新藤黄酸通过调控PI3K/Akt/mTOR信号通路诱导黑色素瘤B16细胞凋亡的研究

目的:探讨新藤黄酸诱导黑色素瘤B16细胞凋亡的机制。方法:MTT试验检测新藤黄酸对B16细胞增殖抑制的作用;采用Hochest 33258荧光染色观察新藤黄酸对B16细胞的影响;透射电镜观察新藤黄酸对B16细胞的超微结构的改变;Western blot法检测PI3K,p-PI3K,Akt,p-Akt,p-mTOR,PTEN蛋白的变化以探讨新藤黄酸诱导B16细胞凋亡的分子机制。结果:新藤黄酸对黑色素瘤细胞B16生长和增殖有明显地抑制作用,并随着新藤黄酸浓度的增加和作用时间的延长细胞活力明显下降;Hochest 33258染色结果显示,新藤黄酸处理的细胞中呈现明显的凋亡特征;透射电镜观察发现新藤黄酸作用B16细胞后,细胞发生明显凋亡的形态学变化;Western blot检测表明p-PI3K蛋白表达呈时间依赖性下降;p-Akt蛋白表达呈时间依赖性下降,而PI3K,Akt蛋白表达量基本不变,p-mTOR蛋白的表达随着时间的延长有所下降,PTEN蛋白的表达呈时间依赖性增加。结论:新藤黄酸在一定的时间和浓度范围内能够抑制黑色素瘤B16细胞的增殖,诱导细胞凋亡,其诱导细胞凋亡的机制可能与调控PI3K/Akt/mTOR信号通路有关。     

老鼠簕生物碱A对肝纤维化大鼠PI3K/Akt/m TOR/p70S6K信号通路的影响

目的观察老鼠簕生物碱A(HBOA)对四氯化碳(CCl4)致肝纤维化大鼠磷酸肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)/p70核糖体蛋白S6激酶(p70S6K)信号通路的影响,探讨HBOA抗肝纤维化的作用机制。方法大鼠随机分成对照组、模型组及HBOA高、中、低剂量(100、50、25 mg/kg)组和秋水仙碱(0.4 mg/kg)组。除对照组外,其余各组ig给予50%CCl4橄榄油溶液,每周2次,连续12周,诱导肝纤维化大鼠模型。于造模第9周起,给药组分别ig相应的受试药物,每天1次,给药4周。实验结束后,计算各组大鼠体质量变化、肝脏指数;检测各组大鼠肝匀浆中丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)活性;蛋白免疫印迹法(Western blotting)法检测肝组织P-PI3K、p-Akt、p-mTOR、p-p70S6K蛋白的表达。结果与模型组比较,各给药组大鼠体质量增加量显著升高,均能降低肝脏指数及肝组织中ALT、AST活性;此外,HBOA高、中剂量组均能显著抑制p-PI3K、p-Akt、p-m TOR、p-p70S6K蛋白的表达。结论 HBOA对肝纤维化大鼠具有一定的保护作用,其机制可能与抑制PI3K/Akt/m TOR/p70S6K信号通路有关。     

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats

ObjectiveTo investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs).MethodsEight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta.ResultsEA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects.ConclusionEA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.     

