mRNA Expression Of PI3K Research Articles

Anti-type I allergic effects of Jing-Fang powder extracts via PI3K/Akt pathway in vitro and in vivo

Jing-Fang powder (Schizonepeta tenuifolia Briq. and Saposhnikovia divaricata (Turcz.) Schischk.) was used to treat chronic bronchitis, asthma and chronic urticaria. Based on the preliminary results of screening research on the antiallergic effective parts of Jing-Fang powder, its ethyl acetate extract fractions (JFEE) and isolate D (JFEE-D) showed the best anti-allergic effect. RBL-2H3 cell activation degranulation model and mice passive cutaneous anaphylaxis (PCA) reaction model were used to investigate the effects and mechanisms of JFEE and JFEE-D on IgE-mediated type I allergic reactions. LC–MS was utilized to determine the composition of JFEE and JFEE-D. We found that JFEE and JFEE-D significantly reduced β-HEX, histamine, IL-4, IL-6 levels in cell supernatants, and improved the degree and morphology of cell degranulation. JFEE and JFEE-D significantly inhibited the increase of ear vascular permeability and abnormal increase of serum IgE, TNF-α, IL-6 levels. JFEE and JFEE-D inhibited mRNA expression of PI3K and Akt and down-regulated protein expression of PI3K, Akt, p-Akt, and PLCγ1 in sensitized RBL-2H3 cells. The combined use of JFEE and JFEE-D with pathway inhibitor Wortmannin revealed synergistic down-regulation of PI3K, Akt, and p-Akt protein expression. The combined use of pathway agonist IGF-1, JFEE and JFEE-D down-regulated increase of p-Akt/Akt protein expression. Moreover, JFEE and JFEE-D significantly inhibited protein expression of PI3K, p-Akt and PLCγ1 in PCA model mice. These results show that JFEE and JFEE-D inhibit type I allergic reactions by inhibiting PI3K/Akt signaling pathway.

To study the mechanism of Cuscuta chinensis Lam. And Lycium barbarum L. in the treatment of asthenospermia based on network pharmacology

Ethnopharmacological relevanceCuscuta chinensis Lam. and Lycium barbarum L. (SC-FL) is a commonly used kidney tonic Chinese medicine combination that is widely used in the clinical treatment of oligoasthenospermia.However, its specific mechanism remains unclear and requires in-depth study. Aim of the studyTo explore the potential targets of SC-FL in the treatment of oligoasthenospermia using network pharmacology, and to verify the results with in vivo and in vitro experiments. Materials and methodsA herb-compound-target-disease network and PPI network were constructed with Cytoscape software. The targets of SC-FL for the treatment of male sterility were introduced into a bioinformatics annotation database, and the GO and KEGG databases were used for pathway enrichment analysis. Subsequently, Tripterygium wilfordii Hook. f. (GTW) polyglycoside was used to induce a spermatogenic dysfunction model in GC-1 spg cells and SD male rats in in vitro and in vivo experiments, respectively. The SC-FL and PI3K pathway inhibitor LY294002 was used to intervene in the spermatogenic dysfunction model to detect the expression of proteins and mRNA related to the PI3K pathway and to detect the indicators related to proliferation and apoptosis. ResultsIn in vitro experiments, the percentage of spermatogenic cells and the proportion of GC-1 spg cells at G0/G1 and G2/M stages in the model group (GTW group) and the inhibitor group (LY group) were significantly decreased (P < 0.01) compared with the blank control group (NC group). The apoptosis rate of the GTW group was significantly increased (P < 0.01). The ultrastructures of GC-1 spg cells in the GTW group and LY group were obviously destroyed. Compared with the GTW group, the SC-FL group had a significantly reduced apoptosis rate of GC-1 spg cells, reduced percentage of cells in S phase, and a significantly improved mitochondrial membrane potential. SC-FL can repair the ultrastructure of GC-1 spg cells damaged by GTW. The above effects of SC-FL are closely related to up-regulation of GFRa1, RET, PI3K, p-AKT, and Bcl-2 and down-regulation of BAD and BAX proteins and mRNA expression. In vivo, compared with the GTW group, the body mass, testicular mass, and epididymal weight of the GTW + SC-FL group were significantly increased (P < 0.01). Sperm concentrations and the PR + NP of GTW + SC-FL were significantly higher than in the GTW group (P < 0.01 or P < 0.05). FSH, LH, and T levels in the GTW + SC-FL and LY + SC-FL groups were significantly higher than those in the GTW and LY group (P < 0.01 or P < 0.05). HE staining results showed that the morphology of testicular tissue in the GTW + SC-FL and LY + SC-FL groups was superior to that in the GTW and LY group. The above effects of SC-FL are closely related to the up-regulation of proteins and mRNA expression of PI3K, p-AKT, and Bcl-2. ConclusionThrough the PI3K/Akt signaling pathway, SC-FL up-regulates GFRa1, RET, PI3K, p-AKT, and Bcl-2, and down-regulates the expression of BAD and BAX proteins and mRNA, thus reducing the percentage of GC-1 spg cells in S-phase, significantly increasing the mitochondrial membrane potential, significantly reducing cell apoptosis, and improving sperm counts and viability.

