Ginsenoside Rb1 protects dopaminergic neurons from inflammatory injury induced by intranigral lipopolysaccharide injection

Accumulating studies suggest that neuroinflammation characterized by microglial overactivation plays a pivotal role in the pathogenesis of Parkinson’s disease.As such,inhibition of microglial overactivation might be a promising treatment strategy to delay the onset or slow the progression of Parkinson’s disease.Ginsenoside Rbl,the most active ingredient of ginseng,reportedly exerts neuroprotective effects by suppressing inflammation in vitro.The present study aimed to evaluate the neuroprotective and anti-inflammatory effects of ginsenoside Rbl in a lipopolysaccharide-induced rat Parkinson’s disease model.Rats were divided into four groups.In the control group,sham-operated rats were intraperitoneally administered normal saline for 14 consecutive days.In the ginsenoside Rbl group,ginsenoside Rb1(20 mg/kg)was intraperitoneally injected for 14 consecutive days after sham surgery.In the lipopolysaccharide group,a single dose of lipopolysaccharide was unilaterally microinjected into the rat substantial nigra to establish the Parkinson’s disease model.Lipopolysaccharide-injected rats were treated with normal saline for 14 consecutive days.In the ginsenoside Rbl +lipopolysaccharide group,lipopolysaccharide was unilaterally microinjected into the rat substantial nigra.Subsequently,ginsenoside Rbl was intraperitoneally injected for 14 consecutive days.To investigate the therapeutic effects of ginsenoside Rbl,behavioral tests were performed on day 15 after lipopolysaccharide injection.We found that ginsenoside Rbl treatment remarkably reduced apomorphine-induced rotations in lipopolysaccharide-treated rats compared with the lipopolysaccharide group.To investigate the neurotoxicity of lipopolysaccharide and potential protective effect of ginsenoside Rbl,contents of dopamine and its metabolites in the striatum were measured by high-performance liquid chromatography.Compared with the lipopolysaccharide group,ginsenoside Rbl obviously attenuated the lipopolysaccharide-induced depletion of dopamine and its metabolites in the striatum.To further explore the neuroprotective effect of ginsenoside Rbl against lipopolysaccharide-induced neurotoxicity,immunohistochemistry and western blot assay of tyrosine hydroxylase were performed to evaluate dopaminergic neuron degeneration in the substantial nigra par compacta.The results showed that lipopolysaccharide injection caused a large loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra and a significant decrease in overall tyrosine hydroxylase expression.However,ginsenoside Rb1 noticeably reversed these changes.To investigate whether the neuroprotective effect of ginsenoside Rbl was associated with inhibition of lipopolysaccharide-induced microglial activation,we examined expression of the microglia marker Iba-1.Our results confirmed that lipopolysaccharide injection induced a significant increase in Iba-1 expression in the substantia nigra;however,ginsenoside Rbl effectively suppressed lipopolysaccharide-induced microglial overactivation.To elucidate the inhibitory mechanism of ginsenoside Rb1,we examined expression levels of inflammatory mediators(tumor necrosis factor-a,interleukin-1β,inducible nitric oxide synthase,and cyclooxygenase 2)and phosphorylation of nuclear factor kappa B signaling-related proteins(IκB,IKK)in the substantia nigra with enzyme-linked immunosorbent and western blot assays.Our results revealed that compared with the control group,phosphorylation and expression of inflammatory mediators IκB and IKK in the substantia nigra of lipopolysaccharide group rats were significantly increased;whereas,ginsenoside Rbl obviously reduced lipopolysaccharide-induced changes on the lesioned side of the substantial nigra par compacta.These findings confirm that ginsenoside Rbl can inhibit inflammation induced by lipopolysaccharide injection into the substantia nigra and protect dopaminergic neurons,which may be related to its inhibition of the nuclear factor kappa B signaling pathway.This study was approved by the Experimental Animal Ethics Committee of Shandong University of China in April 2016(approval No.KYLL-2016-0148).