基于PI3K/Akt/eNOS信号通路探讨补阳还五汤对大鼠蛛网膜下腔出血后脑血管痉挛的影响

目的:探讨补阳还五汤改善蛛网膜下腔出血后脑血管痉挛的相关分子机制。方法:将雄性SD大鼠80只随机分为假手术组、模型组、补阳还五汤低、高剂量组(13,26 g·kg~(-1)·d~(-1)),每组20只。按照10 mL·kg~(-1)每天灌胃给药2次,连续7 d。采用二次枕大池注血法制作蛛网膜下腔出血模型,评估各组大鼠1,3,5,7 d的神经功能评分,利用苏木素-伊红(HE)染色对各组大鼠基底动脉直径进行测量,蛋白免疫印迹法(Western blot)检测基底动脉脑组织磷酸化磷脂酰肌醇3-激酶(p-PI3K),磷酸化蛋白激酶B(p-Akt),内皮型一氧化氮合酶(eNOS),神经型一氧化氮合酶(n NOS)蛋白的表达;酶联免疫吸附测定(ELISA)检测大鼠脑脊液中一氧化氮(NO),内皮素-1(ET-1)的表达。结果:与假手术组比较,模型组大鼠神经功能评分明显下降(P 0. 05),基底动脉直径明显缩小(P 0. 05),基底动脉脑组织p-PI3K,p-Akt,eNOS,n NOS蛋白的表达明显降低(P 0. 05),脑脊液中NO明显减少(P 0. 05),ET-1明显升高(P 0. 05);与模型组比较,不同剂量补阳还五汤组(26,13 g·kg~(-1)·d~(-1))在治疗后3～5 d后大鼠神经功能评分均有不同程度升高,基底动脉直径明显增宽(P 0. 05),p-PI3K,p-Akt,eNOS蛋白的表达明显升高(P 0. 05),NO明显升高(P 0. 05),ET-1明显下降(P 0. 05);与低剂量组比较,经过7 d的补阳还五汤治疗后高剂量组神经功能评分明显升高(P 0. 05),基底动脉直径增宽更明显(P 0. 05),p-PI3K,p-Akt,eNOS蛋白的表达更高(P 0. 05),NO升高更明显(P 0. 05),ET-1下降更明显(P 0. 05)。结论:补阳还五汤对蛛网膜下腔出血后脑血管痉挛的保护作用可能与上调PI3K/Akt/eNOS信号通路中的p-PI3K,p-Akt,eNOS的表达,从而增加NO产生有关。     

罗布麻叶提取物的抗高血压作用及其机制研究

目的: 研究罗布麻叶提取物(extracts from leaves of Apocynum venetum,ELA)的抗高血压作用及其激活磷脂酰肌醇3激酶(PI3K)和(或)蛋白激酶B(Akt)信号转导通路促进一氧化氮释放的作用。 方法: 采用双肾双夹法建立Beagle犬高血压模型,随后按血压值将Beagle犬随机分为模型组(ig,生理盐水)、马来酸依那普利组(ig,1.66 mg·kg^-1)、ELA低剂量组(ig 23 mg·kg^-1)、高剂量组(ig ELA 46 mg·kg^-1)。受试动物在单次给予上述药物后,每隔1 h记录1次动物血压,连续8 h,观察ELA的降压作用;采用细胞培养法,观察ELA对内皮细胞(EAhy 926)一氧化氮(NO)产量和内皮型一氧化氮合酶(eNOS)活性的影响;采用Western blot法,观察ELA对eNOS,磷酸化内皮型一氧化氮合酶(p-eNOS)、磷酸化磷脂酰肌醇3激酶(p-PI3K)以及磷酸化蛋白激酶B (p-Akt)表达量的影响。 结果: 给药后,ELA高、低剂量组Beagle犬收缩压分别为(140.4±7.5),(136.2±6.8) mmHg;舒张压分别为(63.5±3.3),(72.2±4.4) mmHg;平均血压分别为(88.9±4.7),(93.3±5.2)mmHg,与给药前相比均显著降低(P<0.01);ELA可显著增加EA Hy926细胞NO的释放量(P<0.01),提高eNOS活性(P<0.01);Western blot结果表明,ELA可显著增加p-eNOS,p-PI3K以及p-Akt的表达量,与对照组相比,具有显著差异(P<0.01)。 结论: ELA具有明显的降压作用。激活血管内皮细胞内PI3K/Akt信号转导途径,从而促进eNOS磷酸化,提高eNOS活性、增加NO可能是ELA降压作用的机制之一。     

益气扶正复方联合依维莫司对三阴性乳腺癌MDA-MB-468细胞株裸鼠移植瘤的影响

目的研究益气扶正复方联合依维莫司对三阴性乳腺癌MDA-MB-468细胞株裸鼠移植瘤的抑制作用及其作用机制。方法体外培养三阴性乳腺癌MDA-MB-468细胞株,将细胞悬液接种于雌性BALB/C裸鼠第二乳腺乳垫处,建立皮下移植瘤模型。将造模成功的裸鼠随机分为对照组、益气扶正复方(中药复方)组(40mg/kg)、依维莫司组(2mg/kg)和联合组,每组6只。各组灌胃给予相应药物干预,连续30d。比较各组移植瘤体积和质量的变化,计算各组肿瘤抑制率。采用Real-time PCR检测各组mTor、Akt、P70s6k、4Ebp-1 mRNA的表达水平,Western blot检测各组Akt、p-Akt、m-TOR、p-mTOR、PTEN及mTOR下游底物p-4EBP-1、p-P70S6K的蛋白表达水平。结果与中药复方组相比,联合组移植瘤体积和质量显著减小(P0.05,P0.01),肿瘤抑制率明显升高,且联合组mTor和P70s6k mRNA表达及p-mTOR和P70S6K蛋白表达水平明显低于中药复方组,而联合组PTEN蛋白表达高于中药复方组(P0.05)。结论益气扶正复方与依莫维司联用能抑制MDA-MB-468荷瘤裸鼠的肿瘤组织生长,与益气扶正复方单用相比效应更为显著,其作用机制可能与抑制p-mTOR、p-Akt、p-P70S6K活性,提高p-4EBP-1、PTEN表达有关。     

