电项针对全脑缺血VD模型大鼠PI3K/AKT/GSK-3β信号通路的影响

目的研究电项针对全脑缺血血管性痴呆(vascular dementia,VD)模型大鼠磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸蛋白激酶/糖原合成酶激酶-3β(Phosphatidylinositol-3 kinase/serine-threonine kinase/glycogen synthase kinase-3β,P13K/AKT/GSK-3β)信号通路的影响。方法采用四血管阻断方法制备VD模型大鼠,电项针组取双侧风池穴、供血穴,电针30 min/次,1次/d,治疗14d。采用Y迷宫评价大鼠学习记忆能力;荧光定量PCR(RT-PCR)、Western blot法检测大鼠海马组织中磷酸化蛋白激酶B(phosphorylatedproteinkinaseB,p-AKT)、磷酸化糖原合成酶激酶-3β(Phosphorylated GSK-3β,P-GSK-3β)mRNA和p-AKT、p-GSK-3β蛋白的表达。结果与模型组比较,电项针组可显著提高VD大鼠Y迷宫学习与记忆正确次数(P<0.01)。与模型组比较,电项针组大鼠海马组织中p-AKT、p-GSK-3βmRNA和p-AKT、p-GSK-3β蛋白表达均有不同程度的升高(P<0.01)。结论电项针能够改善VD模型大鼠学习记忆能力,具体机制可能是激活PI3K/AKT/GSK-3β信号通路,发挥抗凋亡作用,起到对缺血海马神经元的保护作用。

Active compounds of Caodoukou(Semen Alpinia Katsumadai)inhibit the migration,invasion and metastasis of human pancreatic cancer cells by targeting phosphoinosmde-3-kinase/protein kinase B/mammalian target of rapamycin pathway

OBJECTIVE:To detect the effects of active compounds of Caodoukou(Semen Alpinia Katsumadai)(ACAK)on the proliferation,migration and invasion of pancreatic cancer,and explain the possible molecular mechanism of ACAK interacting with these processes.Methods:Cell counting kit-8 method,cell scratch repair experiment,Transwell migration and invasion experiment,immunohistochemistry,western blot assay and real-time polymerase chain reaction experiment were used to evaluate the effect of ACAK on the proliferation,migration and invasion of pancreatic cancer cells.The levels of active molecules involved in the phosphoinosmde-3-kinase(PI3K)/Akt/the mammalian target of rapamycin(m TOR)signal transduction were detected by Western blot assay.In addition,the function of ACAK in vivo was evaluated by xenotransplantation tumor model in nude mice.Results:The inhibitory effect of ACAK on the proliferation of pancreatic cancer cells showed certain time-dose dependence.The results of scratch repair test,Transwell test,Western blotting and real time polymerase chain reaction assay showed that ACAK could inhibit the migration and invasion of pancreatic cancer cells in vitro.In addition,the regulatory effect of ACAK on epithelialmesenchymal transition(EMT)is partly attributed to PI3K/Akt/mT OR signaling pathway.The experimental results in vivo showed that ACAK regulated the development of pancreatic cancer.Conclusions:ACAK can partly inhibit the activity of EMT and matrix metallopeptidases by down-regulating the downstream proteins of PI3K/Akt/mTOR signal pathway,thus inhibiting the ability of migration and invasion of pancreatic cancer.

Ligustroflavone reduces necroptosis in rat brain after ischemic stroke through targeting RIPK1/RIPK3/MLKL

