCP1412)对体外培养的人肝星状细胞LX-2增殖、细 胞周期、凋亡及活化的作用, 并探索其潜在机制。方法 在大肠埃希菌BL21中采用原核表达方式表达rSjCP1412蛋白, 并通过Ni-NTA亲和层析法及尿素梯度复性透析制备高纯度可溶性rSjCP1412蛋白。使用12.5、25.0、50.0µg rSjCP1412 蛋白分别于37 °C酶解酵母RNA2、3、4h, 酶解产物采用1.5%琼脂糖凝胶电泳分析, 观察rSjCP1412蛋白RNA酶活性。采 用CCK-8法检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞增殖, 采用Annexin V-FITC/PI细胞凋亡双染法检测经 不同浓度rSjCP1412蛋白刺激48 h后LX-2细胞凋亡, 采用DAPI染色法检测经不同浓度rSjCP1412蛋白刺激48 h后LX-2 细胞周期中G0/G1、S、G2/M期细胞百分比, 采用荧光实时定量PCR(qPCR)检测经不同浓度rSjCP1412蛋白刺激48 h后 LX-2细胞中I型胶原、III型胶原和α-平滑肌肌动蛋白(α-SMA)mRN4表达水平;采用Western blotting法检测经rSjCP1412 蛋白刺激LX-2细胞48 h后I型胶原、(α-SMA和Smad4/7蛋白表达水平, 以明确LX-2细胞活化。以上实验均设可溶性虫 卵抗原(SEA)阳性对照和正常组(不含rSjCP1412蛋白的PBS)。采用rSjCP1412蛋白单独刺激或与转化生长因子屮1 (TGF-β1)共同刺激LX-2细胞48 h后, 采用Western blotting检测LX-2细胞中I型胶原、(α-SMA和Smad4蛋白表达水平, 分 析rSjCP1412作用于LX-2细胞的信号通路。结果 成功表达并制备了高纯度可溶性的rSjCP1412蛋白, 且rSjCP1412蛋 白具有RNA酶活性。与正常组相比, 12.5、25.0、50.0µg/mL rSjCP1412和SEA处理48 h后, LX-2细胞存活率均显著下降 (F = 22.417、20.448, P 均 < 0.05); 12.5、25.0、50.0 µg/mL rSjCP1412蛋白处理48 h后, LX-2 细胞凋亡率显著增加(F = 11.350, P < 0.05); 12.5、25.0、50.0 µg/mL rSjCP1412 蛋白处理48 h 后, 可导致 LX-2 细胞 G0/G1 期阻滞(F = 20.710, P < 0.05)。12.5、25.0、50.0µg/mL rSjCP1412蛋白处理可导致LX-2细胞中I型胶原、III型胶原和α-SMA mRNA表达水平均显 著下降(F = 11.340, 456.600, 23.100, P均 <0.05)。rSjCP1412 与 SEA 均可导致LX-2 细胞中 I 型胶原、α-SMA 和 Smad4蛋 白表达水平下调, 活化水平下降(F = 1 302.000、49.750、52.420, P均<0.05)。此外, rSjCP1412蛋白抑制TGF-β1激活的 LX-2细胞中I型胶原、α-SMA和Smad4蛋白表达水平(F = 66.290, 31.300, 27.010, P均<0.05)。结论 SjCP1412在体外 能诱导肝星状细胞LX-2凋亡, 对LX-2细胞增殖、细胞周期及活化均具有一定抑制作用, 其作用机制与下调Smad4信号分 子表达水平相关。.
Keywords:
Hepatic stellate cell; Ribonuclease;