Qili Qiangxin,a compound herbal medicine formula,alleviates hypoxia-reoxygenation-induced apoptotic and autophagic cell death via suppression of ROS/AMPK/mTOR pathway in vitro

ObjectiveQili Qiangxin (QLQX), a compound herbal medicine formula, is used effectively to treat congestive heart failure in China. However, the molecular mechanisms of the cardioprotective effect are still unclear. This study explores the cardioprotective effect and mechanism of QLQX using the hypoxia-reoxygenation (H/R)-induced myocardial injury model.MethodsThe main chemical constituents of QLQX were analyzed using high-performance liquid chromatography-evaporative light-scattering detection. The model of H/R-induced myocardial injury in H9c2 cells was developed to simulate myocardial ischemia–reperfusion injury. Apoptosis, autophagy, and generation of reactive oxygen species (ROS) were measured to assess the protective effect of QLQX. Proteins related to autophagy, apoptosis and signalling pathways were detected using Western blotting.ResultsApoptosis, autophagy and the excessive production of ROS induced by H/R were significantly reduced after treating the H9c2 cells with QLQX. QLQX treatment at concentrations of 50 and 250 μg/mL caused significant reduction in the levels of LC3II and p62 degradation (P < 0.05), and also suppressed the AMPK/mTOR signalling pathway. Furthermore, the AMPK inhibitor Compound C (at 0.5 μmol/L), and QLQX (250 μg/mL) significantly inhibited H/R-induced autophagy and apoptosis (P < 0.01), while AICAR (an AMPK activator, at 0.5 mmol/L) increased cardiomyocyte apoptosis and autophagy and abolished the anti-apoptotic effect of QLQX. Similar phenomena were also observed on the expressions of apoptotic and autophagic proteins, demonstrating that QLQX reduced the apoptosis and autophagy in the H/R-induced injury model via inhibiting the AMPK/mTOR pathway. Moreover, ROS scavenger, N-Acetyl-L-cysteine (NAC, at 2.5 mmol/L), significantly reduced H/R-triggered cell apoptosis and autophagy (P < 0.01). Meanwhile, NAC treatment down-regulated the ratio of phosphorylation of AMPK/AMPK (P < 0.01), which showed a similar effect to QLQX.ConclusionQLQX plays a cardioprotective role by alleviating apoptotic and autophagic cell death through inhibition of the ROS/AMPK/mTOR signalling pathway.     

连翘苷经PI3K/Akt信号通路干预肾细胞癌的机制研究

目的探讨连翘苷抑制人肾细胞腺癌786-0细胞生长、迁移及侵袭的作用机制。方法体外培养786-0细胞,在其中加入不同浓度连翘苷进行干预。MTT法检测细胞生存活力,AO/EB法检测细胞凋亡,划痕实验与Transwell实验分别考察细胞迁移、侵袭能力。Western blotting法检测细胞内PI3K、p-PI3K、Akt、p-Akt、FOXO3a、p-FOXO3a、p21、p27、Fasl、Bim、MMP-2及MMP-9蛋白表达情况。结果连翘苷可有效抑制肾癌细胞生长、促进凋亡,干扰细胞周期,上调p21、p27、Fasl及Bim表达水平,与对照组比较差异明显(P0.05、0.01);与对照组比较,不同浓度连翘苷可明显抑制肾癌细胞迁移与侵袭,减少MMP-2、MMP-9合成(P0.05、0.01);同时连翘苷能明显抑制PI3K、Akt、FOXO3a磷酸化(P0.05、0.01),且呈浓度依赖性。结论连翘苷可通过PI3K/Akt信号通路调控786-0细胞凋亡与细胞周期、抑制肾癌细胞生长;同时连翘苷经PI3K/Akt通路可有效削弱肾癌细胞迁移与侵袭能力。     

