Mechanism of melatonin regulation of PI3K/Akt/mTOR signaling in hypothalamus delaying the initiation of puberty in female mice / 解剖学报

Objective To explore the effect of melatonin ( MLT) on the initiation of puberty in female mice and on the expression level of phosphatidylinositol-3-kinases ( PI3K)/protein kinase B ( Akt)/mammalian target of rapamycin (mTOR) signaling pathway in the frypothalamus. Methods Seventy-eight 20-day-old female KM mice were randomly divided into melatonin (MLT) group and normal saline (NS) group, with 39 mice in each group. Starting at 22 days of age, the MLT group was given a subcutaneous injection of 1 mg/kg melatonin and the NS group was given an equal volume of saline. Thirty-two days of age were selected as the sampling point before puberty initiation and 13 mice were executed in each of the two groups, while 37 and 42 days of age were selected as the sampling point after puberty initiation and 13 mice were executed in each of the two groups. Observation of vaginal opening time in mice, weighing of ovaries and uterus to calculate organ indices. HE staining to observe the number of ovarian corpora lutea. The levels of serum luteinizing hormone (LH)were determined by ELISA. The mRNA and protein expression levels of PI3K/Akt/mTOR pathway in frypothalamus were detected by Real-time PCR and Western blotting. Results Compared with the normal saline group, mice in the melatonin group had significantly delayed vaginal opening time ( P < 0. 05 ) , decreased significantly ovarian and uterine volume and index (P<0. 05) , decreased significantly serum LH levels (P<0. 05) , and decreased significantly mRNA and protein expression levels of the frypothalamic PI3K/Akt/mTOR pathway (P<0. 05). Conclusion Melatonin delays puberty initiation in mice by a mechanism that ma)' be related to inhibition of the hypothalamic PI3K/Akt/mTOR signalling pathway.

Effects of silicon dioxide exposure on lipid deposition and PI3K/AKT/mTOR signaling pathway in MLE-12 cells / 环境与职业医学

Background Lipid metabolism imbalance is tightly linked to the development and progression of multiple diseases. The phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway is important for the regulation of lipid metabolism. However, whether silicosis is associated with lipid metabolic abnormalities has yet to be explored. Objective To observe the changes of lipid deposition, cholesterol, and phosphorylated proteins of PI3K/AKT/mTOR pathway in silicon dioxide (SiO2)-induced MLE-12 cells and to explore potential mechanism of lipid composition regulated though the pathway. Methods (1) MLE-12 cells were stimulated with 50 mg·L−1 SiO2 suspension, and divided into fourgroups: a control group and three SiO2 groups (12, 24, and 48 h of stimulation). (2) Cellproliferation was detected to determine an optimal dose of LY294002, an inhibitor of PI3K protein. LY294002 at 5 μmol·L−1 was used for further study, in which MLE-12 cells cultured for 48 h were divided into four groups: a control group; a 50 mg·L−1 SiO2 suspension stimulation group; a 50 mg·L−1 SiO2 suspension and 5 μmol·L−1 LY294002 treatment group; a 5 μmol·L−1 LY294002 treatment group. Total cholesterol (TC), free cholesterol (FC), cholesterol ester (CE; total cholesterol minus free cholesterol), and triglycerides (TG) were measured with enzyme assay kits. Lipid deposition was observed using Oil Red O staining. The expressions of p-PI3K, p-AKT, and p-mTOR proteins were detected by Western blotting. Results (1) The contents of TC, FC, and CE in the 50 mg·L−1 SiO2-induced MLE-12 cells were increased compared to those of the control group in a time-dependent manner by trend analysis, and the increment at 24 and 48 h were significant. By 48 h, the contents of cholesterol indicators were all elevated: TC from (2.242±0.181) mg·g−1 to (5.148±0.544) mg·g−1, FC from (1.923±0.158) mg·g−1 to (4.168±0.433) mg·g−1, and CE from (0.318±0.067) mg·g−1 to (0.978±0.134) mg·g−1, compared with the control group (P<0.01). The changes of TG were not significant (P>0.05). The SiO2 suspension induced orange-red particle deposition in the MLE-12 cells, especially at 48 h (P<0.01). The protein expression levels of p-PI3K, p-AKT, and p-mTOR in SiO2-stimulated MLE-12 cells were higher than those of the control groups with the prolongation of stimulation time, which peaked at 48 h (P<0.01). (2) The contents of TC, FC, and CE in MLE-12 cells of the SiO2 + LY294002 group were decreased, comparing to those of the SiO2 stimulation only group (P<0.01), companied with less orange-red lipid deposition, and suppressed protein expression levels of p-PI3K, p-AKT, and p-mTOR (P<0.01). Conclusion SiO2 could induce increases of cholesterol and lipid deposition through activation of PI3K/AKT/mTOR signaling pathway in MLE-12 cells.

