Lingo‐1 Inhibited by RNA Interference Promotes Functional Recovery of Experimental Autoimmune Encephalomyelitis

Lingo‐1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo‐1 expression, and the impact of Lingo‐1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo‐1 short hairpin RNA (LV/Lingo‐1‐shRNA) were constructed to inhibit Lingo‐1 expression. LV/Lingo‐1‐shRNA of different titers were transferred into myelin oligodendrocyte glycoprotein‐induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo‐1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining.LV/Lingo‐1‐shRNA administration via ICV injection could efficiently down‐regulate the Lingo‐1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P < 0.01), especially in the 5 × 10⁸ TU/mL and 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups. The locomotor function score in the LV/Lingo‐1‐shRNA treated groups were significantly lower than the untreated or LVCON053 group from day 7 on. The 5 × 10⁸ TU/mL LV/Lingo‐1‐shRNA group achieved the best functional improvement (0.87 ± 0.11 vs. 3.05 ± 0.13, P < 0.001). Enhanced myelination/remyelination was observed in the 5 × 10⁷, 5 × 10⁸, 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups by LFB staining (P < 0.05, P < 0.01, and P < 0.05).The data showed that administering LV/Lingo‐1‐shRNA by ICV injection could efficiently knockdown Lingo‐1 expression in vivo, improve functional recovery and enhance myelination/remyelination. Antagonism of Lingo‐1 by RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE. Anat Rec, 297:2356–2363, 2014. © 2014 Wiley Periodicals, Inc.     

On the benefit of galls of Quercus brantii Lindl. in murine colitis: the role of free gallic acid

Introduction

In this study we investigated the effect of gall of Quercus brantii Lindl., a traditional Iranian medicine, in a murine model of experimental colitis induced in male rats by rectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS).

Material and methods

Quantification of the main active components was done for estimation of total phenolic content and free gallic acid. Gall of Quercus brantii Lindl. in two forms (gall powder and gall hydro alcoholic extract) was gavaged for 10 days (500 mg/kg). Ten days after induction of colitis, colonic status was examined by macroscopic, microscopic and biochemical analyses. Colonic tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were analyzed as biomarkers of inflammatory condition. To determine the role of oxidative stress (OS) in colitis, the levels of cellular lipid peroxidation (LPO), total antioxidant power (TAP) and myeloperoxidase (MPO) were measured in colon tissues.

Results

TNBS-induced colitis exhibited a significant increase in colon MPO activity and concentrations of cellular LPO, TNF-α and IL-1β, while TAP was significantly reduced. Microscopic evaluations of the colonic damage in the colitis group revealed multifocal degenerative changes in the epithelial lining and areas of necrosis, extensive mucosal and sub-mucosal damage with congested blood vessels, edema and hemorrhages along with extensive infiltration of inflammatory cells. Parameters including macroscopic and microscopic scores, TNF-α, IL-1β, LPO, TAP and MPO improved by both gall extract and gall powder of Quercus brantii Lindl. and reached close to normal levels. The level of total phenols (GAE/100 g of sample) and free gallic acid were estimated to be 88.43 ±7.23 (mean ± SD) and 3.74% of dry weight, respectively.

Conclusions

The present study indicates that the gall of Quercus brantii Lindl. is able to exert antioxidative and anti-inflammatory effects on the biochemical and pathological parameters of colitis.     

免疫抑制剂干扰联合瘤细胞接种构建肝癌移植瘤小鼠模型

目的：探讨构建肝癌移植瘤动物模型的最佳方法。方法选用BALB/c小鼠160只,随机分为地塞米松组、肝癌细胞注射组、地塞米松加肝癌细胞注射组及空白对照组,每组40只,雌、雄各半。地塞米松组在小鼠腹腔内注射地塞米松;肝癌细胞注射组于不同时间在肝脏直接接种H22肝癌细胞株3次;地塞米松加肝癌细胞注射组将上述2种方法联合使用;空白对照组注射生理盐水。1个月后,颈椎脱臼处死小鼠,取其肝组织,计算肝指数,苏木精-伊红染色（HE）后观察肝组织病理变化。结果地塞米松组、空白对照组未发现肝组织癌变,肝癌细胞注射组、地塞米松加肝癌细胞注射组癌变的百分比分别为71.4%（20/28）、91.2%（21/23）。结论肝癌细胞直接接种小鼠肝脏及联合地塞米松干扰均可建立肝癌移植瘤动物模型,但联合地塞米松干扰建模的成功率明显升高。     