健脾消癌方对结肠癌细胞中Akt/mTOR信号通路的影响

目的:探讨健脾消癌方含药血清对人结肠癌HCT116细胞蛋白激酶B/哺乳动物雷帕霉素靶蛋白(Akt/mTOR信号通路相关蛋白表达的影响。方法:以15%健脾消癌方含药血清处理结肠癌HCT116细胞,采用Transwell侵袭实验检测HCT116细胞迁移、侵袭作用,采用蛋白质印迹法(Western blot)检测HCT116细胞中Akt,磷酸化蛋白激酶B(p-Akt),mTOR,磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR),核糖体S6蛋白激酶(S6K1),磷酸化核糖体S6蛋白激酶(p-S6K1),真核细胞始动因子4E结合蛋白(4EBP1),磷酸化真核细胞始动因子4E结合蛋白(p-4EBP1)等蛋白的表达,正常血清组设立相同浓度正常血清组(15%),10%胎牛血清组(FBS)。结果:与正常血清组比较,健脾消癌方含药血清组迁移细胞数与侵袭细胞数均显著降低(P 0. 01);与正常血清组比较,Akt蛋白表达无明显下调,p-Akt蛋白表达显著下调(P 0. 01);与正常血清组比较,mTOR蛋白表达无明显下调,p-mTOR蛋白表达显著下调(P 0. 01);与正常血清组比较,S6K1蛋白表达无明显下调,p-S6K1蛋白表达显著下调(P 0. 01);与正常血清组比较,4EBP1蛋白表达无明显下调,p-4EBP1蛋白表达显著下调(P 0. 01)。结论:健脾消癌方抗结肠癌复发转移的机制可能与抑制Akt/mTOR信号通路的激活有关。     

姜黄素对APP/PS1双转基因小鼠海马PI3K及p-PI3K表达的影响

目的:观察姜黄素对AD模型APP/PS1双转基因小鼠磷酯酰肌醇-3激酶(phosphatidylinositol-3-kinase,PI3K)和磷酸化磷酯酰肌醇-3激酶(phosphorylated phosphatidylinositol-3-kinase,p-PI3K)表达的影响.方法:将3月龄的APP/PS1双转基因小鼠随机分为模型组、罗格列酮组(10 mg· kg-1·d-1)、姜黄素高、中、低剂量组(400,200,100 mg·kg-1·d-1),对照组为同月龄同背景的非转基因小鼠.连续灌胃3个月后,应用免疫组织化学和Western blot方法检测小鼠海马中PI3K和p-PI3K的蛋白表达.结果:免疫组化染色模型组小鼠大脑海马CA1区PI3K和p-PI3K阳性细胞均较对照组明显减少(P＜0.05);与模型组相比,各干预组PI3K及p-PI3K阳性细胞均有不同程度的增加,而以罗格列酮组与姜黄素中剂量组阳性细胞恢复最为显著(P＜0.01).Western blot检测结果与免疫组化结果基本一致.结论:姜黄素可以使APP/PS1双转基因小鼠海马中已减少的PI3K和p-PI3K蛋白有所恢复,提示姜黄素可能通过调节PI3K途径的胰岛素信号转导通路发挥抗AD作用.     

解毒化浊方对HER-2阳性乳腺癌细胞PI3K/Akt信号通路相关蛋白表达的影响

目的观察解毒化浊方血清对HER-2阳性乳腺癌细胞PI3K/Akt信号通路相关蛋白表达的影响,探讨解毒化浊方治疗HER-2阳性乳腺癌的作用机制。方法将雌性Wistar大鼠分为对照组、中药组、西药组、联合组4组,分别给药制备含药血清,再以给药血清干预SK-BR-3细胞,采用Western blot检测HER-2、p-HER-2蛋白表达以及PI3K/Akt通路相关蛋白p-Akt、Bad、p-Bad表达。结果 Western blot检测显示,中药组、西药组、联合组HER-2、p-HER-2灰度比较对照组均降低;西药组、联合组HER-2、p-HER-2、p-Akt灰度比较对照组明显降低,西药组、联合组较对照组HER-2、p-HER-2、p-Akt表达含量差异有统计学意义(P0.05)。中药组、西药组、联合组Bad、p-Bad灰度比较对照组降低,总Bad表达含量各组间差异无统计学意义(P0.05)。西药组、联合组p-Bad表达含量差异有统计学意义(P0.05)。结论解毒化浊方具有下调PI3K/Akt信号通路中相关蛋白表达,协同抑制剂发挥减毒增效的抗肿瘤作用。     