Effect of Huangqisan Pellets on PI3K/Akt/mTOR Signaling Pathway and Autophagy in Kidney of Diabetic Nephropathy Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Huangqisan pellets （HQS） on the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway and autophagy in the kidney of diabetic nephropathy （DN） rats. MethodDN rat model was established through high-fat diet combined with intraperitoneal injection of streptozotocin （35 mg·kg-1）. DN rats were randomly assigned into model group， irbesartan （0.027 g·kg-1） group， low-dose HQS （0.54 g·kg-1） group and high-dose HQS （1.08 g·kg-1） group. The levels of 24 h urinary total protein （UTP）， serum albumin （Alb）， serum creatinine （SCr）， urea nitrogen （BUN）， triglyceride （TG） and total cholesterol （TC） were measured after 12 weeks of continuous administration. The pathological changes of renal tissue were observed via hematoxylin-eosin （HE） staining. The expression of podocyte split diaphragm proteins nephrin and podocin in the renal tissue were detected by immunohistochemistry. The protein levels and phosphorylation of key proteins in PI3K/Akt/mTOR signaling pathway， as well as the expression of yeast Atg6 homolog （Beclin1） and microtubule-associated protein 1 light chain 3 （LC3） in the renal tissue were analyzed by Western blot. ResultCompared with the control group， the model group showcased increased 24 h UTP， SCr， BUN， TG， and TC levels and decreased Alb level （P<0.01）. After modeling， the rats showed granulosity of epithelial cells of renal tubules， thickening of capillary basement membrane， proliferation of mesangial cells， and sclerosis of glomerulus. Furthermore， modeling down-regulated the expression of nephrin and podocin in the podocyte hiatus of glomerulus （P<0.01） as well as the protein levels of p-PI3K， p-Akt， and p-mTOR and the autophagy markers LC3 and Beclin1 in renal tissue （P<0.01）. Compared with model group， irbesartan and HQS decreased the 24 h UTP， Cr， BUN， TG， and TC levels， increased the Alb level， and alleviated the pathological damage of kidney. Moreover， they up-regulated the expression of Nephrin and Podocin in the podocyte hiatus of glomerulus， as well as the protein levels of p-PI3K， p-Akt， p-mTOR， LC3， and Beclin1 in renal tissue （P<0.05， P<0.01）. ConclusionHQS may inhibit the PI3K/Akt/mTOR signaling pathway to enhance podocyte autophagy and protect the glomerulus， thus slowing down the development of DN.

