The influence of Phosphatidylinositol 3-kinase/Akt Pathway on the Ischemic Injury during Rat Liver Graft Preservation

We aimed to investigate the role of phosphatidylinositol 3 (PI3)-kinase/Akt pathway on ischemic injury. Rat liver grafts were preserved in UW solution with different treatments and were compared by 1-week survival rates and morphological changes with those of the control group. PI3-kinase/Akt was significantly activated at the sites of Thr 308 and Ser 473 in the preserved grafts. Downstream target proteins, glycogen synthase kinase-3beta (GSK-3beta) and caspase-9, were inactivated. However, survival signal transduction from Akt to Bad was blocked by calcium release after activation of PI3-kinase/Akt. Significant activation of caspase-12, -3 and -7 contributed to cell apoptosis and severe ischemic injury was shown after 7 h of preservation by UW solution with insulin. Downregulation of phospho-Akt at Thr 308 and Ser 473 was due to partial inhibition of PI3-kinase/Akt pathway by LY294002. Activation of GSK-3beta and inactivation of caspase-12 and Bad could be found in the LY294002 groups in which the liver grafts showed less ischemic injury. Higher 1-week survival rates in the heparin, LY294002, and glucagon groups confirmed the dysregulation of the pathway. In conclusion, PI3-kinase/Akt pathway was dysregulated and contributed to ischemic injury during preservation. Heparin and LY294002 could improve graft viability by maintaining calcium homeostasis during preservation.     

Dual efficacy of Fasudil at improvement of survival and reinnervation of flap through RhoA/ROCK/PI3K/Akt pathway

Fasudil is reported to be effective at protecting against ischaemic diseases, and at augmenting axon growth. In this study, we aim to evaluate its efficacy in promoting flap survival and reinnervation. Ninety‐two Institute of Cancer Research (ICR) mice were used and divided into the control, Fasudil, LY294002, Fasudil+LY294002 groups, receiving a daily intraperitoneal injection of normal saline, Fasudil (10 mg/kg), LY294002 (5 mg/kg), and Fasudil (10 mg/kg) + LY294002 (5 mg/kg), respectively. On days 0 and 5, the blood perfusion and diameter of the iliolumbar artery in the pedicle of the flaps in the four groups were evaluated using laser speckling contrast imaging (LSCI). On day 5, the flaps were photographed and the necrosis rate of the flaps was calculated using Photoshop CS6. In addition, tissues were harvested from the flaps and divided into two parts. One part underwent routine cryosection and immunofluorescent staining using the antibody against CD31 for evaluation of the microvascular density in the four groups. In the other part, the expression of RhoA, ROCK1+2, p‐CPI‐17, p‐MYPT, p‐PTEN, p‐PI3K, p‐Akt, and vascular endothelial growth factor (VEGF) within the flaps were determined using western blotting. Moreover, at days 0, 7, 15, and 30 after flap surgery, the axons within the flaps were evaluated using immunofluorescent staining with the antibody against Neurofilament‐200. It turned out that the necrosis rate was (24.4 ± 7.7)%, (5.2 ± 1.6)%, (29.8 ± 4.2)%, and (30.9 ± 7.1)%, respectively, in the control, Fasudil, LY294002, LY294002+Fasudil groups. There was a significant reduction in the necrosis rate of the flaps in the Fasudil group (P < .001). The LSCI and immunofluorescent staining demonstrated that Fasudil could significantly expand the diameter of the iliolumbar artery in the pedicle, boost the overall blood perfusion, and increase the microvascular density of the flaps in the Fasudil group (P < .05), which could all be abolished by PI3K inhibitor LY294002. On day 5, the expression of p‐CPI‐17, p‐MYPT, and p‐PTEN were downregulated, whereas pPI3K, p‐Akt, and VEGF were upregulated in the Fasudil group (P < .001). As for reinnervation, Neurofilament‐200 fluorescent staining revealed that at days 15 and 30 after flap harvest, only in the Fasudil group could new axons be observed. It can be concluded that Fasudil could simultaneously improve the survival and axon growth after flap harvest, a dual efficacy achieved by inhibition of the RhoA/ROCK pathway, which in turn activates /PI3K/AKT pathway.     