桂枝茯苓丸调节PI3K/Akt/mTOR通路对PCOS-IR大鼠排卵障碍的影响

目的:探讨桂枝茯苓丸对来曲唑联合高脂乳剂导致的多囊卵巢综合征伴胰岛素抵抗(PCOS-IR)大鼠排卵障碍的影响.方法 :将72只雌性SD大鼠随机分为空白组、模型组、二甲双胍组和桂枝茯苓丸低、中、高剂量组,每组12只.除空白组外,其余大鼠每日予来曲唑0.001 g·kg1结合高脂乳剂15 mL?kg1连续灌胃30 d建立P...     

Aerobic exercise suppresses hepatocellular carcinoma by downregulating dynamin-related protein 1 through PI3K/AKT pathway

ObjectiveExercise, as a common non-drug intervention, is one of several lifestyle choices known to reduce the risk of cancer. Mitochondrial division has been reported to play a key role in the occurrence and transformation of hepatocellular carcinoma (HCC). This study investigated whether exercise could regulate the occurrence and development of HCC through mitosis.MethodsBioinformatics technology was used to analyze the expression level of dynamin-related protein 1 (DRP1), a key protein of mitochondrial division. The effects of DRP1 and DRP1 inhibitor (mdivi-1) on the proliferation and migration of liver cancer cells BEL-7402 were observed using cell counting kit-8, plate colony formation, transwell cell migration, and scratch experiments. Enzyme-linked immunosorbent assay, Western blot and real-time polymerase chain reaction were used to detect the expression of DRP1 and its downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. A treadmill exercise intervention was tested in a nude mouse human liver cancer subcutaneous tumor model expressing different levels of DRP1. The size and weight of subcutaneous tumors in mice were detected before and after exercise.ResultsThe expression of DRP1 in liver cancer tissues was significantly upregulated compared with normal liver tissues (P < 0.001). The proliferation rate and the migration of BEL-7402 cells in the DRP1 over-expression group were higher than that in the control group. The mdivi-1 group showed an inhibitory effect on the proliferation and migration of BEL-7402 cells at 50 μmol/L. Aerobic exercise was able to inhibit the expression of DRP1 and decrease the size and weight of subcutaneous tumors. Moreover, the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) decreased in the exercise group. However, exercise could not change p-PI3K and p-AKT levels after knocking down DRP1 or using mdivi-1 on subcutaneous tumor.ConclusionAerobic exercise can suppress the development of tumors partially by regulating DRP1 through PI3K/AKT pathway.     

铁筷子挥发油对慢性脑缺血致血管性认知功能障碍大鼠的作用及机制研究

目的 探究苗药铁筷子[腊梅Chimonanthus praecox及山腊梅C. nitens的干燥根]挥发油对慢性脑缺血（chronic cerebral hypoperfusion,CCH）致血管性认知功能障碍（vascular cognitive impairment,VCI）大鼠的作用及机制。方法 采用改良的双侧颈总动脉结扎（bilateral common carotid artery occlusion,BCCAO）法建立CCH模型,将造模成功大鼠随机分为模型组及铁筷子挥发油高、中、低剂量（80、40、20 mg/kg）组和丁苯酞（63 mg/kg）组,另设假手术组,给予相应药物干预28 d,采用水迷宫实验检测各组大鼠学习记忆能力;采用旷场实验、Y迷宫实验检测各组大鼠自主活动性和新奇事物探索能力;采用苏木素-伊红（HE）染色、Nissl染色、TUNEL染色观察各组大鼠大脑皮层及海马CA1区神经细胞结构变化、尼氏小体以及凋亡细胞数量变化;采用ELISA法检测各组大鼠血清中脑源性神经营养因子（brain-derived neurotrophic factor,BDNF）水平;采用比色法检测各组大鼠海马组织中乙酰胆碱酯酶（acetylcholinesterase,AChE）和乙酰胆碱转移酶（cholineacetyltransferase,ChAT）的活性;采用Western blotting法检测各组大鼠海马组织中BDNF、酪氨酸激酶受体B（tyrosine kinase receptor B,TrkB）、磷酸化TrkB（phosphorylated TrkB,p-TrkB）、磷脂酰肌醇-3-激酶（phosphatidylin-ositol-3-kinase,PI3K）、蛋白激酶B（Akt）、p-Akt和半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3,Caspase-3）蛋白表达。结果 与模型组比较,铁筷子挥发油组大鼠逃避潜伏期缩短（P＜0.05、0.01）,穿越平台次数和跨格次数提高（P＜0.05、0.01）,在新异臂所待时间延长（P＜0.05）;神经细胞结构的变性得到改善,脑组织中尼氏小体的数量提高（P＜0.05）,细胞凋亡减少（P＜0.05）;血清中BDNF水平及海马组织中ChAT活性升高（P＜0.05、0.01）,海马组织中AChE活性降低（P＜0.05）;海马组织中BDNF、TrkB、p-TrkB、PI3K和p-Akt蛋白表达水平均显著升高（P＜0.05、0.01）,Caspase-3蛋白表达水平降低（P＜0.05）。结论 苗药铁筷子挥发油可以改善CCH致VCI大鼠认知功能障碍,其作用机制可能与激活BDNF/TrkB/PI3K/Akt信号通路有关。     