Effect of Sishenwan on PI3K/Akt/mTOR Signal Pathway in Colonic Tissue of Rats with Ulcerative Colitis Model of Spleen Kidney Yang Deficiency / 中国实验方剂学杂志

Objective:To discuss the effect of Sishenwan on phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin（PI3K/Akt/mTOR） signaling pathway related genes and proteins in colon tissue and interleukin-1<italic>β</italic>（IL-1<italic>β</italic>） and interleukin-10（IL-10） expression levels in serum of ulcerative colitis （UC） model rats with spleen kidney Yang deficiency. Method:The 120 SPF Wistar rats were randomly divided into normal group and model group after 7 days of adaptive feeding in SPF laboratory. The model group were given dinitrobenzene sulfonate （DNBS）/ethanol solution enema+hydrocortisone subcutaneously injection+senna leaf gavage to establish UC model of spleen kidney yang deficiency. The rats who successfully established the model were randomly divided into five groups：model group， mesalazine （0.36 g·kg^-1） group， and Sishenwan high， medium and low dose （3.2，1.6，0.8 g·kg^-1） groups， the volume of which was 10 mL·kg^-1. The model group and the blank group were given distilled water of the same volume. Once a day for 21 days. Observe the general conditions of the rats daily， and record the weight， fecal traits and occult blood of the mice for disease activity index （DAI） scoring.Take the rat colon tissue to observe the gross morphology and colon injury， and score the colon mucosal injury index （CMDI）. Hematoxylin-eosin （HE） staining was used to observe pathological changes. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was used to detect the expression level of PI3K， Akt， mTOR mRNA in colon tissue. The levels of IL-1<italic>β</italic> and IL-10 in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression level of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， mTOR， p-mTOR protein in colon tissue. Result:Compared with blank group， the general survival status of the rats in model group was relatively poor， the DAI score and the CMDI index were significantly increased （<italic>P<</italic>0.01）. The intestinal mucosa partially disappears， the glands disappear， and a large number of inflammatory cells infiltrate and gather in the mucosal layer and the base layer in the pathological sections of the model group. The expression levels of PI3K， Akt， and mTOR mRNA were significantly increased （<italic>P</italic><0.01）. The IL-1<italic>β</italic> content was significantly increased and the IL-10 content was significantly decreased （<italic>P</italic><0.01）. The expression levels of p-PI3K，p-Akt and p-mTOR protein were significantly increased （<italic>P</italic><0.01）. Compared with the model group， the DAI scores of Sishenwan high and medium dose groups and mesalazine group decreased （<italic>P</italic><0.05）. The CMDI index of mesalazine group and the high， middle and low dose groups of Sishenwan was significantly reduced （<italic>P</italic><0.01）. Pathological sections of rats showed that the inflammatory cells in the drug group decreased， and the mucosal layer structure returned to normal to varying degrees. The mesalazine group and the Sishenwan medium-dose group had the best effects， and the mucosal structure was close to the blank control group. The expression levels of PI3K， Akt， mTOR mRNA in the high and medium dose groups of Sishenwan， mesalazine group and Akt mRNA in low dose group of Sishenwan were significantly reduced （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of PI3K and mTOR mRNA in low-dose group of Sishenwan decreased， but the difference was not statistically significant. The IL-1<italic>β</italic> content was significantly reduced and the IL-10 content was significantly increased in high， medium dose groups of Sishenwan and mesalazine groups （<italic>P</italic><0.05，<italic>P</italic><0.01）. The level of IL-1<italic>β</italic> decreased and the level of IL-10 increased in the low-dose group of Sishenwan， but the difference was not statistically significant. The expression levels of p-PI3K， p-Akt and p-mTOR protein in the high， medium， and low dose groups of Sishenwan and mesalazine group decreased to varying degrees， and the differences were statistically significant（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Sishenwan has the effect of improving the general condition and intestinal mucosal damage of ulcerative colitis model rats with spleen and kidney Yang deficiency. The mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

Research progress in relationships between mTOR signaling pathway and drug addiction in central nervous system / 中国药理学与毒理学杂志

Drug addiction is a chronic recurrent brain disease. Repeated drug exposure may lead to neuroadaptive changes in the brain loop, and obsessive-compulsive drug seeking behaviors. In vivo, the signal transduction cascade amplification pathway mediates the remodeling of the reward loop of the central nervous system. Related studies have shown that the mammalian target of rapamycin (mTOR) signaling pathway is directly related to drug addiction, inducing neural adaptation and reward effects. Therefore, the research progress in mTOR signaling pathway and relationships between drug addiction were reviewed, and the prospects of research on the molecular mechanism of drug addiction were discussed.