血管紧张素II通过PI3K/Akt/FOXO1信号通路诱导骨骼肌细胞萎缩

目的探讨血管紧张素II(Ang II)对骨骼肌细胞的调控作用及其作用机制。方法使用Ang II及其1型受体(AT1R)拮抗剂(ARB)奥美沙坦干预C2C12成肌细胞,分为Control、Ang II和Ang II+ARB三组。使用2%马血清诱导成肌细胞分化为肌管细胞。免疫荧光染色检测肌管细胞的平均面积改变,Western blot检测肌管细胞泛素连接酶、生肌调节因子(MRFs)和PI3K/Akt/FOXO1信号通路蛋白表达的变化。结果免疫荧光染色显示,Ang II干预后,肌管细胞的平均直径和面积减小(P0.001);使用奥美沙坦干预后,肌管细胞平均直径和面积明显增大(P 0.01)。Western blot显示,Ang II干预后,肌管细胞pPI3K/PI3K(P0.01),p-Akt/Akt (P 0.001)和p-FOXO1/FOXO1 (P 0.01)的比率降低; MURF1 (P 0.05)和MAFbx (P 0.001)蛋白表达明显升高,MHC(P0.01)和MyoD(P 0.05)蛋白表达明显降低;使用奥美沙坦处理后,能够减轻Ang II对肌管细胞的干预作用。结论 Ang II能够通过抑制PI3K/Akt/FOXO1信号通路的磷酸化,促进蛋白的降解,抑制蛋白的合成,诱导骨骼肌细胞萎缩。     

右归丸通过PI3K/Akt/mTOR信号通路对膝骨关节炎模型鼠软骨组织保护作用的研究

目的研究右归丸对膝骨关节炎(knee osteoarthritis,KOA)模型大鼠磷脂酰肌醇3-激酶(phosphatidyl inositol 3-kinase,PI3K)、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)信号通路的影响。方法采用改良Hulth法制备大鼠KOA模型,模型制备成功6周后用右归丸进行干预,灌胃2个月后取材。HE法观察各组大鼠软骨组织病理形态改变并进行Mankin评分,实时荧光定量PCR法检测各组大鼠软骨组织白细胞介素-1β(interleukin-1β,IL-1β)、基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)和基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)的表达变化,Western blot法检测各组大鼠软骨组织磷脂酰肌醇3-激酶(phosphatidyl inositol 3-kinase,PI3K)、磷酸化的蛋白激酶B(phosphorylated protein kinase B,pAkt)、磷酸化的哺乳动物雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,pmTOR)和Beclin1的蛋白表达。结果与假手术组比较,模型组大鼠软骨组织Makin评分明显升高,软骨组织IL-1β、MMP-3和MMP-13的基因表达均明显升高,软骨组织PI3K、pAkt和pmTOR的蛋白表达均明显升高(P0. 01);而模型组大鼠软骨组织Beclin1的蛋白表达明显降低(P0. 01);模型组关节软骨边缘严重破坏,软骨细胞排列紊乱。与模型组相比,右归丸组大鼠软骨组织Makin评分明显降低,软骨组织IL-1β、MMP-3和MMP-13的基因表达均明显降低,软骨组织PI3K、pAkt和pmTOR的蛋白表达均明显降低,Beclin1的蛋白表达明显升高(P0. 05或P0. 01),其软骨结构趋于正常,软骨细胞分布偶见不均,关节软骨表面欠光滑。结论右归丸可能是通过抑制IL-1β、MMP-3、MMP-13、PI3K、p Akt、pmTOR的表达和增强Beclin1的表达来达到治疗KOA的目的。     

Cinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155

BackgroundTo prove the internal connection, we deciphered the effect of cinnamaldehyde on kidney senescence through establishing animal and cell models.MethodsIn vivo, a rat senescence model was constructed using D-galactose (D-gal), and the modeled rats were further treated with cinnamaldehyde. In vitro, rat renal tubular epithelial cells (NRK-52E) were transfected with miR-155 mimic or inhibitor and then treated with cinnamaldehyde, D-gal or PI3K inhibitor (LY294002). The serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) of the rats were measured by an automatic biochemical analyzer. Pathological changes of kidney were determined by hematoxylin-eosin staining. The senescence and viability of NRK-52E cells were assessed by SA-β-gal staining and CCK-8 assay, respectively. The levels of miR-155, p-PI3K/PI3K, p-Akt/Akt, LC3B (LC3-II and LC3-I) and Beclin1 were detected by qRT-PCR, immunohistochemistry, or western blot.ResultsD-gal elevated the levels of BUN, Scr and miR-155 in the kidney, induced the renal pathological damage, inhibited the cell viability, increased the numbers of SA-β-gal-, LC3B- and Beclin1-positive cells and upregulated the levels of LC3-II/LC3-I and Beclin1 both in the kidney and cells. Cinnamaldehyde reversed D-gal-induced effects on the kidney and cells, and moreover, the cinnamaldehyde-induced anti-D-gal effects on cells could be suppressed by miR-155 mimic but promoted by miR-155 inhibitor. LY294002 potentiated D-gal-induced effects, and reversed cinnamaldehyde- and miR-155 inhibitor-caused impacts on the PI3K/Akt pathway and LC3-II/LC3-I level in D-gal-induced cells.ConclusionCinnamaldehyde attenuates kidney senescence and injury through PI3K/Akt pathway-mediated autophagy via downregulating miR-155.     

P38MAPK和P13K/Akt信号通路在大鼠糖尿病神经病理性疼痛中的交互作用

目的 观察p38MAPK和P13K/Akt信号通路在大鼠糖尿病神经病珲性疼痛中的交互作用.方法 Wistar大鼠腹腔单次注射链脲菌素65 mg/kg制作糖尿病神经病理痛模型.4周后用von frey纤维测双后足机械痛阈,痛阈明显下降为糖尿病神经病理性疼痛造模成功.将96只成模大鼠随机均分为三组:糖尿病神经病理痛组(D组),PI3K抑制药组(E组)和p38MAPK抑制药组(F组).另取同窝大鼠32只作为对照组(C组).成模后的每周周一,E组和F组大鼠分别静脉注射P13K抑制药Wortmannin 0.5 mg/kg和p38MAPK抑制药SB203580 1 mg/kg,直至处死大鼠.于给药前(T1)和给药后第2周末(T2)、第4周末(T3)、第6周末(T4)分别随机取8只大鼠,检测机械缩足反应阈值(MWT)、神经传导速度(NCV)、脊髓和背根神经节(DRG)磷酸化Akt(p-Akt)水平和磷酸化p38MAPK(p-p38MAPK)水平.结果 与C组比较,D、E和F组在T1～T4时MWT下降,NCV减慢,p-Akt和p-p38MAPK水平升高(P<0.05);与D组比较,E和F组在T2～T4时MWT升高,NCV增快,E和F组p-Akt明显下降,F组p-p38MAPK水平明显下降(P<0.05).结论 P38MAPK通过激活其下游的P13K/Akt信号通路参与了糖尿病大鼠神经病理痛的形成和维持.     

软骨细胞凋亡与PI3K/Akt途径及相关信号分子的研究进展

骨性关节炎（osteoarthritis,OA）是指关节面软骨发生原发性或继发性退变及结构紊乱,伴随软骨下骨质增生、软骨剥脱,从而使关节逐渐破坏、畸形,最终发生关节功能障碍的一种退行性疾病。在生理状态下,软骨细胞的分解代谢和合成代谢处于平衡状态,维持软骨细胞外基质结构和功能的完整性,而软骨细胞凋亡既可以维持这种平衡,也可以破坏这种平衡。因此,软骨细胞的凋亡作用已成为当今医学领域中的研究课题之一。软骨细胞凋亡是一种由基因控制多信号调控的、主动去除细胞的程序性死亡过程。     

