Changes in collagen fiber content and hepatic stellate cell distribution in the liver of chick embryos and growing chickens

The content of collagen and the distribution of hepatic stellate cells (HSCs) were studied to elucidate the occurrence of sex‐dependent variations in the liver of developing embryos and growing chickens. Chick embryos from embryonic days (e) 12 to e20 and chicks at 1, 4 and 8 weeks were analyzed. Liver tissue was processed using NaOH maceration and freeze‐dried to obtain the collagen fiber specimens. HSCs were identified by double fluorescent immunohistochemistry for desmin and vimentin. There were no sex‐dependent variations in the percentage of collagen fiber per liver weight and HSC area during embryonic stages. However, the content of collagen fiber increased during embryonic development in both sexes. On the other hand, the area of HSCs significantly increased in growing males but did not show any change in females. Importantly, sex differences were observed in both collagen fiber content and HSC area in the liver at 8 weeks. These results indicate that the occurrence of collagen content variations takes place at 8 weeks in chicken liver, suggesting that a sex‐dependent hormone may play an important role on the collagen production of HSCs in the growing chicken liver.     

Notch1 regulates activation of pancreatic stellate cells by non-classical Notch signaling pathway

AIM: To investigate the effect of Notch1 on the activation of pancreatic stellate cells (PSCs). METHODS: The expression of Notch1 in pancreatic duct adenocarcinoma (PDAC) tissues was detected by the immunohistochemical and immunofluorescence double staining. The PSCs were isolated, cultured, and identified by oil red O staining, Western blot and RT-qPCR. The expression of Notch1 and HES1 was detected by Western blot and RT-qPCR. After transfection of Notch1 siRNA to PSCs, Western blot was used to detect the protein expression of α-smooth muscle actin (α-SMA), fibronectin and collagen type Ⅰ (ColⅠ) in activated PSCs. The expression of Notch1 and HES1 was also detected by Western blot. The effects of Notch1 siRNA on migration ability and viability of PSCs were determine by scratch test and CCK-8 assay. RESULTS: The results of immunohistochemical and immunofluorescence double staining showed that Notch1 expressed in α-SMA positive cells in PDAC stroma. The mouse PSCs were successfully cultured, and the expression of α-SMA, fibronectin, ColⅠ, Notch1 and HES1 in activated PSCs were significantly increased compared with unactivated PSCs (P<0.01). After transfection of Notch1 siRNA to mouse PSCs, the expression of α-SMA and ColⅠ was significantly reduced compared with negative groups, but the expression of fibronectin and HES1 did not change significantly. After knock-down of Notch1 expression in activated PSCs, the migration ability and viability of PSCs were significantly reduced compared with negative group. CONCLUSION: Notch1 is involved in regulating the activation of PSCs. Knock-down of Notch1 expression inhibits the expression of the markers of activated PSCs, α-SMA and ColⅠ, reduces the activation of PSCs, and attenuates the migration capacity and viability of PSCs. Notch1 regulates the activation of PSCs without relying on the classic Notch signaling pathway.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Oxymatrine inhibits hepatic stellate cell (HSC) autophagy during HSC activation induced by arsenic

AIM: To investigate the effect of oxymatrine (OM) on the autophagy of hepatic stellate cells (HSC) during HSC activation induced by arsenic (As). METHODS: Supernatant of human LO2 hepatocytes cultured with 100 μmol/L NaAsO₂ for 24 h was collected. Human HSC line LX-2 was cultured for 24 h before mingling culture supernatant of LO2 cells with arsenic exposure and normal culture media in a 1:4 ratio, and then treated with low dose (0.25 g/L) and high dose (1 g/L) of OM. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in culture medium were measured by biochemical method. The level of transforming growth factor-β1 (TGF-β1) in the culture supernatant of LO2 cells with arsenic exposure was detected by ELISA. The cell viability was examined by MTT assay. The protein levels of α-smooth muscle actin (α-SMA), autophagy-related gene 12 (Atg12), autophagy-related gene 5 (Atg5) and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blot. Lipid droplets were observed by oil red O staining under microscope. RESULTS: The levels of AST and ALT were obviously increased in the culture supernatant of LO2 cells with arsenic exposure (P<0.05). The viability of LX-2 cells was obviously enhanced while adding supernatant of LO2 cells with arsenic exposure into the culture media of LX-2 cells (P<0.05), the number of lipid drops decreased, and the expression of α-SMA was increased (P<0.05). Co-incubation with supernatant of LO2 cells and arsenic exposure obviously increased the expression of Atg12, Atg5, LC3-Ⅱ at protein level in the LX-2 cells as compared with control group (P<0.05). Low dose and high dose of OM inhibited the viability of LX-2 cells caused by co-incubation of the supernatant of LO2 cells with arsenic exposure (P<0.05) and decreased the protein expression of α-SMA, Atg12, Atg5 and LC3-Ⅱ in the LX-2 cells (P<0.05). High dose of OM was more effective in expression of LC3-Ⅱ than low dose (P<0.05). The number of lipid droplets in the LX-2 cells treated with OM was increased obviously as compared with the supernatant of LO2 cells with arsenic exposure. CONCLUSION: Arsenic exposure promotes the activation of HSC by damage of hepatocytes. Autophagy participates in the activation of HSC, and the mechanism is concerned with providing energy by degrading the lipid droplet of HSC. Oxymatrine intervention alleviates liver fibrosis by inhibiting autophagy and activation of HSC.     