Effect of raddeanin A on autophagy and apoptosis of colon cancer HCT116 cells through regulating mTOR pathways / 重庆医学

Objective To explore raddeanin A(RA) inhibiting the proliferation of HCT116 cells and its related mechanism.Methods The MTT method was used to observe the RA inhibition on the proliferation of gastric cancer cells HCT116.Flow cytometry was used to detect the RA effects on cell apoptosis.Transmission electron microscope(TEM) was used to observe RA induced autophagy.Western blot was used to test expression of apoptosis and autophagy related proteins.Results RA could significantly inhibit the proliferation of HCT16 cells with concentration and time dependence;double layer membrane of autophagosome was detected by TEM;FCM showed that RA induced apoptosis in HCT116 cells,moreover the apoptosis rate was increased with the concentration increase;Western blot showed that the expression of autophagy related proteins(Beclin1 and LC3) was increased,the expression of apoptosis inhibition protein Bcl-2 was decreased.On the contrary,the expression of apoptosis promotion proteins(Bax,Cleaved-caspase-3,Cleaved-PARP) was increased,the expression of mTOR protein in the mTOR signal pathway was increased,while the p-mTOR protein expression was decreased.When the mTOR pathway inhibitor rapamycin(RAPA) was added to cells,Beclin-1,LC3,Cleaved-caspase-3,Cleaved-PARP proteins were increased,and apoptosis rate was also increased.Conclusion RA can inhibit the proliferation of HCT116 cells and can induce cellular autophagy and apoptosis,its mechanism may be realized by regulating the mTOR signal pathway.

Influence of Phosphatidylinositol-3-Kinase/Protein Kinase B-Mammalian Target of Rapamycin Signaling Pathway on the Neuropathic Pain Complicated by Nucleoside Reverse Transcriptase Inhibitors for the Treatment of HIV Infection / 中华医学杂志(英文版)

Background@#Nucleoside reverse transcriptase inhibitors (NRTIs) are the earliest and most commonly used anti-human immunodeficiency virus drugs and play an important role in high active antiretroviral therapy. However, NRTI drug therapy can cause peripheral neuropathic pain. In this study, we aimed to investigate the mechanisms of rapamycin on the pain sensitization of model mice by in vivo experiments to explore the effect of mammalian target of rapamycin (mTOR) in the pathogenesis of neuropathic pain caused by NRTIs.@*Methods@#Male Kun Ming (KM) mice weighing 20-22 g were divided into control, 2 mg/kg rapamycin, 12 mg/kg stavudine, and CMC-Na groups. Drugs were orally administered to mice for 42 consecutive days. The von Frey filament detection and thermal pain tests were conducted on day 7, 14, 21, 28, 35, and 42 after drug administration. After the last behavioral tests, immunohistochemistry and western blotting assay were used for the measurement of mTOR and other biomarkers. Multivariate analysis of variance was used.@*Results@#The beneficial effects of rapamycin on neuropathic pain were attributed to a reduction in mammalian target of rapamycin sensitive complex 1 (mTORC1)-positive cells (70.80 ± 2.41 vs. 112.30 ± 5.66, F = 34.36, P < 0.01) and mTORC1 activity in the mouse spinal cord. Mechanistic studies revealed that Protein Kinase B (Akt)/mTOR signaling pathway blockade with rapamycin prevented the phosphorylation of mTORC1 in stavudine-intoxicated mice (0.72 ± 0.04 vs. 0.86 ± 0.03, F = 4.24, P = 0.045), as well as decreased the expression of phospho-p70S6K (0.47 ± 0.01 vs. 0.68 ± 0.03, F = 6.01, P = 0.022) and phospho-4EBP1 (0.90 ± 0.04 vs. 0.94 ± 0.06, F = 0.28, P = 0.646).@*Conclusions@#Taken together, these results suggest that stavudine elevates the expression and activity of mTORC1 in the spinal cord through activating the Akt/mTOR signaling pathway. The data also provide evidence that rapamycin might be useful for the treatment of peripheral neuropathic pain.