PI3K/Akt信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用

目的 探讨磷脂酰肌醇-3激酶/蛋白激酶B(phosphoinositide-3 kinase/Akt,PI3K/Akt)信号通路在吡咯喹啉醌促雪旺细胞增殖过程中的作用.方法 体外分离培养雪旺细胞,S-100免疫荧光鉴定;Western blot检测PI3K下游因子Akt磷酸化激活形式(p-Akt)的表达,并通过PI3K激酶抑制剂(wortmannin)阻断该通路后p-Akt的表达情况.结果 毗咯喹啉醌可使雪旺细胞发生形态学变化,加入吡咯喹啉醌后30 min即可检测到p-Akt的表达,4 h达高峰,12 h基本无表达;吡咯喹啉醌在1～100 nmol/L范围内可使p-Akt表达增加;加入wortmannin阻断PI3K后p-Akt上调表达消失(P<0.05).结论 吡咯喹啉醌可使雪旺细胞发生形态学变化,PI3K/Akt信号通路在吡咯喹啉醌促雪旺细胞增殖过程中发挥重要作用.     

Methylprednisolone Inhibits Autophagy of Vascular Endothelial Cells in Rat Femoral Head Via PI3K/Akt/mTOR Pathway

ObjectiveTo study the relationship between vascular endothelial cells (VEC) and autophagy, and its regulatory mechanism in steroid‐induced avascular necrosis of the femoral head (SANFH).MethodsIn cell experiment, VEC were isolated and cultured from the femoral head of Sprague–Dawley rats and divided into three groups: blank control group (Ctrl), methylprednisolone group (MP), and methylprednisolone+mTOR‐shRNA group (MP + shmTOR). The autophagy formation was observed by transmission electron microscope. The mRNA expression of PI3K, Akt, mTOR, Beclin1 and MAP1LC3 was detected by RT‐PCR and the protein expression was detected by Western blot and immunofluorescence. Expression of the damage marker 6‐keto‐PGF1α was detected by the ELISA method. In vivo experiment, after establishing the model, the grouping method was the same as cell experiment. Autophagosomes were observed by same method, and the expression of related factors was detected by the same method in cell experiment.ResultsIn the cell experiment, autophagosomes in the MP group were significantly lower than in the Ctrl group, and the autophagosomes in the MP + shmTOR group were intermediate between two groups (P < 0.05). The mRNA expression levels of PI3K, Akt and mTOR in the MP group were significantly higher than in the Ctrl group, while the MP+ shmTOR group presented intermediate levels between these groups (average gray value were 3837.90, 2996.30, 3005.60, F = 428.64, P < 0.05). MRNA expression levels of Beclin1 and MAP1LC3 in the MP group were significantly lower than that in Ctrl group (P < 0.05). The content of 6‐keto‐PGF1α in the MP + shmTOR group was higher than in the Ctrl group and lower than in the MP group at the evaluated time intervals (average absorbance value were 104.98, 206.83, 145.91, F = 352.83, P < 0.01). In vivo experiment, the content of 6‐Keto‐PGF1α in the hormone group increased as time went on; the mTOR‐si group was higher than that in control group, but lower than that in the hormone group (P < 0.01). The mRNA expressions of Beclin1 and MAP1LC3 in the control group were higher than those in the hormone group, while the mRNA expressions of PI3K, Akt and mTOR were lower than those in the mTOR‐si group (P < 0.05).ConclusionThe steroid inhibited the physiological protective effect of autophagy on SANFH by increasing the expression of PI3K/Akt/mTOR signaling pathway related factors and decreasing the expression of Beclin1 and MAP1LC3 in the femoral head VEC.     