Intra-Cellular Calcium Signaling Pathways (PKC, RAS/RAF/MAPK, PI3K) in Lamina Cribrosa Cells in Glaucoma.

The lamina cribrosa (LC) is a key site of fibrotic damage in glaucomatous optic neuropathy and the precise mechanisms of LC change remain unclear. Elevated Ca2+ is a major driver of fibrosis, and therefore intracellular Ca2+ signaling pathways are relevant glaucoma-related mechanisms that need to be studied. Protein kinase C (PKC), mitogen-activated MAPK kinases (p38 and p42/44-MAPK), and the PI3K/mTOR axis are key Ca2+ signal transducers in fibrosis and we therefore investigated their expression and activity in normal and glaucoma cultured LC cells. We show, using Western immune-blotting, that hyposmotic-induced cellular swelling activates PKCα, p42/p44, and p38 MAPKs, the activity is transient and biphasic as it peaks between 2 min and 10 min. The expression and activity of PKCα, p38 and p42/p44-MAPKs are significantly (p < 0.05) increased in glaucoma LC cells at basal level, and at different time-points after hyposmotic stretch. We also found elevated mRNA expression of mRNA expression of PI3K, IP3R, mTOR, and CaMKII in glaucoma LC cells. This study has identified abnormalities in multiple calcium signaling pathways (PKCα, MAPK, PI3K) in glaucoma LC cells, which might have significant functional and therapeutic implications in optic nerve head (ONH) fibrosis and cupping in glaucoma.

Long-term co-administration of sodium nitrite and sodium hydrosulfide inhibits hepatic gluconeogenesis in male type 2 diabetic rats: Role of PI3K-Akt-eNOS pathway

ObjectiveA deficiency in hydrogen sulfide (H2S) and nitric oxide (NO) contributes to the development of type 2 diabetes (T2D). An inhibitory effect on liver gluconeogenesis has been reported in rats with T2D with co-administration of sodium nitrite and sodium hydrosulfide (NaSH); the underlying mechanisms have however not yet been elucidated. The aim of this study is to determine the long-term effects of co-administering sodium nitrite and NaSH on expression of genes involved in liver gluconeogenesis in rats with T2D. MethodsT2D was induced using a high fat diet combined with low-dose of streptozotocin (30 mg/kg). Rats were divided into 5 groups (n = 7/group): Control, T2D, T2D + nitrite, T2D + NaSH, and T2D + nitrite+NaSH. Nitrite (50 mg/L) and NaSH (0.28 mg/kg) were administered for 9 weeks. Intraperitoneal pyruvate tolerance test (PTT) was performed at the end of the ninth week and mRNA expressions of PI3K, Akt, eNOS, PEPCK, G6Pase, and FBPase were measured in the liver. ResultsCo-administration of nitrite and NaSH decreased elevated serum glucose concentrations during PTT. Compared to T2D + nitrite, co-administration of nitrite and NaSH resulted in significant increases in mRNA expression of PI3K, Akt, and eNOS and significant decreases in mRNA expression of G6Pase and FBPase but had no effect on PEPCK expression. ConclusionLong-term NaSH administration at low-dose, potentiated the inhibitory effects of nitrite on mRNA expression of key liver gluconeogenic enzymes in rats with T2D. This inhibitory effect of nitrite and NaSH co-administration on gluconeogenesis were associated with increased gene expression of PI3K, Akt, and eNOS in the liver.