Effect of Atorvastatin on apoptosis of HL-60 leukemic cells via the regulation of the phosphatidylinositol 3-kinase/serine/threonine protein kinase/mammalian target of rapamycin signaling pathway / 中华实用儿科临床杂志

Objective To investigate the effect of Atorvastatin on the proliferation and apoptosis of leukemic HL-60-cell line,and to explore the possible role of phosphatidylinositol 3-kinase/serine/threonine protein kinase /mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway in this process.Methods HL-60 cells were incubated with different concentrations of Atorvastatin (1,5,10 μmol/L),and HL-60 cells without any treatment were used as controls.The proliferation of HL-60 which was investigated by four methyl thiazolyl tetrazolium assay when cells were cultured for 12,24,48 hours.The apoptosis was detected by flow cytometry after cells were incubated for 48 hours.The mRNA and protein expressions of AKT,PI3K and mTOR were detected by reverse transcription-polymerase chain reaction and Western blot methods,respectively.Results The results indicated that Atorvastatin could inhibit the proliferation and induce the apoptosis of HL-60 cells.When treated with 10 μmol/L Atorvastatin after 48 h,the proliferation inhibition of HL-60 was observed most obviously,with a high rate of (39.77 ± 3.01) ％,compared with the control group,it had statistical significance (t =4.016,P ＜ 0.01),meanwhile,the apoptosis of HL-60 was most notable,at a rate of (43.29 ±3.91)％,compared with the control group,it had statistical significance (t =3.625,P ＜ 0.05).There were basal expression of AKT,PI3K and mTOR in the control group.When treated with 10 μmol/L Atorvastatin after 48 h,the mRNA expression of PI3K,AKT and mTOR were down-regulated most obviously,at a decrease of (37.05 ± 4.11) ％,(53.79 ± 3.27) ％,(40.63 ± 2.42) ％ (t =4.805,3.799,4.312,all P ＜ 0.05),respectively,in comparison with the control group.At the same condition,the protein expression of PI3K,AKT and mTOR were decreased most visibly,with a decline of (41.09 ± 3.17) ％,(45.67 ± 2.92) ％,(63.41 ± 3.59) ％ (t =3.576,4.727,4.902,all P ＜ 0.05) respectively in comparison with the control group.Conclusions Atorvastatin can inhibit the proliferation and induce the apoptosis of leukemic cell HL-60,and the mechanism may be associated with the PI3K/ AKT/mTOR signal pathway.

Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway and epilepsy / 中华实用儿科临床杂志

Study of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway has been becoming more and more popular.This pathway widely exists in kinds of cells of human being.As one main anti-apoptic and enhancing survival pathway in cells, it plays an important role in cellular growth (increased cell size), proliferation (increased cell number), apoptosis, cell survival and migration.At the same time,the pathway regulates many major cellular processes and is implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes, and neurodegeneration disease, epilepsy.In recent years,many studies have shown that the dysfunction of PI3K/Akt/mTOR signaling pathway can lead to neurodevelopmental disease.Loss of tuberous sclerosis complex (TSC)1/2 or phosphatase ad tensin homologue deleted on chromosome 10 (PTEN), or environmental stimuli such as inflammation, epilepsy, or hypoxia may stimulate mTOR-dependent protein synthesis,resulting in a host of cellular, structural, and physiological responses that culminate in clinical symptoms.Study the role of mTOR signaling pathway in early-onset epileptic encephalopathy, discuss the intervention and therapy in early-onset epileptic encephalopathy have important clinical meanings.In this article, the components, physiological functions,information were elucidated relative to the PI3 K/Akt/mTOR signaling pathway, and the interaction of the signaling pathway and epilepsy was discussed.