新疆草鹅植物性神经的研究——Ⅰ.交感神经功能性形态

新疆草鹅的交感神经系统,前端起自两侧颈前神经节,后端止于第四对尾节,形成纵贯脊柱两侧的交感干;第二至十五颈椎两侧的交感干,完全在推动脉管内。胸部节间交感干,均分为两支围绕着肋骨头。椎旁神经节共有46对,其中颈节19对,胸节8对,腰荐节15对,尾节4对。椎前神经节共有9个,其中包括腹腔神经节2,肠系膜前神经节2,肾上腺神经节2,肾动脉神经节2,肠系膜后神经节1。另外,还有数目不定的中间神经节(包括直——迥肠节状神经干节)。交感干和椎旁神经节发出的纤维,除形成脏器相应的神经丛外,还沿主动脉形成了四个较大的神经丛,如腹腔神经丛、主动脉神经丛、腹下神经丛和肠系膜后神经丛等。     

穴位与内脏相关的初级传入联系途径

本文综述了穴位解剖组织学、针感及循经感传实质、穴位与内脏的初级传入神经在脊髓前、脊髓内汇聚以及穴位实质,穴位与内脏初级传入神经和针刺镇痛的关系。神经科学的研究表明,针感和内脏感觉的信号初级传入途径的研究,是探明穴位作用的特异性、内脏牵能与针刺镇痛机制的第一步,本文对研究和解释这个问题提供了可靠的形态学资料,为今后人们对内脏机能的深入研究提供了新的理论依据。     

GnRH受体在山羊结状神经节的分布

取山羊结状神经节5对,采用免疫组织化学SP法观察促性腺激素释放激素（Gonadotropin releasing hormon,GnRH）受体在结状神经节的分布特点。结果显示,GnRH受体在结状神经节上广泛分布,神经元、卫星细胞、神经纤维、血管肉皮细胞均有不同程度的免疫阳性染色。在神经元胞体中,细胞膜和细胞质有GnRH受体强阳性产物分布,核不着色;卫星细胞、血管内皮细胞也有强阳性产物分布;神经纤维存在弱阳性产物分布。图像分析结果表明,神经元中GnRH受体的相对表达量与其他非神经结构相比差异性显著（P〈0．05）。结果表明,山羊结状神经节中的GnRH受体主要存在于神经元中,山羊结状神经节中的内脏感觉传入神经元对GnRH具有反应性,提示GnRH结状神经节内脏感觉传入神经元的GnRH受体可能作为GnRH对内脏器官的内分泌调节和自主神经对内脏器官的神经调节这2种途径协调作用的节点。     

Effects of NGF on collagen secretion and morphology of hepatic stallete cells

AIM: To investigate the effects of nerve growth factor (NGF) on the changes of collagen secretion and morphology of hepatic stallete cells(HSCs). METHODS: Rat HSCs were incubated with different concentrations of NGF for 24 h. Collagen I and III in the supernatants of culture medium secreted by HSCs were detected by enzyme-linked immunosorbent assay. The morphological changes of HSCs were observed under inverted microscope with acridine orange staining and under transmission electronic microscope. RESULTS: When HSCs was incubated with NGF at concentrations of 100, 200 or 400 μg/L for 24 h, the content of collagen I and collagen III in the culture supernatants were significantly reduced compared with control group (P<0.05). After stimulated with NGF at the concentration of 100 μg/L for 24 h, the growth of the HSCs was inhibited and the morphous of the cells became round or oval gradually. The morphological changes of apoptotic cells were also observed by acridine orange staining and transmission electronic microscopy. CONCLUSION: NGF inhibits HSCs to synthesize collagen I and collagen III. Inhibition of collagen production and promotion of apoptosis in HSCs may be the possible mechanisms of NGF to reverse liver fibrosis.