雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及mTOR信号通路的影响

为了探究雷帕霉素对糖尿病肾病大鼠足细胞生物学行为及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin,mTOR）信号通路的影响,采用链脲霉素腹腔注射构建糖尿病肾病大鼠模型,将正常大鼠体内取出的足细胞设为对照组,模型大鼠体内取出的足细胞设为糖尿病肾病模型组（DN组）,取2 mg·kg^-1雷帕霉素干预DN组足细胞,并将其设为雷帕霉素组（RAPA组）。采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐[3-（4,5-dimethylthiahiazo-z-y1）-2,5-diphenytetrazoliumromide,MTT]法检测足细胞增殖水平,Transwell检测细胞迁移和侵袭能力,流式细胞术检测细胞凋亡水平,Western blot法检测上皮-间充质转化标志物[E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）、波形纤维蛋白（vimentin）]、mTOR和核糖体S6激酶1（S6K1）蛋白表达水平。结果显示,与对照组相比,DN组细胞增殖水平显著被抑制,细胞迁移、侵袭水平显著升高,细胞凋亡率显著增加,上皮-间充转标志物E-cadherin表达显著下调,N-cadherin和Vimentin表达显著上调,mTOR/S6K1信号通路被显著活化（P<0.05）。与DN组相比,RAPA组细胞增殖水平显著升高,细胞迁移、侵袭水平显著降低,细胞凋亡率显著降低,E-cadherin表达显著上调,N-cadherin和Vimentin表达显著下调,mTOR和S6K1的蛋白表达显著被抑制（P<0.05）。结果表明,雷帕霉素通过抑制mTOR信号通路,促进足细胞体外增殖,抑制细胞迁移、侵袭、凋亡和上皮-间充质转化,发挥对糖尿病肾病大鼠足细胞的保护作用。     

Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats treated with 1.0 mg/ kg rapamycin exhibited fewer and milder side effects than those treated with 3.0 mg/kg. In summary, all these data suggest that there is not a rapamycin dose that can inhibit mTOR for epilepsy without causing any side effects, but 1 mg /kg may be the optimal dose for young rats for suppressing mTOR with relatively few side effects.     

丙泊酚对转化生长因子-β1诱导肝星状细胞激活的影响及其机制

目的: 观察丙泊酚对转化生长因子-β1(TGF-β1)诱导的肝星状细胞系HSC2-T6细胞激活的影响并探讨其可能的作用机制。方法: 实验分为对照组、TGF-β1组、丙泊酚组、TGF-β1+丙泊酚组、雷帕霉素组、TGF-β1+丙泊酚+雷帕霉素组。先用含雷帕霉素(5 μmol/L)DMEM培养液培养1 h,用丙泊酚(100 μmol/L)处理1 h,然后再加入TGF-β1(5 ng/ml)继续共同培养24 h。细胞的增殖水平通过MTT法测量,细胞培养液上清中透明质酸(HA)、IV型胶原(IV-C)和层粘连蛋白(LN)的浓度采用ELISA法测量,细胞的超微结构采用透射电镜观测,α-平滑肌肌动蛋白(α-SMA)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化mTOR(p-mTOR)及自噬相关基因Beclin 1、微管相关蛋白1轻链3(LC3)和p62的表达通过Western blot测量。结果: 与对照组比较,TGF-β1组细胞增殖、α-SMA蛋白的表达、培养液上清中HA、IV-C和LN的浓度、自噬体数量、Beclin-1和LC3-Ⅱ的蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值显著性增加(P均＜0.05),p-mTOR蛋白的表达和p-mTOR/mTOR比值及p62的蛋白表达显著性降低(P均＜0.05)。与TGF-β1组比较,丙泊酚+TGF-β1组细胞增殖、α-SMA蛋白的表达、培养液上清中HA、IV-C和LN的浓度、自噬体数量、Beclin-1和LC3-Ⅱ的蛋白表达及LC3-Ⅱ/LC3-Ⅰ比值均显著性降低(P均＜0.05),p-mTOR蛋白表达和p-mTOR/TOR比值及p62的蛋白表达均显著性增加(P均＜0.05)。mTOR抑制剂雷帕霉素部分逆转丙泊酚的作用。结论: 丙泊酚抑制TGFβ1诱导的肝星状细胞激活,其机制涉及mTOR-自噬途径。     