OBJECTIVE To explore the effect of ligustroflavone on ischemic brain injury in stroke rat and the under⁃lying mechanisms.METHODS A rat model of ischemic stroke was established by middle cerebral artery occlusion(MCAO).Administration of ligustroflavone(10,30,60 mg·kg-1,ig)15 min before ischemia,after which neurological deficit score and infarct volume were detected by longa score and TTC stain.The cell viability and necrosis rate of hypoxia-cultured PC12 cells(O2/N2/CO2,1:94:5,8 h)were evaluated by MTS and LDH release rate.Flow cytometry further verified the mortality rate of PC12 cells.Necroptosis-associated proteins(RIPK1,RIPK3 and MLKL/p-MLKL)were detected by Western blotting.The interaction between RIPK3 and RIPK1 or MLKL were confirmed by immunoprecipitation.Operating Environ⁃ment(MOE)program demonstrated the possible combination of ligustroflavone with RIPK1,RIPK3 and MLKL.RESULTS Ischemic injury(increase in neurological deficit score and infarct volume)and upregulation of necroptosis-associated proteins were showed in rat MCAO model.Administration of ligustroflavone(30 mg·kg^-1,ig)evidently improved neurological func⁃tion,reduced infarct volume,and decreased the levels of necroptosis-associated proteins except the RIPK1.Consistently,hypoxia-cultured PC12 cells caused cellular injury(LDH release and necroposis)concomitant with up-regulation of necroptosis-associated proteins,and these phenomena were blocked in the presence of ligustroflavone(25μmol·L^-1)except the elevated RIPK1 levels.Using the Molecular Operating Environment(MOE)program,we identified RIPK1,RIPK3,and MLKL as potential targets of ligustroflavone.Further studies showed that the interaction between RIPK3 and RIPK1 or MLKL was significantly enhanced,which was blocked in the presence of ligustroflavone.CONCLUSION Ligus⁃troflavone protects rat brain from ischemic injury,and its beneficial effect is related to the prevention of necroptosis through a mechanism involving targeting RIPK1,RIPK3,and/or MLKL.

RIP3/MLKL-mediated neuronal necroptosis induced by methamphetamine at 39℃

Methamphetamine is one of the most prevalent drugs abused in the world.Methamphetamine abusers usually present with hyperpyrexia (39℃),hallucination and other psychiatric symptoms.However,the detailed mechanism underlying its neurotoxic action remains elusive.This study investigated the effects of methamphetamine + 39℃ on primary cortical neurons from the cortex of embryonic Sprague-Dawley rats.Primary cortex neurons were exposed to 1 mM methamphetamine + 39℃.Propidium iodide staining and lactate dehydrogenase release detection showed that methamphetamine + 39℃ triggered obvious necrosis-like death in cultured primary cortical neurons,which could be partially inhibited by receptor-interacting protein-1 (RIP1) inhibitor Necrostatin-1 partially.Western blot assay results showed that there were increases in the expressions of receptor-interacting protein-3 (RIP3) and mixed lineage kinase domain-like protein (MLKL) in the primary cortical neurons treated with 1 mM methamphetamine + 39℃ for 3 hours.After pre-treatment with RIP3 inhibitor GSK’872,propidium iodide staining and lactate dehydrogenase release detection showed that neuronal necrosis rate was significantly decreased;RIP3 and MLKL protein expression significantly decreased.Immunohistochemistry staining results also showed that the expressions of RIP3 and MLKL were up-regulated in brain specimens from humans who had died of methamphetamine abuse.Taken together,the above results suggest that methamphetamine + 39℃ can induce RIP3/MLKL regulated necroptosis,thereby resulting in neurotoxicity.The study protocol was approved by the Medical Ethics Committee of the Third Xiangya Hospital of Central South University,China (approval numbers: 2017-S026 and 2017-S033) on March 7,2017.

PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis,apoptosis,and nec roptosis,which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases.Although our previous lite rature mining study suggested that PANoptosis might occur in neuronal ischemia/repe rfusion injury,little experimental research has been reported on the existence of PANoptosis.In this study,we used in vivo and in vitro retinal neuronal models of ischemia/repe rfusion injury to investigate whether PAN optosis-like cell death(simultaneous occurrence of pyroptosis,apo ptosis,and necroptosis)exists in retinal neuronal ischemia/repe rfusion injury.Our results showed that ischemia/repe rfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo.Ischemia/repe rfusion inju ry also significantly upregulated caspase-1,caspase-8,and NLRP3 expression,which are important components of the PANoptosome.These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Electroacupuncture Pretreatment at Neiguan (PC 6) attenuates autophagy in rats with myocardial ischemia reperfusion through the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway

OBJECTIVE: To investigate the protective efficacy of electroacupuncture(EA) pretreatment at Neiguan(PC6) on myocardial ischemia-reperfusion(I/R) in rats.METHODS: Fifty rats were randomly divided into five groups(n = 10): sham operation group, model group(underwent in vivo myocardial I/R), EA pretreatment group [EA at Neiguan(PC 6) 1 week before I/R], wortmannin group(1 week before I/R, the PI3K inhibitor, wortmannin, was injected), EA pretreatment + wortmannin group(both pretreatments were performed simultaneously). After establishing the I/R model, 2,3,5-triphenyltetrazolium chloride(TTC) staining was used to analyze the weight and area of the myocardial infarction tissue.The biosignal and pressure test system was used to determine the left ventricular systolic mean pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), fractional shortening(FS), and ejection fraction(EF). Ultraviolet spectrophotometry was used to determine the expression of creatine kinase(CK)-MB, inducible nitric oxide synthase(i NOS), and total antioxidant capacity(T-AOC) in the serum. The expression of autophagy-related protein 13(ATG13), mammalian target of rapamycin(m TOR), and phosphatidylinositol 3-kinase(PI3K) in cardiac muscle cells was determined by immunofluorescence. Hematoxylin and eosin staining was used to observe autophagy-related pathological changes in rat cardiomyocytes, and ultrastructural changes of cardiomyocytes were examined by transmission electron microscopy.RESULTS: In this study, the infarction size and tissue weight of the EA pretreatment group were decreased compared with the model group(P <0.0001). Furthermore, compared with the model group, the LVEDP values of the EA pretreatment group were significantly reduced(P = 0.0091), and the LVSP, FS, and EF values were slightly increased (P = 0.0007, 0.0020, 0.0031). EA pretreatment also significantly decreased the expression of CK-MB and i NOS, while it increased the expression of T-AOC in the serum of rats with I/R injury(P <0.0001). Furthermore, EA pretreatment slightly widened the myocardial fiber space, reduced necrosis and myocardial cell swelling and maintained the nucleus and mitochondria structure intact.CONCLUSION: EA pretreatment promoted autophagy flux and alleviated myocardial I/R injury through the PI3K-Akt-m TOR pathway.

Efficacy of self-made Gengnian decoction on phosphatidylinositol 3-kinases/protein kinase B/mammalian target of rapamycin signaling pathway in perimenopausal rats

OBJECTIVE:To investigate the efficacy of self-made Gengnian decoction on expressions of phosphatidylinositol 3-kinase(PI3 K),protein kinase B(Akt)and mammalian target of rapamycin(m TOR)in ovarian tissues of perimenopausal rats.They were identified with symptom pattern of kidney-Yang deficiency in terms of Traditional Chinese Medicine.METHODS:Female Sprague-Dawley rats aged10-12 months were selected.Estrous cycle was observed by vaginal smears of keratinocytes to screen the perimenopausal model rats.The chosen rats were randomly divided into five groups,including perimenopausal model of kidney-Yang deficiency group(24 rats),self-made Gengnian decoction of high-dose group(24 rats),self-made Gengnian decoction of middle-dose group(24 rats),self-made Gengnian decoction of low dose group(24 rats)and tibolone control group(24 rats).In addition,rats aged 4-6 months were selected as young control group.The perimenopausal model rats of kidney-Yang deficiency were prepared by alternative intramuscular injection of hydrocortisone 5 mg·kg^-1·d^-1The successfully prepared models in self-made Gengnian decoction of high-dose,middle-dose and low-dose groups and tibolone control group were given self-made Gengnian decoction 26.4,13.2 and 6.6 mg·kg^-1·d^-1,and tibolone tablets solvent 0.22 mg·kg^-1·d^-1,respectively,through intragastric administration.Models group and young control group were given the same dose of normal saline,1 time a day for 15 consecutive days.24 h after the last administration,blood and ovarian tissues were collected after anesthesia with 20%ethyl carbamate.The follicles of different levels in ovarian tissue were observed and counted by histopathological hematoxylin-eosin staining.Enzyme linked immunosorbent assay was applied to test insulin-like growth factor-1(IGF-1)level in the serum of experimental rats.The expression levels of PI3 K,phosphorylated-Akt(p-Akt)and phosphorylated-m TOR(p-m TOR)m RNA in ovarian tissue were detected by quantitative real-time polymerase chain reaction.RESULTS:The total follicle counts of perimenopausal model rats with kidney-Yang deficiency were significantly reduced,and the number of follicles(mainly increased in preantral follicles and antral follicles)in perimenopausal model rats with kidney-Yang deficiency was significantly increased after intervention of high and middle doses of Gengnian decoction and tibolone(P<0.05).Compared with normal rats in young control group,the levels of IGF-1 in serum of perimenopausal rats with kidney-Yang deficiency were significantly decreased(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated.The relative expression levels of PI3 K,p-Akt,p-m TOR m RNA in ovarian tissues of perimenopausal rats with kidney-Yang deficiency were significantly lower than those of young rats(P<0.01),and those intervened by high dose of Gengnian decoction and tibolone were significantly up-regulated(P<0.05).CONCLUSION:Self-made Gengnian decoction can increase the levels of IGF-1,PI3 K,Akt and m TOR m RNA expression in serum.