Effect of transcutaneous auricular vagus nerve stimulation on fasting blood glucose and serum insulin concentration in Zucker diabetes fatty rats: 经皮耳迷走神经刺激对Zucker糖尿病大鼠血糖及血清胰岛素浓度的影响

ObjectiveTo observe the influence of transcutaneous auricular vagus nerve stimulation (taVNS) on fasting plasma glucose (FPG) and serum insulin (INS) in Zucker diabetes fatty (ZDF) rats and explore the regulatory effect of taVNS on blood glucose in ZDF rats.MethodsA total of 20 male ZDF rats were randomized into a model group and a taVNS group, 10 rats in each one. Besides, the other 10 Zucker Lean (ZL) rats were selected to be a control group. The rats in the control group were fed with common forage and those in the model group and the taVNS group were fed with high-sugar and high-fat forage. The intervention of electric stimulation was applied in the rats of taVNS group, with 2/15 Hz in frequency, disperse-dense wave and 2 mA in intensity. Each intervention lasted 30 min, once a day, consecutively for 12 weeks. No any intervention was given in the control group and the model group. The body mass and FPG level were recorded once every 2 weeks in the rats. After the experiment, the rats were sacrificed and blood sample was collected. Enzyme linked immunosorbent assay (ELISA) was adopted to determine the level of serum INS in the rats.ResultsAfter the modeling, compared with the control group, the body mass and FPG level were all higher (P < 0.05, P < 0.01) and the concentration of serum INS was lower (P < 0.01) in the rats of the model group. After intervention, compared with the model group, the body mass was lower in week 6 to 10 (P < 0.01, P < 0.05), FPG level was lower in week 8 to 12 (P < 0.01, P < 0.05) and serum INS concentration was higher (P < 0.01) in the rats of the taVNS group.ConclusiontaVNS apparently improves in hyperglycemia in ZDF rats and increases serum insulin concentration in the rats.     

Tangshenping granule inhibits pyroptosis in a rat model of streptozotocin-induced diabetic nephropathy via the NLRP3/caspase-1/GSDMD pathway

ObjectiveTo explore the inhibitory effect of Tangshenping (TSP) on pyroptosis in a streptozotocin-induced diabetic nephropathy (DN) rat model.MethodsDN was established in Sprague–Dawley rats. Rats were randomly divided into DN (model group), irbesartan, and TSP low-, medium-, and high-dose groups, besides the control group. The 24 h albuminuria content, and serum content of TC, TGs, Scr, IL-1β, UREA, LDLs, and IL-18 were assessed. Hematoxylin & eosin and Mallory staining were performed to examine pathological changes in the kidney. The mRNA and protein expression of NLRP3, caspase 1, and GSDMD in the kidney were also examined.ResultsThe 24 h albuminuria content was obviously lower in the treatment groups compared to the model group (all P < .01). Levels of TC, TGs, Scr, UREA, LDLs, and IL-18 after drug interventions were obviously lower compared to the model group (all P < .05). The serum content of IL-1β in the TSP medium- and high-dose groups were much lower compared to the model group (P = .013 and P = .001, respectively). Through immunohistochemistry and western blotting, we observed that the protein expressions of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 were lower after drug interventions compared to the model group (all P < .05). Using qPCR, we observed that the mRNA expressions of caspase-1, IL-1β, IL-18, and GSDMD after drug interventions were significantly lower compared to the model group (all P < .05). The mRNA expressions of NLRP3 in the TSP medium- and high-dose groups were both lower compared to the model group (all P < .05).ConclusionTSP downregulated mRNA and protein expressions of NLRP3, caspase-1, and GSDMD. Our findings demonstrate that the beneficial effects of TSP on renal function are at least partly mediated by the inhibition of micro-inflammation and modulation of the expression of pyroptosis-related factors.     