黄芪注射液对缺血性心肌病大鼠心肌自噬及心肌重塑的影响*

目的: 研究黄芪注射液对缺血性心肌病大鼠心肌重塑、网腔钙结合蛋白(calumenin)及自噬影响。方法: 36只雄性SD大鼠分为正常对照组(n=12)、缺血性心肌病组(n=12)、黄芪注射液组(n=12),3组大鼠术前行心电图及心脏彩超检查后,正常对照组不做任何处理,而缺血性心肌病组和黄芪注射液组大鼠开胸结扎冠状动脉20 min后,解开结扎线行再灌注后关闭胸腔建立心肌缺血模型,黄芪注射液组术后每次注射黄芪注射液10 g/kg体重,每周注射1次,共注射4次。3组大鼠术后4周行心脏彩超检查后处死大鼠取心脏行HE染色、VG染色,观察心肌病理改变,用Western blot技术检测各组大鼠心肌细胞calumenin、LC3-I、LC3-II表达变化及LC3-I /LC3-II比值变化。结果: 与缺血性心肌病组比较,黄芪注射液组大鼠心脏彩超及心肌病理得到明显改善;同时,calumenin表达增加LC3-I /LC3-II比值表达降低(P＜0.01)。结论: 黄芪注射液对缺血性心肌病大鼠心室重塑及心肌细胞自噬有明显抑制作用,该作用可能是通过calumenin所介导的。     

甲胺磷和17β－雌二醇对萼花臂尾轮虫实验种群动态的影响

对暴露于不同浓度的甲胺磷(0.01、0.1、1.0、10.0、100.0、1 000.0和10 000.0 μg·L-1)和17β-雌二醇(0.001、0.01、0.1、1.0、10.0、100.0、和1 000.0 μg·L-1)溶液中的萼花臂尾轮虫的种群动态进行了研究.结果表明:甲胺磷和17β-雌二醇对轮虫种群动态的影响存在差异.甲胺磷和17β-雌二醇对实验期间轮虫的平均种群增长率均有显著影响,但对轮虫种群中的混交雌体受精率的平均值无显著影响;甲胺磷对实验期间轮虫种群中的携卵雌体数/非携卵雌体数和混交雌体百分率的平均值均有显著影响,而17β-雌二醇则无显著影响;甲胺磷对实验期间轮虫的休眠卵总产量无显著影响,而17β-雌二醇的影响较显著.与对照相比,10.0～10 000.0 μg·L-1的甲胺磷和100.0 μg·L-1的17β-雌二醇均使轮虫的平均种群增长率显著提高;0.1～10 000.0和10.0 μg·L-1的甲胺磷分别显著地降低了实验期间轮虫种群中的携卵雌体数/非携卵雌体数和混交雌体百分率的平均值;1 000.0 μg·L-1的17β-雌二醇显著降低了轮虫的休眠卵总产量.在实验浓度范围内,轮虫平均种群增长率(Y,d-1)与甲胺磷浓度(X,μg·L-1)之间的关系为Y=-2×10-8X2 0.0002X 0.3474.种群增长率可用于监测和评价甲胺磷对轮虫种群动态的影响.     