乙酰胆碱受体对胞壁酰二肽激活小鼠巨噬细胞NLR2/RIP2通路的调控作用

目的 评价乙酰胆碱受体对胞壁酰二肽（MDP）激活小鼠巨噬细胞Nod样受体2/受体相互作用蛋白2（NLR2/RIP2）通路的调控作用.方法 RAW264.7细胞长至对数生长期时,接种于12孔培养板（细胞密度1＆#215;106个/ml,2 ml/孔）,108个培养孔.采用随机数字表法,将其分为3组（n=36）,正常对照组（C组）常规培养;M组加入MDP,终浓度为10 μg/ml;G组同时加入MDP和α7烟碱型乙酰胆碱受体特异性激动剂GTS-21,终浓度分别为10、50 μg/ml.分别于MDP孵育1、6、24h时取12个培养孔,取细胞悬液,采用实时荧光定量PCR法检测NLR2 mRNA表达,采用Western blot印迹法检测RIP2表达,采用ELISA法检测培养液TNF-α和高迁移率族蛋白-1（HMGB1）的浓度.结果 与C组比较,M组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平升高（P＜0.05）;与M组比较,G组不同时点NLR2 mRNA、RIP2、TNF-α和HMGB1的水平降低（P＜0.05）.结论 乙酰胆碱受体可抑制MDP激活小鼠巨噬细胞NLR2/RIP2通路转导.

Bushenhuoxue improves cognitive function and activates brain-derived neurotrophic factor-mediated signaling in a rat model of vascular dementia

OBJECTIVE:To explore the protective mechanisms of the Traditional Chinese Medicine Bushenhuoxue(BSHX)in a rat model of vascular dementia(VD).METHODS:A rat model of VD was developed using bilateral common carotid artery occlusion(BCCAO).Rats were administered BSHX(10.14 or 5.07 g/kg),nimodipine(11.06 mg/kg;positive control),or saline(control)by gavage daily for 30 d post-surgery.Learning and memory abilities were assessed using the Morris water maze.Morphological changes in the hippocampus were observed using light microscopy(hematoxylin and eosin staining)and transmission electron microscopy(TEM).The m RNA and protein expression levels of brain-derived neurotrophic factor(BDNF),tyrosine receptor kinase B(Trk B),phosphatidyl inositol 3-kinase(PI3 K),serine/threonine kinase(AKT),and c AMP response element binding protein(CREB)were measured by real-time polymerase chain reaction(RT-PCR)and Western blot,respectively.RESULTS:Compared with the sham group,rats with BCCAO exhibited impaired learning and memory abilities(Morris water maze)and showed abnormalities in neuronal morphology(light microscopy)and ultrastructure(TEM)in the hippocampus.They also had decreased m RNA and protein expressions of BDNF,Trk B,PI3 K,AKT,and CREB in hippocampal tissue(all P<0.05).In rats with BCCAO,administration of BSHX attenuated deficits in learning and memory,improved the morphology and ultrastructure of hippocampal neurons,and enhanced m RNA and protein expression levels of BDNF,Trk B,PI3 K,AKT,and CREB(all P<0.05).CONCLUSION:BSHX may protect hippocampal neurons and improve learning and memory abilities,at least in part via the activation of BDNF/Trk B/PI3 K/AKT/CREB signaling.