Involvement of mTOR/Survivin signaling pathway in TUA(2β, 3β, 23-trihydroxy-urs-12-ene-28-olic acid)-induced apoptosis in human gastric cancer cell line BGC823 cells

Ethno-pharmacological relevanceA natural ursolic compound, 2β,3β,23-trihydroxy-urs-12-ene-28-olic acid (TUA) was isolated from the root of Actinidiafulvicoma Hance. (A.fulvicoma Radix), which is used as a traditional hebal medicine to cure innominate inflammation of unknown origin of the digestive tract in the She nationality.Aim of the study: The aim of present study was to investigate the effects of TUA on gastric cancer and to clarify the potential mechanisms in human gastric cancer cell line BGC823 cells in vitro and in vivo. Materials and methodsCell proliferation, apoptosis, cell cycle, autophagy were all measured by MTS assay, flow cytometry following exposure to TUA. The mRNA expressions of PI3K, AKT, mTOR, P70S6K, Survivin and the protein expressions of p-PI3K, p-AKT, p-mTOR, p-P70S6K, Survivin were determined by qRT-PCR and Western blotting analysis, respectively. In vivo antitumor activity of TUA was assessed in a xenograft model. ResultsIn vitro studies showed that TUA significantly suppressed the viability of BGC823 cells in a concentration- and time-dependent manner but not GES-1 non-tumorigenic human gastric epithelial cells. TUA also significantly increased the apoptosis rate and the sub G2 population by cell cycle analysis in a concentration dependent manner. Exposure to TUA decreased PI3K, AKT, mTOR, P70S6K, Survivin mRNA, inhibited the phosphorylation of major receptors involved in autophagy and apoptosis, such as PI3K, AKT, mTOR and P70S6K, while reduced the expression of Survivin in BGC cells. In vivo studies showed that TUA decreased tumor volume and tumor weight and also down regulated the autophagy-related proteins expression. ConclusionsTUA occupies underlying antitumor effects, the potential mechanisms may involve the suppression of mTOR/Survivin pathways connected to autophagy and the activation of apoptotic pathways in gastric cancer cells.

Astilbin-induced inhibition of the PI3K/AKT signaling pathway decelerates the progression of osteoarthritis.

Degeneration and destruction of articular cartilage are the key characteristics of osteoarthritis (OA). In recent studies, the use of astilbin (AST), the primary active ingredient of Astilbe chinensis, has been shown to correlate with a reduction in inflammatory disease symptoms. The present study aimed to investigate the effects and mechanisms of AST on OA. A rat model of OA was constructed and in vivo experiments were performed using the AST, PBS, OA and control groups. The cartilage tissues of each group were assessed by hematoxylin and eosin and toluidine blue staining. The gene expression levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, AKT, PI3K and other related proteins were analyzed by reverse transcription-quantitative PCR and western blot analysis. AST was found to significantly inhibit IL-1β and TNF-α protein expression; this further confirmed that IL-1β, TNF-α and PI3K mRNA expression was downregulated, indicating that the protective mechanism of AST is associated with the PI3K/AKT pathway. Overall, the results of the present study demonstrate that AST can improve OA symptoms by downregulating the PI3K/AKT signaling pathway, and may therefore be a potential therapeutic option for patients with OA.

Comparison of Autophagy mRNA Expression between Chronic Otitis Media With and Without Cholesteatoma.

Background and ObjectivesAutophagy is known to be associated with pathogen infection. However, the expression of autophagy-related proteins has not been studied in chronic otitis media without cholesteatoma (COM) or with cholesteatoma (CholeOM). This study aimed to determine whether there is a difference between COM and CholeOM in autophagy-related gene mRNA expression. Subjects and MethodsFor 47 patients with chronic otitis media, the inflammatory tissues were classified into granulation tissue (COM) or cholesteatoma (CholeOM) according to biopsy results. ResultsPI3K mRNA expression (COM vs. CholeOM, mean±SD, 0.009±0.010 vs. 0.003±0.004; p=0.004) was lower, whereas Beclin-1 mRNA expression (0.089±0.107 vs. 0.176±0.163; p=0.034) was higher in the CholeOM group. Expression of PI3K mRNA in the CholeOM group was lower than that in the COM subgroups with presence of bacteria (0.022±0.019 vs. 0.001±0.001; p=0.001), otorrhea (0.049±0.068 vs. 0.003±0.004; p=0.004), and hearing loss over 40 dB (0.083±0.130 vs. 0.003±0.004; p=0.005). ConclusionsThe data suggested that different autophagy proteins play important roles in chronic otitis media according to the presence or absence of cholesteatoma.

MicroRNA-33a negatively regulates myoblast proliferation by targeting IGF1, follistatin and cyclin D1.