四物汤在卵巢衰老大鼠骨骼肌中的雌激素样作用机制研究

摘要 目的：探讨四物汤通过胰岛素样生长因子-1（Insulin-ike growth factors-1，IGF-1）/磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（AKT）/雷帕霉素靶蛋白（Mammalian target of rapamycin，mTOR）mTOR信号通路发挥对4-乙烯基环己烯二环氧化合物（4-Vinylcyclohexene Diepoxide，VCD）诱导的卵巢衰老大鼠骨骼肌的保护作用及其分子机制。方法：选用28日龄雌性F-344大鼠，随机选取6只作为空白对照组，剩余大鼠连续腹腔注射VCD溶液（160 mg/kg/d）20天，每天阴道涂片检测动情周期，连续观察12 d无角化细胞或仅有少量角化细胞即符合"卵巢衰老"表现，经动情周期筛选出24只造模成功的大鼠，分为模型组（Model）、阳性（戊酸雌二醇，E₂）对照组、四物汤高剂量（SWT-H）组和四物汤低剂量（SWT-L）组，每组6只，灌胃持续3周后取材。取大鼠骨骼肌组织进行HE染色观察病理组织结构；MASSON染色骨骼肌纤维形态变化；RT-PCR检测骨骼肌组织中各基因的mRNA水平。结果：与空白组比较，模型组骨骼肌肌纤维排列疏松，分布不均且有断点，胞浆不均，细胞核增多，出现增生的结缔组织，胶原纤维逐渐增多且肌纤维间距变宽；与模型组相比，E₂组、SWT-H组与SWT-L组肌纤维排列相对整齐，胞浆较为均匀。肌肉形态较规则，纤维间距缩短，胶原纤维减少。RT-PCR结果显示，与空白组相比，模型组IGF-1、PI3K、AKT、mTOR基因的mRNA表达量明显下调，E₂组、SWT-H组与SWT-L组的IGF-1、PI3K、mTOR的表达明显上升，SWT-H组AKT表达无明显变化，无统计学意义，SWT-L组AKT表达相对降低。与模型组相比，E₂组、SWT-H组与SWT-L组的IGF-1、AKT、mTOR的表达明显增加，且具有剂量依赖性，PI3K表达增加，但无明显剂量依赖性。结论：四物汤可以明显改善VCD诱导的卵巢衰老大鼠的肌肉减少情况，其机制可能是通过IGF-1-PI3K-AKT-mTOR信号通路来发挥其抑制肌肉流失的作用。     

Fish oil up-regulates hepatic autophagy in rats with chronic ethanol consumption

In this study, we examined the regulation of autophagy by fish oil in rats under ethanol-containing diets. Thirty male Wistar rats (8-week-old) were divided into six groups and fed a control diet or an ethanol-containing diet, which was adjusted with fish oil to replace 25% or 57% of the olive oil. After 8 weeks, rats in the E (ethanol diet) group showed the significantly higher plasma aspartate transaminase (AST) and alanine transaminase (ALT) activities, protein expression of cytochrome P450 2E1 (CYP2E1), and levels of hepatic inflammatory cytokines. However, all of those items had significantly decreased in the EF25 (ethanol with 25% fish oil) and EF57 (ethanol with 57% fish oil) groups. As to autophagic indicators, protein expressions of mammalian target of rapamycin (mTOR), Unc-51-like autophagy activating kinase 1 (ULK1) and p62 were significantly increased in the E group. Conversely, the protein expressions of light chain 3II (LC3II)/LC3I and Beclin1 were significantly decreased in the E group. On the other hand, protein expressions of phosphorylated Akt, mTOR, ULK1, and p62 were down-regulated, protein expressions of LC3II/LC3I and Beclin1 were conversely up-regulated in the EF25 and EF57 groups. Fish oil activated hepatic autophagy via inhibiting the Akt signaling pathway, which exerted protective effects against ethanol-induced liver injury in rats.     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

丙戊酸钠协同硼替佐米对RPMI8226细胞的促凋亡作用

目的:以骨髓瘤细胞株RPMI8226为实验对象,观察丙戊酸钠(valproic acid,VPA)和硼替佐米(bortezomib,BZ)对此细胞株的增殖及凋亡的诱导情况。方法:实验分组如下:对照组,VPA单药组(1.0 mmol/L),BZ单药A组(10.0 nmol/L),BZ单药B组(20.0 nmol/L),BZ单药C组(35.0 nmol/L),联合用药A组(VPA1.0 mmol/L+BZ10.0 nmol/L),联合用药B组(VPA 1.0 mmol/L+BZ20.0 nmol/L),联合用药C组(VPA 1.0 mmol/L+BZ 35.0 nmol/L)。用MTT技术检测细胞增殖抑制情况;流式细胞仪检测凋亡比例。结果:丙戊酸钠与硼替佐米单用对RPMI8226细胞株细胞增殖有抑制作用,有细胞凋亡,但丙戊酸钠与硼替佐米协同用药A组、B组、C组增殖抑制可达75.1%及凋亡情况可达68.9%(P0.01)。结论:丙戊酸钠与硼替佐米协同用药后对RPMI8226细胞增殖抑制及诱导凋亡作用更显著,丙戊酸钠对硼替佐米有增敏作用。     

Increased leptin by hypoxic-preconditioning promotes autophagy of mesenchymal stem cells and protects them from apoptosis