黑芥ANS基因的克隆和在芸薹属B基因组的PCR鉴别

利用同源克隆方法克隆黑芥ANS基因,获得2个ANS基因拷贝BnANS1和BnANS2,长度分别为1 366bp和1 367bp。这2个基因都有1个76bp的内含子。比较BnANS1和BnANS2基因序列,发现在编码区、5’与3’非编码区和内含子区域都有多态性,5’非编码区有3个碱基的多态性和4个碱基的缺失,编码区有26个碱基的多态性;内含子有2个碱基的多态性;3’非编码区有18个碱基的多态性和3个碱基的插入。这2个ANS拷贝都编码356个氨基酸的蛋白,具有其他物种同样的ANS蛋白保守结构域,属于2OG-Fe(II)加氧酶超家族。BnANS1编码的蛋白质理论分子量为40 448.58Da,等电点为5.05;BnANS2编码的蛋白质理论分子量为40 468.52Da,等电点为5.04。比较这2个ANS基因拷贝编码的蛋白质序列,发现有9个多态性位点。这2个ANS拷贝在黑芥的种皮和胚中均检测到表达。获得了1对ANS引物,能从白菜、黑芥、甘蓝、芥菜型油菜、甘蓝型油菜和埃塞俄比亚芥中,用等位特异PCR方法特异识别来自芸薹属B基因组的ANS基因。

补肾活血颗粒对帕金森病模型大鼠黑质纹状体bcl-2、bax表达的影响

目的探讨补肾活血颗粒对帕金森病模型大鼠黑质纹状体中B淋巴细胞瘤-2蛋白(B-cell lymphoma-2,bcl-2)、bax表达的影响。方法 33只雄性SD大鼠随机分为生理盐水正常组、6-羟基多巴胺模型组和补肾活血颗粒治疗组,应用免疫组织化学染色技术观察大鼠黑质纹状体中bcl-2、bax表达的变化情况。结果模型组bcl-2在黑质和纹状体的细胞阳性表达率增殖指数(proliferation index,PI)分别为0.43±0.022、1.14±0.057,均低于正常组(均P<0.01)和治疗组(均P<0.01)。模型组bax在黑质和纹状体的PI值分别为2.07±0.104、2.86±0.143,均高于正常组(均P<0.01)和治疗组(均P<0.01)。结论补肾活血颗粒治疗帕金森病的疗效机制可能与上调帕金森病大鼠黑质纹状体系统bcl-2蛋白表达及下调bax蛋白表达有关。

磷脂酰肌醇3激酶、蛋白激酶B、哺乳动物雷帕霉素靶蛋白在帕金森病模型大鼠黑质区的表达

目的观察磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路相关蛋白在帕金森病模型大鼠黑质区的表达及变化情况。方法 96只健康Sprague-Dawley大鼠随机分为假手术组、模型组、PI3K抑制剂LY294002组及p-m TOR抑制剂雷帕霉素组,每组又分为模型制备成功后4 d和8 d两个亚组,每个亚组12只。后三组颈背部皮下注射鱼藤酮制作帕金森病大鼠模型,相应时间点免疫组化及Western blotting检测大鼠黑质区PI3K、p-Akt、p-m TOR蛋白表达。结果与假手术组比较,模型组各时间点PI3K、p-Akt和p-m TOR蛋白表达增加(P<0.05),8 d亚组高于4 d亚组(P<0.05)。与模型组比较,LY294002组各时间点p-Akt、p-m TOR蛋白表达减少(P<0.05),PI3K蛋白表达无明显变化(P>0.05);雷帕霉素组各时间点p-m TOR蛋白表达下降(P<0.05),PI3K、p-Akt蛋白表达无明显变化(P>0.05)。结论黑质PI3K/Akt/m TOR信号通路激活可能是帕金森病发生、发展的重要机制之一。

埃塞俄比亚芥与诸葛菜属间杂种的基因组原位杂交分析

用幼胚培养方法重新获得的埃塞俄比亚芥BrassicacarinataA.Braun,2n=34与诸葛菜Orychophragmusviolaceus L.O.E.Schulz,2n=24的属间杂种仍为混倍体2n=14～34,2n=34细胞的频率最高;绝大多数花粉母细胞PMCs表现正常的17个二价体配对和17∶17的分离.基因组原位杂交分析结果表明,在所有体细胞和PMCs中不含有整条的诸葛菜染色体,2n=34的体细胞和PMCs中包含了来自黑芥B.nigra L.Koch,2n=16的16条染色体.这些具有完全或部分埃塞俄比亚芥染色体组成的细胞,可能来源于以前提出的杂种细胞在有丝分裂中完全或部分亲本染色体组分开和染色体复制,并伴随诸葛菜染色体的消除.

