牦牛颈上神经节对其生境适应的形态学机制

本研究旨在研究牦牛颈上神经节(SCG)对其生境适应的形态学特征,以探讨高原动物交感神经系统对青藏高原生态环境适应的形态学机制.运用常规HE及尼氏染色法,并采用形态计量学和SPSS16.0统计软件,对成年牦牛和黄牛SCG的形态学特征进行比较研究.结果表明:牦牛SCG的长、宽和厚度均显著地小于黄牛的相应数据(P＜0.01);牦牛的SCG主要由表面被膜、神经节单元、神经纤维、血管及结缔组织组成,被膜较黄牛的薄,结缔组织所占比例较黄牛的少,但节内组织较黄牛的致密;牦牛的神经节单元主要包含神经节细胞、卫星细胞、成纤维细胞、毛细血管和神经纤维,该组成特征与黄牛的相似;其神经节单元单个视野内节细胞、卫星细胞及成纤维等细胞与黄牛的基本相同(P=1),但牦牛节细胞间的神经纤维较黄牛的少,而血管数显著多于黄牛的(P＜0.01);牦牛SCG的头部以神经节单元为主,尾部以神经纤维为主,其特征与黄牛的相似.结果提示,在青藏高原高寒、低氧的极端生态环境中,牦牛在半放野、全年放牧的状态下,其SCG经长期进化形成了体积较小,神经节细胞、卫星细胞及其细胞间神经纤维较少,但血管十分丰富等形态学特征以适应极端生境.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.     

利用免疫组化方法初步鉴定雌性三疣梭子蟹视神经节GnRH受体

为了研究促性腺激素释放激素(GnRH)受体在甲壳动物视神经节中的存在及分布情况,采用MaxVisionTM免疫组织化学方法,以兔抗人GnRH受体的多克隆抗体,对不同发育期雌性三疣梭子蟹的视神经节进行了免疫组织化学定位。结果显示,GnRH受体的免疫阳性物质在视神经节的多个部位,视神经层、视外髓、视内髓、视端髓及X器中都有较为广泛的存在,在视外髓、视内髓与X器处的神经分泌细胞中尤为明显。不同发育期的雌性三疣梭子蟹视神经节GnRH受体的免疫阳性分布位置相似,免疫阳性强度存在一定的差异。三疣梭子蟹视神经节存在的GnRH受体免疫阳性物质,为GnRH参与视神经节调节作用提供了形态学依据。     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

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.     

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 EXPERIMENTAL NERVE ROOT COMPRESSION ON ARTERIAL BLOOD FLOW VELOCITY IN THE SEVENTH LUMBAR SPINAL GANGLION OF THE DOG: MEASUREMENT USING INTRAOPERATIVE DOPPLER ULTRASONOGRAPHY

Intraoperative Doppler ultrasonography was used to measure the effects of four experimental nerve root compression treatments (central compression, central-plus-lateral compression, lateral compression, and compression release) on arterial blood flow velocities in the seventh lumbar spinal ganglion of three dogs. Graphed blood flow velocity changes (change = treatment value − pretreatment value) were below baseline during the first three compression treatments and above baseline following compression release. Mean blood flow velocity changes for both central-plus-lateral compression and lateral compression differed (p ≤ 0.05) from changes for central compression. Changes for central-plus-lateral compression did not differ (p > 0.05) from changes for lateral compression. Changes among the first three compression treatments differed (p ≤ 0.05) from changes for compression release. No histologic abnormalities were identified in compressed nerve tissues, compared to contralateral controls. These findings indicate that stenosis within the L7-S1 intervertebral foramen may cause ischemia of the L7 spinal ganglion in dogs.     

青海省湟中县牦牛病毒性腹泻5种相关病原的检测与分析

为了掌握湟中县牦牛病毒性腹泻病原的流行现状,对湟中县内11个乡镇的138份腹泻牦牛粪便样品进行了牛病毒性腹泻病毒(BVDV)、牛肠道病毒(BEV)、牛轮状病毒(BRV)、牛冠状病毒(BCV)和牛星状病毒(BAstV) 5种病毒性腹泻致病原进行检测与分析。结果:BVDV、BEV、BRV、BCV和BAstV的平均感染率分别为44. 93%、21. 74%、8. 70%、5. 07%和6. 52%,5种病原中BVDV和BEV在所有乡镇均有流行,BRV、BCV、BAstV在部分乡镇呈散发性流行; 5种病原在1～6月龄犊牦牛中的检出率明显高于6月龄以上成年牦牛。138份腹泻牦牛中存在BVDV、BEV、BRV、BCV、BAstV的单独感染和混合感染,总单感率为53. 62%;共存在10种混感型,总混感率为15. 22%。表明湟中县牦牛存在BVDV、BEV、BRV、BCV和BAstV的感染,且混合感染情况复杂,应引起高度重视。