Autophagy is the basic catabolic progress involved in cell degradation of unnecessary or dysfunctional cellular components.It has been proven that autophagy could be utilized for cell survival under stresses.Hypoxic-preconditioning(HPC)could reduce apoptosis induced by ischemia and hypoxia/serum deprivation(H/SD)in bone marrow-derived mesenchymal stem cells(BMSCs).Previous studies have shown that both leptin signaling and autophagy activation were involved in the protection against apoptosis induced by various stress,including ischemia-reperfusion.However,it has never been fully understood how leptin was involved in the protective effects conferred by autophagy.In the present study,we demonstrated that HPC can induce autophagy in BMSCs by increased LC3-II/LC3-I ratio and autophagosome formation.Interestingly,similar effects were also observed when BMSCs were pretreated with rapamycin.The beneficial effects offered by HPC were absent when BMSCs were incubated with autophagy inhibitor,3-methyladenine(3-MA).In addition,down-regulated leptin expression by leptin-shRNA also attenuated HPC-induced autophagy in BMSCs,which in turn was associated with increased apoptosis after exposed to sustained H/SD.Furthermore,increased AMP-activated protein kinase phosphorylation and decreased mammalian target of rapamycin phosphorylation that were observed in HPC-treated BMSCs can also be attenuated by down-regulation of leptin expression.Our data suggests that leptin has impact on HPC-induced autophagy in BMSCs which confers protection against apoptosis under H/SD,possibly through modulating both AMPK and mTOR pathway.     

骨髓基质干细胞移植改善慢性酒精中毒大鼠脑损害的相关机制研究

目的:探讨骨髓基质干细胞(bone marrow stormal cells, BMSCs)静脉移植对慢性酒精中毒大鼠脑保护作用的相关机制。方法:体外分离、培养、扩增SD大鼠BMSCs。成年雄性SD大鼠随机分为慢性酒精中毒组、BMSCs回输组、磷酸缓冲盐溶液(phosphate buffer saline,PBS)回输组和对照组,每组7只。前三组用酒精灌胃8周建立慢性酒精中毒动物模型,对照组不造模(给予蔗糖灌胃),BMSCs回输组和PBS回输组于造模7周时一次性经尾静脉回输BMSCs或PBS。免疫印迹法检测海马Bcl-2、Bax、NGF、BDNF以及信号转导分子p-Akt的表达;反转录PCR检测海马神经生长因子(nerve growth factor, NGF)和脑源性神经营养因子(brain derived neurotrophic factor, BDNF)。结果:BMSCs回输组海马抗凋亡蛋白Bcl-2表达高于其余三组(P0.05);促凋亡蛋白Bax表达低于慢性酒精中毒组(P0.01),与对照组无统计学差异(P=0.989)。BMSCs回输组鼠海马内NGF和BDNF m RNA和蛋白表达、p-Akt蛋白表达均高于其余三组(P0.05)。结论:静脉移植BMSCs能够明显改善慢性酒精中毒大鼠海马的细胞凋亡;其可能与自或旁分泌BDNF和NGF营养因子有关,且可能部分是通过激活PI3K/Akt通路实现。     

Rapamycin Ameliorates Inflammation and Fibrosis in the Early Phase of Cirrhotic Portal Hypertension in Rats through Inhibition of mTORC1 but Not mTORC2

Objective

Hepatic stellate cells (HSCs) transdifferentiation and subsequent inflammation are important pathological processes involved in the formation of cirrhotic portal hypertension. This study characterizes the pathogenetic mechanisms leading to cholestatic liver fibrosis and portal hypertension, and focuses on mammalian target of rapamycin (mTOR) pathway as a potential modulator in the early phase of cirrhotic portal hypertension.

Methods

Early cirrhotic portal hypertension was induced by bile duct ligation (BDL) for three weeks. One week after operation, sham-operated (SHAM) and BDL rats received rapamycin (2 mg/kg/day) by intraperitoneal injection for fourteen days. Vehicle-treated SHAM and BDL rats served as controls. Fibrosis, inflammation, and portal pressure were evaluated by histology, morphometry, and hemodynamics. Expressions of pro-fibrogenic and pro-inflammatory genes in liver were measured by RT-PCR; alpha smooth muscle actin (α-SMA) and antigen Ki67 were detected by immunohistochemistry; expressions of AKT/mTOR signaling molecules, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, and interleukin-1 beta (IL-1β) were assessed by western blot.

