Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

猪肝星状细胞内质网应激模型的建立及对泛素化的影响

试验对衣霉素（tunicamycin,TM）处理猪肝星状细胞的浓度和培养时间进行筛选,以期成功构建内质网应激（endoplasmic reticulum stress,ERS）模型,并在此基础上探究ERS下猪肝星状细胞泛素化的变化。试验采用浓度为0、1、2、5、10和15 μg/mL的TM处理猪肝星状细胞,培养2、4、8、16、24和36 h。通过检测细胞抑制率对TM浓度和培养时间进行初筛;通过检测细胞周期及ERS标志基因和蛋白表达对TM浓度和培养时间进行终选,以确定是否成功构建ERS模型,同时,在ERS下检测猪肝星状细胞泛素化相关基因的表达变化。结果表明：由细胞抑制率检测结果初步确定TM浓度5 μg/mL、培养8或24 h后有可能出现ERS。5 μg/mL TM在培养8或24 h时,ERS标志基因表达均极显著上调（P < 0.01）。在培养8 h时,ERS标志蛋白中仅ATF4显著上调（P < 0.05）,细胞周期中仅G2/M期细胞比例显著下降（P < 0.05）;在培养24 h时,ERS标志蛋白均显著上调（P < 0.05）,细胞周期在G1期出现阻滞,并导致S期和G2/M期细胞比例极显著下降（P < 0.01）。以上结果说明,5 μg/mL TM培养24 h可成功构建细胞ERS模型。在ERS下,细胞泛素化相关基因UBA2和UBE2E的表达均极显著升高（P < 0.01）。综上,猪肝星状细胞经5 μg/mL TM处理24 h,可成功建立ERS模型,并启动泛素化机制。     

催产素受体在雌性山羊颈胸神经节的分布

为探讨催产素(OT)是否可以影响雌性山羊颈胸神经节的活动.采用免疫组化SP法观察催产素受体在雌性山 羊颈胸神经节的分布特点.催产素受体在颈胸神经节分布广泛,节内的神经细胞、卫星细胞和过路纤维均有OTR 免疫阳性产物分布;OTR主要在神经细胞中表达,相对表达量与其他非神经结构相比差异性显著(P＜0.05);在神经 细胞中,OTR免疫阳性产物主要存在于胞膜、胞质、核仁,核膜不着色.雌性山羊颈胸神经节神经元对OT具有反应 性,提示OT可能通过影响颈胸神经节神经元的活动,从而经其发出的交感节后神经这一途径调节其所支配的靶器 官如心血管、汗腺、呼吸等的生理活动.     

MR IMAGING FINDINGS IN A DOG WITH LUMBAR GANGLION CYSTS

Intraspinal cysts of the L6-L7 and L7-S1 articular process joints in a six-year-old neutered female German Shepherd Dog were diagnosed using magnetic resonance (MR) imaging. Histopathology provided a diagnosis of ganglion cysts. Clinical, laboratory, radiographic and MR imaging findings are described. Briefly, radiographic findings revealed lumbarization of the first sacral vertebra, and fusion of the first caudal vertebra to the sacrum. In addition, spondylosis and articular process osteoarthrosis occurred at L6-L7 and L7-S1. MR imaging revealed multiple, well encapsulated structures ranging in size from 3-10 mm in diameter which were found to arise from the L6-L7 and L7-S1 articular process joints. These cysts had signal intensities that varied from hyperintense to the cerebrospinal fluid (CSF) on T1 weighted images to isointense to CSF on T2 weighted images. Decompressive surgery in conjunction with arthrodesis of these joints resulted in resolution of clinical signs. The dog remained pain-free 1 1/2 years following surgical therapy.     

兔减压神经的纤维成分—HRP法

艇ＨＲＰ法对兔减压神经进行了研究，标记的传入神经元绝大多数位于迷走神经结状节尾侧半部偏一侧，少数见于颈静脉神经节，其中枢突标记终末见于延髓闩以上的孤束核内侧部，腹侧部以及迷背核内。在颈前节也出现标记细胞，表明减压神经中也含有起源于 前节的交感节后纤维成分。主动脉压力感受器有经减压神经来的感觉神经交感节后神经纤维支配。提示减压神经是含有一般内脏传入和一般内脏传出纤维的混合神经，为研究心血管生理提供了     

Actions of Tefluthrin on Rat Na(v)1.7 Voltage-Gated Sodium Channels Expressed in Xenopus Oocytes

In rats expression of the Na_v1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Na_v1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 μM modified of Na_v1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked “late current” at the end of a 40 ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to twofold by the application of a train of up to 100 5 ms depolarizing prepulses. These effects of tefluthrin on Na_v1.7 channels were qualitatively similar to its effects on rat Na_v1.2, Na_v1.3 and Na_v1.6 channels assayed previously under identical conditions. However, Na_v1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Na_v1.3 and Na_v1.6 channels. It is likely that Na_v1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Na_v1.3, Na_v1.6 and Na_v1.8) and two pyrethroid-resistant isoforms (rat Na_v1.2 and Na_v1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.     

青果作用后大鼠肝星状细胞蛋白质组双向电泳技术的建立

[目的]建立青果作用后大鼠肝星状细胞蛋白质组双向电泳分离体系,提高其分辨率和重复性。[方法]青果作用于大鼠肝星状细胞,提取全蛋白,按标准条件对蛋白质进行双向电泳分离,并对各关键因素进行优化。[结果]最终选择的裂解液配方是1%TBP、4%CHAPS、0.2%Bio-Lyte、40 mmol/L Tris、8 mol/L尿素、2 mol/L硫脲;使用pH 4～7的IPG胶条进行被动水化上样,等电聚焦采用缓慢升压模式,电泳参数根据Bio-Rad公司的预设方案进行调整;使用改良硝酸银法进行蛋白质斑点染色,从而获得了满意的蛋白质组双向电泳图谱。[结论]成功建立了青果作用后大鼠肝星状细胞蛋白质组双向电泳分离体系,具有较高的分辨率和重复性,为进一步较为系统、全面、准确的评价青果及其复方制剂的作用机制和药效提供方法学基础。     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.