柠檬草水提物对高血糖模型小鼠降糖作用的实验研究

目的探讨柠檬草水提物对高血糖模型小鼠的降血糖作用。方法采用高脂饮食加小剂量链脲佐菌素建立高血糖小鼠模型,随机分为三组：高血糖模型组（模型组）、消渴丸组[给予1 g/（kg·d）消渴丸灌胃]、柠檬草水提物组[给予0.1 mL/（10 g·d）柠檬草水提物灌胃],每组8只,雌雄各半,连续给药28 d;同时随机抽取10只小鼠（雌雄各半）作为正常对照组。在实验的第7、14、21、28天测定各组小鼠的体质量和血糖值,在实验的第28天取小鼠肝、肾、脾、胸腺称其质量,比较各组小鼠体质量、血糖水平及内脏质量的变化。结果与正常对照组比较,模型组小鼠体质量下降,血糖升高,胸腺萎缩,胸腺质量下降,差异均有统计学意义（P〈0.05）。与模型组比较,消渴丸组和柠檬草水提物组小鼠体质量变化率减小,消渴丸组血糖值下降,差异均有统计学意义（P〈0.05）;柠檬草水提物组血糖值与模型组比较,差异无统计学意义（P〉0.05）。结论通过高脂饮食加链脲佐菌素成功复制了高血糖小鼠模型,与模型组比较,消渴丸对该模型小鼠的体质量下降和血糖升高有对抗作用;柠檬草水提物可对抗小鼠体质量的下降,但对造模引起的血糖升高无对抗作用。     

两种雌激素对去卵巢大鼠子宫内膜的影响

目的：探讨戊酸雌二醇和炔雌醇对去卵巢大鼠子宫内膜的作用,为绝经后妇女雌激素补充治疗提供理论依据。方法选用3月龄雌性Sprague-Dawley（ SD）大鼠40只,实验分为Sham组（假手术组）、Ovx组（去卵巢组）、A组（炔雌醇组）、B组（戊酸雌二醇组）。除Sham组外,其余各组均切除双侧卵巢建立骨质疏松动物模型。去势后4周开始A组炔雌醇（0.1 mg·kg-1·d-1）、B组戊酸雌二醇（0.4 mg·kg-1·d-1）灌胃。4组均在90 d对大鼠称重,取大鼠子宫进行称重,子宫内膜行病理切片检查。结果用药12周后各组间大鼠体重无统计学差异。 B 组子宫湿重及子宫指数明显低于A组（P〈0.05）,与Ovx组比较,无显著性差异（P〉0.05）。但A组子宫湿重及子宫指数明显升高,与Ovx组比较差异有统计学意义（P〈0.05）。两组用药组与Ovx组相比均显示子宫内膜增厚,上层单层柱状,肌层肌纤维排列规则一致,浆膜层光滑。 B组可见内膜增厚,但低于A组,两组均未见内膜增生过长、核异性和非典型增生样改变。结论戊酸雌二醇与炔雌醇用于绝经后雌激素补充治疗,虽对子宫内膜均有生长作用,但均未见过度生长及异型性改变。     

An ultrastructural study of chondroptosis: programmed cell death in degenerative intervertebral discs in vivo

Apoptosis has been regarded to mediate intervertebral disc degeneration (IDD); however, the basic question of how the apoptotic bodies are cleared in the avascular intervertebral disc without phagocytes, which are essential to apoptosis, remains to be elucidated. Our goals were to investigate the ultrastructure of nucleus pulposus (NP) cells undergoing chondroptosis, a variant of apoptotic cell death, in a rabbit annular needle‐puncture model of IDD. Experimental IDD was induced by puncturing discs with a 16‐G needle in New Zealand rabbits. At 4 and 12 weeks after puncture, progressive degeneration was demonstrated by X‐ray, magnetic resonance imaging and histological staining. TUNEL staining suggested a significant increase in the apoptosis index in the degenerated NP. However, the percentage of apoptotic cells with the classic ultrastructure morphology was much less than that with chondroptotic ultrastructure morphology under transmission electron microscopy (TEM). The chondroptotic cells from the early to late stage were visualized under TEM. In addition, the percentage of chondroptotic cells was significantly enhanced in the degenerated NP. Furthermore, ‘paralyzed’ cells were found in the herniated tissue. Western blotting revealed an increase in caspase3 expression in the degenerated NP. The expression of the Golgi protein (58K) was increased by the fourth week after puncture but decreased later. These findings indicate that chondroptosis is a major type of programmed cell death in the degenerated rabbit NP that may be related to the progressive development of IDD.