MiR-33a is found as a regulator of cell proliferation in many cancer cells. However, it remains unknown if and how miR-33a plays a role in myoblast proliferation. To investigate the effect of miR-33a on myoblast proliferation, miR-33a mimic or inhibitor was co-administered with or without insulin-like growth factor 1 (IGF1) to simulation myoblasts. Our study showed that up-regulation of miR-33a impaired myoblast proliferation, while down-regulation of miR-33a enhanced myoblast proliferation. Mechanistically, we examined that miR-33a can inhibit the transcription of IGF1, follistatin (FST) and cyclin D1 (CCND1) by targeting their 3′UTR region in both HEK293T cells and duck myoblasts. Moreover, up-regulation of miR-33a decreased and its down-regulation increased the mRNA expression of PI3K, Akt, mTOR and S6K. Importantly, the decreased PI3K, Akt, mTOR and S6K expression by miR-33a mimics was abrogated by co-administered with IGF1. Altogether, our results demonstrated that miR-33a may directly target IGF1, FST and CCND1 to inhibit myoblast proliferation via PI3K/Akt/mTOR signaling pathway. In conclusion, miR-33a is a potential negative regulator of myoblast proliferation and by modulating its expression could promote the early development of skeletal muscle.

Immune-related genes expression analysis of Western mosquitofish (Gambusia affinis) challenged with Aeromonas hydrophila

The great Gambusia affinis (G. affinis) is considered as an important animal model to study the endocrine disruption, ecological behavior, and environmental pollutant. The present study aims to build a new promising infection model with Aeromonas hydrophila (A. hydrophila) in aquaculture. The mRNA expression of Rac1, MyD88, IRAK4, TAK1, IKKβ, and IL-1β in G. affinis were significance higher (P < 0.05) in the liver of G. affinis than that of brain and intestine. And the PI3K mRNA expression level was significant lower (P < 0.05) in the intestine than that of brain and liver. The mRNA levels of AKT3 were significant higher (P < 0.05) in the brain than that of liver and intestine. And then the brain, liver, and intestine were collected at different time points (0 h, 3 h, 9 h, 18 h, 24 h, 48 h) after post injection of LD50 of A. hydrophila. The 0.85% NaCl was used as a negative control for the LD50 of A. hydrophila. The RT-PCR results showed that mRNA expressions of TLR2/4 pathway downstream genes MyD88, IRAK4, TAK1, Rac1, IKKβ, and IL-1β were firstly significantly up-regulated (P < 0.05) and were then backed to the 0 h group levels in three tissues. In contrast, mRNA expressions of TLR2/4 pathway downstream genes PI3K and AKT3 were firstly significantly decreased (P < 0.05) and were then increased to the 0 h group levels in brain and intestine. In summary, the results indicated that A. hydrophila could cause inflammatory reaction in intestinal and brain. In addition, the liver showed a provocative reaction when infected with A. hydrophila.

Experimental Study on the Effect of Aconite and Angelica sinensis on Myocardial Ischemia Rats with Yang Deficiency and Blood Stasis.

Objective To investigate the intervention effect and mechanism of Aconite and Angelica sinensis on myocardial ischemia rats with Yang deficiency and blood stasis. Methods SPF-class SD rats were randomly divided into low-dose and high-dose groups. Each group was divided into control group, model group, and drug-administered group (FZ, DG, FG; 1 : 0.5, 1 : 1, 1 : 2). A rat model was prepared by intraperitoneal injection of hydrocortisone and isoproterenol plus cold stimulation. Each group was given corresponding decoction or distilled water for 14 days. The behavioral changes of rats in each group were observed. The morphological changes of rats cardiomyocytes were observed by HE staining. The average optical density (MOD value) and percentage of positive cells of Bcl-2, Bax, and Akt were determined by immunohistochemical staining method, and PEIs were calculated. Western blot and RT-PCR were used to determine the expression of PI3K, Caspase-3, Akt protein, and gene expression. Results The compatibility of Aconite and Angelica sinensis improved the morphology of rat cardiomyocytes, increased the PEI values of Akt and Bcl-2 protein, and decreased the PEI values of Bax protein (P < 0.01). The compatibility reduced the expression of Caspase-3 protein of rat myocardium and increased the protein expression of p-Akt, PI3K, and p-PI3K (P < 0.01). The compatibility also significantly reduced the expression of Caspase-3 mRNA and increased the expression of PI3K mRNA and Akt mRNA (P < 0.05 or P < 0.01), and the effect of high-dose FG (1 : 2) group is the best. Conclusions The method of preparing a rat model of myocardial ischemia with Yang deficiency and blood stasis was feasible. The compatibility of Aconite and Angelica sinensis reduced myocardial fibrosis and inflammatory reaction, protected ischemic cardiomyocytes, and reduced myocardial injury, whose mechanism may be related to the regulation of PI3K/Akt pathway. The compatible group had better intervention effects than Aconite or Angelica sinensis alone. The best one was high-dose FG (1 : 2).