MPTP对小鼠不同脑区神经递质代谢酶基因表达的影响

1-甲基 -4 -苯基 -1,2 ,3 ,6-四氢吡啶 (MPTP)能够诱发黑质纹状体系统多巴胺能神经元损伤并产生与帕金森病类似的症状。本实验通过 MPTP注射诱导 C5 7BL小鼠神经元损伤 ,利用逆转录 PCR方法检测酪氨酸羟化酶 (TH)、单胺氧化酶 B(MAO-B)以及胆碱乙酰基转移酶 (Ch AT)三种神经递质代谢相关基因的表达变化。结果发现 ,在小鼠黑质与纹状体中 ,MPTP导致 TH基因表达显著降低 ,而 MAO-B与 Ch AT基因的表达变化在黑质与纹状体则有所不同 ,在黑质 MPTP导致 MAO-B与 Ch AT基因表达的上升 ,在纹状体则略有下降。

大鼠大脑皮层－尾壳核和尾壳核－黑质间的往返定位投射

用霍乱毒素Ｂ亚单位（ＣＢ）追踪法系统地研究了大鼠大脑皮层与尾壳核和尾壳核与黑质之间的定位投射。将ＣＢ分别泳入尾壳核不同节段的不同区域后，大脑皮层内的逆标胞体和顺标终末主要见于额叶１区和２区、顶叶１区和２区以及颗粒岛皮质；黑质内的顺标终末主要见于网状带的不同部位，而逆标胞体则主要见于致密带和腹侧被盖区。本研究结果说明在大鼠大脑皮层－尾壳核和尾壳核－黑质之间存在着且有点对点定位特征的往返联系。这种定位投射对躯体运动的精细调节提供了形态学基础。

大白菜和黑芥种间杂种的获得及鉴定

为丰富大白菜的抗病基因类型,特别是培育根肿病抗性种质,以品质优良的大白菜(Brassica campestrisL.pekinensis)自交系为母本,具有黑腐、根肿病抗性的野生黑芥(B.nigra)为父本,通过种间杂交,获得了13株杂种植株。利用形态学、细胞学和分子标记3种方法对杂种进行了鉴定,并分析了杂种的花粉活力及其育性。结果表明:杂种表型介于白菜和黑芥双亲之间,SRAP分子标记结果的聚类分析表明杂种在DNA水平上更趋向于白菜母本。F1雄蕊发育不好,花粉育性低,13株杂种中,仅获得杂种H4的回交后代。杂种H4的部分细胞染色体数目超过18条,约48%的细胞染色体数目为26条,超过了预期杂种染色体的数目。不育F1植株染色体数目等于和少于18条,结果提示杂种育性和细胞染色体数目有一定关系,染色体数目的增加提高了杂种的育性。

不同时程吗啡依赖对大鼠中脑黑质多巴胺神经细胞的影响

目的观察不同时程吗啡依赖对大鼠中脑黑质(Substantia nigra,SN)多巴胺能神经细胞的影响。方法按逐日递增原则背部皮下注射盐酸吗啡,建立吗啡依赖大鼠模型,取脑组织经10%福尔马林液固定后石蜡包埋,组织连续切片,免疫组织化学和免疫荧光染色,观察多巴胺能神经细胞特异性标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)蛋白表达变化;Fluoro-Jade B染色观察吗啡依赖不同时程黑质多巴胺能神经细胞变性坏死情况。结果 TH免疫组化结果显示随着吗啡依赖时间的延长黑质阳性细胞逐渐减少,吗啡依赖6周组阳性细胞减少明显。Fluoro-Jade B染色结果证实多巴胺能神经细胞发生了变性坏死。结论较长时程吗啡依赖大鼠黑质多巴胺能神经细胞损伤明显。