黄连素通过PI3K/Akt/mTOR信号通路抑制高糖条件下的肾脏系膜细胞增殖

目的：探讨黄连素（berberine,BBR）对高糖条件下的大鼠肾脏系膜细胞 HBZY-1 cells （MCS）增殖的影响及其作用机制。方法将对数生长期细胞分成正常糖处理组（NG 组）,高糖组（HG组）和黄连素组（BBR组）。NG组细胞体系常规培养培养,HG 组细胞体系加入40 mmol/L 葡萄糖,BBR组细胞培养体系中加入40 mmol/L葡萄糖＋黄连素30μmol/L。各组细胞培养48 h 后,利用MTT检测各组HBZY-1细胞增殖;RT-PCR检测各组细胞p85,Akt和mTOR基因表达;West-ern blotting检测细胞中 p85、p-p85、Akt、p-Akt,mTOR 和 Collagen-Ⅳ蛋白的变化。结果 NG 组与HG组增值率分别为（25%±3%）和（75%±5%）,与 NG组增值率比较,HG组肾脏系膜细胞增殖率明显增高（P〈0.05）,p85与 Akt mRNA表达水平和蛋白水平均无明显变化（P＆gt;0.05）,但 p-p85、p-Akt,Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显增加（P〈0.05）;BBR 组增值率为（42%±5%）,与 HG 组比较,BBR 组肾脏系膜细胞增值率明显降低（P〈0.05）,但 p-p85、p-Akt、Collagen-Ⅳ蛋白表达水平和 mTOR mRNA 表达与蛋白水平均明显下降（P〈0.05）,而 p85和Akt mRNA和蛋白表达水平均无统计学差异（P〈0.05）。结论黄连素能抑制高糖导致的系膜细胞增殖,其作用可能是通过抑制PI3K/Akt/mTOR信号通路激活。     

Role of PI3-kinase/Akt signalling pathway in renal function and cell proliferation after renal ischaemia/reperfusion injury in mice

BACKGROUND: PI3K/Akt pathway has been shown to play a critical role in the regulation of mitogenic signalling, apoptosis, cell proliferation and survival in a variety of cells and tissues. The aim of the present study was to investigate the role of PI3K/Akt pathway in the renal ischaemia/reperfusion. METHODS: Four experimental groups, sham-operative mice, vehicle-delivered and wortmannin-treated ischaemic/reperfusion injury mice, wortmannin-treated normal mice were designed to examined serum blood urea nitrogen level, renal injury, proliferating cell nuclear antigen protein and Akt phosphorylation status at 30 min, 90 min, 24 h, 48 h of reperfusion after ischaemic treatment. Wortmannin or its vehicle was given intraperitoneally at 4 h before surgery. Blood urea nitrogen was measured, and immunohistochemistry and western blotting were used to detect the components of PI3K/Akt pathway in the ischaemic/reperfusion injury kidney. RESULTS: PI3-kinase inhibitor wortmannin imposes a deleterious effect on serum blood urea nitrogen level, renal function after renal ischaemia/reperfusion injury in mice. The renal cell proliferation increased after ischaemia/reperfusion injury in mouse, which could be inhibited by wortmannin. Phosphorylation of Akt was increased after ischaemia/reperfusion in the mouse kidney, and reduced by wortmannin administration. CONCLUSION: This primary study suggests that PI3-kinase/Akt signalling pathway play an important role in the regulation of the renal repair after ischaemia/reperfusion injury.     

miR-155靶向调节PIK3R1对类风湿关节炎大鼠模型PI3K/Akt/mTOR信号通路的影响

目的 探讨miR-155靶向调节PIK3R1对类风湿关节炎（rheumatoid arthritis,RA）大鼠PI3K/Akt/mTOR信号通路的影响。方法 SD大鼠随机分为对照组、模型组、miR-155 agomir组、miR-155 antagomir组、miR-155阴性对照组,诱导RA模型,分组处理后,观察关节症状,检测关节炎指数及后足趾容积;HE染色观察大鼠关节组织病理形态;使用试剂盒检测大鼠关节组织炎性因子IL-6、IL-17及IL-18水平;免疫印迹检测大鼠关节组织PI3K/Akt/mTOR信号通路蛋白表达;qRT-PCR实验检测大鼠关节组织miR-155及PIK3R1 mRNA水平;双荧光素酶报告基因实验检测miR-155对PIK3R1的靶向调控作用。结果 与对照组比较,模型组大鼠关节炎症状明显,关节炎指数、后足趾容积、关节组织炎性因子IL-6、IL-17及IL-18水平、关节组织p-PI3K/PI3K、p-Akt/Akt及p-mTOR/mTOR水平、关节组织miR-155水平明显升高（P＜0.05）,PIK3R1 mRNA水平明显降低（P＜0.05）。与模型组、miR-155阴性对照组比较,miR-155 antagomir组大鼠上述指标得到明显改善（P＜0.05）;miR-155 agomir组与miR-155 antagomir组趋势相反（P＜0.05）。结论 miR-155可靶向下调PIK3R1的表达,激活PI3K/Akt/mTOR信号,加重RA大鼠关节炎症损伤,下调miR-155表达,可抑制PI3K/Akt/mTOR信号激活及炎症反应发生发展,改善关节炎症状。     