淫羊藿素通过Akt/mTOR调控糖酵解抑制肝内胆管癌细胞增殖的作用机制研究

目的 探究淫羊藿素对人肝内胆管癌HuCCT1细胞增殖的影响及其作用机制。方法 CCK-8法检测淫羊藿素对HuCCT1细胞增殖活性的影响;平板克隆法检测淫羊藿素对HuCCT1细胞集落形成能力的影响;流式细胞术检测淫羊藿素对HuCCT1细胞周期的影响;分光光度法检测淫羊藿素对Hu CCT1细胞葡萄糖摄取量、乳酸生成量、三磷酸腺苷(adenosine triphosphate,ATP)生成量以及己糖激酶(hexokinase,HK)和丙酮酸激酶(pyruvate kinase,PK)活性的影响;Western blotting法检测淫羊藿素对HuCCT1细胞中增殖相关蛋白和蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)通路及糖酵解相关蛋白表达的影响;Western blotting检测淫羊藿素对瞬时转染Akt基因质粒的HuCCT1细胞中Akt/mTOR及糖酵解相关蛋白表达的影响。结果 淫羊藿素显著抑制HuCCT1细胞的活力,并呈时间和剂量相关性;淫羊藿素呈剂量相关性地抑制HuCCT1细胞...     

基于mTOR信号通路研究黄芩苷对对乙酰氨基酚诱导肝损伤后肝修复作用

目的 探讨黄芩苷对对乙酰氨基酚（acetaminophen,APAP）诱导的肝损伤后肝再生的作用及机制。方法 C57BL/6小鼠随机分成对照组、模型组和黄芩苷（40、80 mg/kg）组,小鼠ip APAP（300 mg/kg）1 h后ig黄芩苷,ip APAP 24 h或48 h后检测血清中天冬氨酸氨基转移酶（aspartate aminotransferase,AST）、丙氨酸氨基转移酶（alanine aminotransferase,ALT）活性;通过苏木素-伊红（HE）染色法观察肝脏的病理变化;采用qRT-PCR检测肝脏组织中肝细胞生长因子（hepatocyte growth factor,HGF）和表皮生长因子（epidermal growth factor,EGF）mRNA表达;通过BeyoClick^™ EdU法检测肝脏组织细胞增殖情况;通过免疫组化染色法观察肝脏组织中增殖细胞核抗原（proliferating cell nuclear antigen,PCNA）阳性细胞数量;通过Western blotting检测肝脏组织中PCNA、细胞周期蛋白D1（Cyclin D1）、p62、微管相关蛋白1轻链3（microtubule-associated protein 1 light chain,LC3B）、磷酸化哺乳动物雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin,p-mTOR）和p-S6蛋白的表达。给予mTOR抑制剂雷帕霉素后,观察雷帕霉素对黄芩苷促进肝修复作用的影响。结果 黄芩苷可以显著减少APAP诱导的急性肝损伤小鼠的肝坏死面积（P＜0.05、0.01）,上调肝脏HGF、EGF mRNA表达和PCNA、Cyclin D1、p62、p-S6、p-mTOR蛋白表达（P＜0.05、0.01）,下调LC3B蛋白表达（P＜0.05、0.01）,从而激活mTOR信号通路,促进APAP诱导的肝损伤后肝再生。此外,黄芩苷对肝再生的促进作用在给予mTOR抑制剂雷帕霉素后减弱（P＜0.05）。结论 黄芩苷能够通过激活mTOR信号通路,促进肝细胞增殖,并抑制其介导的自噬,从而促进肝修复。     