Results

The AKT/mTOR signaling pathway was markedly activated in the early phase of cirrhotic portal hypertension induced by BDL in rats. mTOR blockade by rapamycin profoundly improved liver function by limiting inflammation, fibrosis and portal pressure. Rapamycin significantly inhibited the expressions of phosphorylated 70KD ribosomal protein S6 kinase (p-P70S6K) and phosphorylated ribosomal protein S6 (p-S6) but not p-AKT Ser473 relative to their total proteins in BDL-Ra rats. Those results suggested that mTOR Complex 1 (mTORC1) rather than mTORC2 was inhibited by rapamycin. Interestingly, we also found that the level of p-ERK1/2 to ERK1/2 was significantly increased in BDL rats, which was little affected by rapamycin.

Conclusions

The AKT/mTOR signaling pathway played an important role in the early phase of cirrhotic portal hypertension in rats, which could be a potential target for therapeutic intervention in the early phase of such pathophysiological progress.     

Effects of SDF-1/CXCR4 on the Repair of Traumatic Brain Injury in Rats by Mediating Bone Marrow Derived Mesenchymal Stem Cells

Our study aims to investigate the effects of the SDF-1/CXCR4 axis on the repair of traumatic brain injury (TBI) in rats by mediating bone marrow derived from mesenchymal stem cells (BMSCs). Healthy male SD rats were collected, their tibiofibulars were removed, cultured, and BMSCs were collected. The expression of cell-surface molecular proteins was examined using flow cytometry. The mRNA and protein expression of CXCR4 in cells were tested using qRT-PCR and western blotting analysis. An electronic brain injury instrument was utilized to build TBI rat models and each rat was assigned into the experiment, positive control and control groups (10 rats in each group). The morris water maze was used to calculate the escape latency and number of times rats in each group crossed the platform. Neurological severity scores (NSS) was calculated to evaluate the recovery of neurological functioning. The distribution of neuronal nuclear antigens was detected using double-labeling immunohistochemistry. The morphological changes in the hippocampal neuronal and the number of BrdU-positive cells were observed through Nissl’s staining and high magnification. The mRNA and protein expressions of CXCR4 were gradually increased as SDF-1 concentration increased. NGF and BDNF positive cells were expressed in each group. The distribution of neuronal nuclear antigens in the experiment group was elevated compared to the control and positive control groups. Among the three groups, the experimental group had the shortest escape latency and the highest number platform crossings. The difference in NSS among the three groups was significant. The experimental group had better cell morphology and a higher number of BrdU-positive cells than the other groups. The present study demonstrates that transplanting BMSCs with SDF-1-induced CXCR4 expression can promote the repair of TBI. This is expected to become a new treatment regimen for TBI.     

耐力运动对增龄大鼠脑皮层突触可塑性的影响及相关调控机制*

目的:探讨规律性耐力运动对脑皮层增龄性老化适应性的作用与机制。方法:将三个不同年龄段的健康SPF级雄性Sprague-Dawley大鼠分为3月龄 (青年,n=20)、13月龄 (中年,n=24)和23月龄 (老年,n=24)组,每组又随机分为静息组和运动组;静息组三组静息,运动组三组实施10周递增负荷规律的中等强度耐力运动:运动方式为跑台运动(坡度0),运动强度从最大摄氧量(V·O2max) 60%～65%逐渐递增到70%～75%,运动时间为10周;取大鼠脑皮层,HE染色测试大鼠脑皮层增龄性形态学变化,检测BDNF和SOD的蛋白表达及突触素-1(SYN1)和CaMK IIα/AMPKα1/ mTOR通路等相关基因。结果:静息各组大鼠的脑皮层结构呈现年龄增龄性衰老变化,脑皮层SOD表达呈增龄性下降趋势,BDNF表达变化呈增龄性上升趋势,SYN1和CaMK IIα表达水平随增龄性趋势变化不大,AMPKα1和SirT2以及 IP3R、AKT1、mTOR mRNA表达水平随年龄变化呈现中年略上升而老年下降趋势;与静息各组大鼠相比,运动各组大鼠脑皮层神经细胞核排列紧密有序,显微镜下观察细胞核的数量明显增加,运动促进大鼠脑皮层SOD、BDNF和突触素SYN1表达水平增加,其中老年大鼠SOD、BDNF表达水平显著上调(P＜0.01),青年和老年大鼠SYN1表达水平显著上调(P＜0.05),运动上调中年和老年大鼠脑皮层CaMK IIα表达水平上调(P＜0.01),而对青年大鼠CaMK IIα表达水平却是下调(P＜0.01),运动可上调青年大鼠脑皮层的AMPKα1表达水平(P＜0.05),而对中年和老年大鼠AMPKα1的影响不显著,运动均可上调各年龄大鼠脑皮层的SirT2表达水平(P＜0.05),运动上调各年龄大鼠脑皮层的IP3R/AKT1/ mTOR表达水平,其中青年IP3R显著上调(P＜0.01),青年和中年mTOR显著上调(P＜0.01),老年mTOR也显著上调(P＜0.05)。结论:耐力运动通过上调BDNF的表达水平,调控CaMK IIα信号、激活AMPK信号通路和IP3R/AKT1/mTOR信号通路,改善脑皮层的突触可塑性。     

缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用*

目的:研究缺血性心肌病大鼠心肌细胞自噬在心肌重塑中的作用。方法:36 只雄性SD大鼠分为正常对照组、假手术组、缺血性心肌病组( n=12),3组大鼠术前行心脏彩超检查,正常对照组大鼠不进行处理;假手术组大鼠开胸后不结扎冠状动脉,关闭胸腔;缺血性心肌病组大鼠开胸结扎冠状动脉20 min后,解开结扎线行再灌注后关闭胸腔,3组大鼠术后4周行心脏彩超检查后处死大鼠取心脏行HE染色、masson染色,观察心肌病理改变,用Western blot技术检测各组大鼠心肌细胞GRP78、LC3-I、LC3-II、Beclin-I表达及LC3-II/LC3-I比值的变化。结果:与正常组及假手术组比较,缺血性心肌病大鼠心室扩大,EF值降低;心肌排列紊乱,心肌纤维化增加;线粒体空泡化严重;内质网应激关键蛋白GRP78上调;自噬相关蛋白LC3-I、LC3-II、Beclin-I及LC3-II/LC3-I比值增加。结论:缺血性心肌病大鼠心肌细胞中内质网应激和自噬可能在心肌重塑中具有重要作用。     

玉米赤霉烯酮在小鼠体内的致突变性研究

用小鼠骨髓细胞微核试验和染色体畸变试验评价了赤霉病麦毒素之一玉米赤霉烯酮的体内致突变效应。玉米赤霉烯酮的试验剂量为0,0.1,1.0,10.0,100.0mg/kg体重,以环磷酰胺为阳性对照。实验结果表明,玉米赤霉烯酮各试验剂量组的微核发生率和染色体畸变率与阴性对照组相比均无显著性统计学差异,亦未发现有明显的性别差异。     

玉米赤霉烯酮在小鼠体内的致突变性研究*

用小鼠骨髓细胞微核试验和染色体畸变试验评价了赤霉病麦毒素之一玉米赤霉烯酮的体内致突变效应。玉米赤霉烯酮的试验剂量为0,0.1,1.0,10.0,100.0mg/kg体重,以环磷酰胺为阳性对照。实验结果表明,玉米赤霉烯酮各试验剂量组的微核发生率和染色体畸变率与阴性对照组相比均无显著性统计学差异,亦未发现有明显的性别差异。     

骨髓间充质干细胞(BMSCs)移植于大鼠肝硬化模型的动物实验研究

目的:探讨骨髓间充质干细胞应用于肝硬化模型的的实验疗效,为临床试验提供数据。方法:制作大鼠肝硬化模型,将肝硬化大鼠随机分为两组,实验组和对照组,每组25只。将骨髓间充质干细胞通过门静脉注射入实验组大鼠肝脏,术后两组大鼠仍然以0.5mL/100g的维持剂量腹腔注射40%CCl4植物油溶液,3周后处死所有实验动物,取其静脉血检测ALT、TBIL、ALB、AFP,并取大鼠肝脏做组织切片观察。结果:实验大鼠肝脏可观察到诱导分化的肝细胞。实验组大鼠死亡3只,对照组死亡2只。对照组与实验组肝功能指标ALT、ALB、TBIL、AFP有显著差异,P<0.05,具有统计学意义,AST差异无统计学意义,P>0.05。结论:BMSCs注入肝硬化大鼠模型中能有效改善肝脏的生理功能,对临床试验有很好的指导作用。     