表没食子儿茶素没食子酸酯通过PI3K/Akt通路抑制胰腺癌细胞糖酵解的实验研究

目的：探讨表没食子儿茶素没食子酸酯（EGCG）对胰腺癌细胞的增殖及糖代谢的影响。方法：用CCK8试剂盒检测不同浓度EGCG对胰腺癌PANC-1细胞增殖的影响,用葡萄糖乳酸试剂盒检测EGCG对糖酵解的影响,Western blot法检测糖酵解酶及PI3K/Akt通路蛋白的表达。结果：CCK8结果显示,EGCG可以抑制PANC-1细胞的增殖,其抑制作用具有剂量依赖性和时间依赖性（P <0.05）。葡萄糖乳酸检测实验表明,EGCG可以抑制PANC-1细胞的葡萄糖消耗和乳酸生成（P <0.05）。Western blot结果表明,EGCG可以抑制糖酵解酶己糖激酶-2(HK-2)、磷酸果糖激酶（PFK）和乳酸脱氢酶（LDH）及磷酸化PI3K和Akt的表达,且EGCG浓度越高,抑制作用越明显（P <0.05）。结论：EGCG抑制人胰腺癌细胞系PANC-1的增殖和糖酵解,其抑制作用可能与抑制PANC-1细胞中PI3K/Akt通路有关。     

Fibroblast growth factor 2-induced cytoplasmic asparaginyl-tRNA synthetase promotes survival of osteoblasts by regulating anti-apoptotic PI3K/Akt signaling

Fibroblast growth factor 2 (FGF2), the potent bone anabolic agent, regulates the bone development, as well as the growth, remodeling and healing of the fracture. The intracellular signaling of FGF2 leads to activation of genes involved in cell proliferation, migration, differentiation and survival. However, little is known about FGF2-regulated proteins in the osteoblasts. Therefore, in this study, protein profiling in FGF2-treated MC3T3-E1 preosteoblast cells was evaluated using proteomic technologies. Six proteins including asparaginyl-tRNA synthetase (NARS), eukaryotic translation termination factor 1 (ETF1), GDP-forming succinyl-CoA synthetase (SUCLG2), heat shock protein 84 (HSP 84), sorting nexin 9 (SNX9) and α glucosidase 2α neutral subunit (GANAB) were increased more than 3-fold after the FGF2 treatment. Also, two proteins including β-tropomyosin and tropomyosin 2 were decreased to 2-folds. Among these proteins, asparaginyl-tRNA synthetase (NARS), a member of aminoacyl-tRNA synthetases (AARS), was strikingly up-regulated more than 900-fold. The overexpression of NARS significantly increased the proliferation of both the MC3T3-E1 and the primary mouse calvarial cells. In contrast, significant reduction of the basal expression of NARS by siNARS remarkably suppressed the proliferation and induced the death of cell. After the siNARS treatment, the resistance to apoptosis induced by serum deprivation was also significantly reduced. The level of p-Akt was also reduced and the activity of caspase 3 significantly enhanced. In addition, NARS-induced protection against apoptosis was abolished by the treatment of PI3K inhibitors, wortmannin and LY294002. In conclusion, we suggest that NARS is one of the important mediators of FGF2 induced survival signaling in osteoblasts through the activation of PI3K/Akt survival pathway.