Hewei Jiangni granule alleviates visceral hypersensitivity in a rat model of non-erosive reflux disease via transient receptor potential channel signaling

ObjectiveTo uncover the underlying mechanism of Hewei Jiangni granule (HWJNG) on non-erosive reflux disease (NERD) treatment by examining histological changes, gastrointestinal neurochemicals release and visceral hypersensitivity-related receptor expression in NERD model rats.MethodsA NERD rat model was established via a combination of basal sensitization and acid perfusion. HWJNG treatments at different doses were then administered. Pathological changes to tissues, mast cell (MC) activation, serum levels of esophageal visceral hypersensitivity-related neurochemicals, and transient receptor potential (TRP) receptor mRNA and protein levels were investigated.ResultsCompared with the control group, the expression of tryptase in MCs, the changes of intercellular space, and the serum levels of substance P (SP), calcitonin gene-related peptides (CGRP) and proteinase-activated receptor 2 (PAR2) increased in the model group (all P < .05). The expression of TRP vanilloid 1 (Trpv1) mRNA decreased in esophagus and dorsal root ganglia (DRG) of the model group (P = .030 & P = .013), and the expression of TRP melastatin channel subfamily member 8 (Trpm8) mRNA decreased in the esophagus of model group (P < .01). The level of esophageal TRPV1 protein increased in the model group (P < .01) and the level of TRPM8 protein decreased in esophagus and DRG of the model group (both P < .05). Compared with the model group, the serum levels of SP, CGRP, and PAR2 in the medium-dose HWJNG group showed significant decreases (all P < .05). The expression of Trpv1 mRNA in esophagus and DRG of the HWJNG groups and the Omeprazole group remarkably decreased (all P < .05), as was the expression of Trpm8 mRNA in esophagus of the HWJNG groups (all P < .05).ConclusionHWJNG alleviated visceral hypersensitivity in NERD model rats by regulating TRP-mediated signaling. Our results indicate that HWJNG has potential as a therapeutic agent for NERD.     

Efficacy of catgut-embedding therapy at “five neck points” on cervical spondylotic arteriopathy and effects on hemodynamics: A randomized clinical trial “颈五针”穴位埋线治疗椎动脉型颈椎病疗效及对血流动力学的影响-随机临床试验

ObjectiveTo observe the effect of catgut-embedding therapy at five neck points in the treatment of cervical spondylotic arteriopathy (CSA) and analyze its effects on the hemodynamics of patients with CSA.MethodsUsing the computer randomization method, 72 patients with CSA were divided into a catgut-embedding therapy group (n = 36, none dropped out) and a western medication group (n = 36, none dropped out). In the catgut-embedding therapy group, the therapy was applied to five neck points, i.e. bilateral J?ngjiāj? (颈夹脊) at C5, bilateral J?ngjiāj? (颈夹脊) at C6, and Dàzhuī (大椎GV14) once a week. One 3-week treatment session was required. In the western medication group, flunarizine hydrochloride 5 mg capsules were administered orally once daily for 3 weeks. Efficacy was assessed before and after 3 weeks treatment using a functional evaluation scale for cervical spondylotic arteriopathy (FS-CSA). Changes in vertebral-basilar arterial hemodynamics, serum nitric oxide (NO) level, and plasma endothelin (ET) level were detected.ResultsEfficacy was assessed when the treatment session was completed. The total efficacy rate was significantly higher in the catgut-embedding therapy group (86.1%) than in the western medication group (61.1%; P < 0.01). The after treatment FS-CSA score was reduced compared with baseline score in both groups (P < 0.05), and the reduction in the catgut-embedding therapy group was superior to that in the Western medication group (P < 0.05). Compared with baseline, the flow velocity and pulsatility index of the bilateral vertebral and basilar arteries increased at each period in both groups (P < 0.05) except for the left systolic velocity of the vertebral artery after treatment. The increase in the catgut-embedding therapy group was greater than that in the western medication group (P < 0.05). After treatment, the serum NO concentration was increased and the plasma ET concentration was reduced compared to baseline in both groups (P < 0.05), while the improvements in the catgut-embedding therapy group were superior to those in the western medication group (P < 0.05).ConclusionThe clinical efficacy in the catgut-embedding therapy group was superior to that in the western medication group. Catgut-embedding therapy at five neck points may effectively improve psychological and functional conditions, promote vertebral basilar arterial supply, and relieve clinical symptoms in patients with CSA.