Effects of plasmin on sperm-oocyte interactions during in vitro fertilization in the pig

The present study was undertaken to examine the effect of plasmin on sperm viability and sperm-oocyte interaction during in vitro fertilization in the pig. Porcine sperm, which were washed in Dulbecco's PBS were re-suspended and incubated in fertilization medium (mTBM; modified Tris-buffered medium) containing 0, 0.1, 1.0, 10.0 or 100.0ng/mL of plasmin. Sperm viability was not affected by plasmin treatment. Addition of plasmin in doses ranging from 0.1 to 100.0ng/mL for 2, 4 or 6h to washed boar sperm resulted in enhancement of acrosome reaction (AR) compared with untreated cells. The concentration of 0.1ng/mL plasmin (95+/-18 sperm/oocyte) had no effect on sperm binding, whereas 1.0ng/mL (123+/-21 sperm/oocyte), 10.0ng/mL (124+/-16 sperm/oocyte) and 100.0ng/mL (124+/-15 sperm/oocyte) of plasmin increased sperm binding compared with the control (83+/-15 sperm/oocyte). The zona pellucida solubility (zona dissolution time) was less in medium with 1.0ng/mL (123+/-24s), 10.0ng/mL (99+/-15s) or 100.0ng/mL (95+/-19s) plasmin compared with control (176+/-27s). When pig oocytes and sperm were co-incubated in various concentrations of plasmin for 6h, the penetration rate was greater in medium with 1.0ng/mL plasmin (77.5+/-3.1%) compared with the control. However, there were no differences in the polyspermic rates and mean number of sperm (MNS)/oocyte between the groups treated with plasmin and control. These results suggest that plasmin might play a role in events related to fertilization.     

Rapamycin preserves the primordial follicle pool during cisplatin treatment in vitro and in vivo

Rapamycin has been proven to effectively inhibit the activation of primordial follicles while cisplatin‐induced the loss of primordial follicles due to the over‐activation of the primordial follicle stockpile. Whether rapamycin could inhibit the loss of primordial follicles induced by cisplatin is still unknown. The ovaries of neonatal Sprague Dawley rats were cultured in vitro in different doses of rapamycin (0.08, 0.16, and 0.32 μg/ml) and cisplatin (0.1, 0.4, and 0.8 μg/ml). The immature BALB/c mice were administered cisplatin with or without rapamycin by intraperitoneal injection. Ovaries were collected to analyze the histomorphology, the messenger RNA (mRNA) expression of anti‐Mullerian hormone (AMH), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) and the expression of key proteins of mammalian target of rapamycin (mTOR) pathway. Growing follicle counts of ovaries cultured in vitro in the R0.16 and R0.32 groups were decreased and the ratio of growing to primordial follicles was also decreased in a dose‐dependent manner. In the C0.8 group, growing follicles were decreased compared with the other groups while the ratio was substantially increased in the C0.4 and C0.8 group. Co‐treatment attenuated primordial follicle loss and reduced the upregulated ratio induced by cisplatin. Ovarian follicle dynamics in vivo was consistent with the in vitro results. Primordial follicles counts were statistically increased and the ratio was reduced in the rapamycin group compared with the control group. Primordial follicle counts were dramatically reduced in the cisplatin group whereas co‐treatment with rapamycin slightly recovered its counts. There was no obvious difference in the number of growing follicles between the cisplatin group and other groups. The ratio was significantly increased in cisplatin‐treated mice whereas decreased in the co‐treatment group. The apoptosis rate of antral follicles in cisplatin‐treated mice was higher than the other groups while the apoptosis rate was decreased in the co‐treatment group in vivo. Compared with the control and rapamycin group, the mRNA expression of AMH, GDF9, and BMP15 were downregulated in the cisplatin group. The co‐treatment group recovered the mRNA expression of BMP15. In addition, the expression of key protein of mTOR pathway rpS6 and its phosphorylated forms were increased in the cisplatin‐treated group while co‐treatment decreased their expression. Rapamycin attenuated the loss of primordial follicles induced by cisplatin through the inhibitory effect of rapamycin on the mTOR pathway. These results suggest that rapamycin may be an effective drug for the protection of ovarian function during chemotherapy.