MiR-365 inhibits duck myoblast proliferation by targeting IGF-I via PI3K/Akt pathway.

miR-365 is found to be involved in cancer cell proliferation and apoptosis. However, it remains unknown if and how miR-365 plays a role in myoblast proliferation. In the present study, we found that overexpression of miR-365 can inhibit duck myoblast proliferation. To uncover the mechanism by which miR-365 inhibits duck myoblast proliferation, we showed that miR-365 can down-regulate insulin-like growth factor-I (IGF-I) by directly targeting its 3′untranslated region (UTR). Moreover, enhanced miR-365 decreased the mRNA expression of PI3K, Akt, mTOR and S6K. Importantly, the enhanced PI3K, Akt, mTOR and S6K expression by miR-365 inhibitor (anti-miR-365) was abrogated by treatment with LY294002, a PI3K inhibitor. Together, our results indicated that miR-365 may target IGF-I to inhibit duck myoblast proliferation via PI3K/Akt pathway.

Carbamazepine Alleviates Retinal and Optic Nerve Neural Degeneration in Diabetic Mice via Nerve Growth Factor-Induced PI3K/Akt/mTOR Activation.

Aim: Diabetic retinopathy causes loss of vision in adults at working-age. Few therapeutic options are available for treatment of diabetic retinopathy. Carbamazepine (CARB), a widely used antiepileptic drug, was recently accounted for its neuroprotective effect. Nerve growth factor (NGF) activates various cascades among which, PI3K/Akt/mTOR pathway has a vital action in NGF-mediated neuronal differentiation and survival. This study evaluated the effect of CARB in the treatment of diabetic retina and unveiled some of the underlying molecular mechanisms.Main Methods: Alloxan diabetes model was induced in 36 albino well-acclimatized mice. After establishment of the diabetic model in 9 weeks, mice were assigned to treatment groups: (1) saline, (2) alloxan-diabetic, (3 and 4) alloxan+CARB (25 or 50 mg per kg p.o) for 4 weeks. After completion of the therapeutic period, mice were sacrificed and eyeballs were enucleated. Retinal levels of NGF and PI3K/Akt were assessed using real-time polymerase chain reaction. Further, total and phosphorylated TrKA, PI3K, Akt, mTOR as well as Caspase-3 were measured by Western blot analysis.Key Findings: Histopathological examination demonstrated that CARB attenuated vacuolization and restored normal thickness and organization of retinal cell layers. In addition, CARB increased pTrKA/TrKA ratio and ameliorated diabetes-induced reduction of NGF mRNA and immunostaining in retina. Additionally, it augmented the mRNA expression of PI3K and Akt, as well as the protein level of the phosphorylated PI3/Akt/mTOR.Significance: Results highlighted, for the first time, the neuronal protective effect for CARB in diabetic retina, which is mediated, at least in part, by activation of the NGF/PI3K/Akt/mTOR pathway.

Protective effect of hydrogen-rich water on oxidative stress cell model and the impact of the phosphatidylinositol 3 kinase/protein kinase B pathway

To explore the protective effect of hydrogen-rich water on the oxidative stress injury of astrocytes in mice and its effect on phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signal pathway. In vitro, mice astrocytes were cultured and the logarithmic growth period cells were taken for experiment. (1) Experiment one: some cells were acted by 1.25, 2.50, 5.00, 10.00 μmol/L hydrogen peroxide (H2O2) for 20 minutes to determine the appropriate concentration required for astrocyte damage induced by H2O2; cultivating 3, 6, 9, and 12 hours with hydrogen-rich water of 25, 50, 100, and 200 μmol/L, respectively, to determine the concentration and time of hydrogen-rich water pretreatment; the 50 μmol/L hydrogen-rich water was cultured together with PI3K/Akt signal pathway inhibitors wortmannin (WM) 200 nmol/L or 400 nmol/L to determine the best inhibition concentration of wortmannin. Astrocyte activity was detected by methyl thiazolyl tetrazolium (MTT) colorimetry. (2) Experiment two: some cells were divided into blank control group, H2O2 injury group, hydrogen-rich water pretreatment group (HW+H2O2 group), and co-culture of hydrogen-rich water and wortmannin pretreatment group (HW+WM+H2O2 group). The mRNA expressions of PI3K and Akt were detected by reverse transcription-polymerase chain reaction (RT-PCR); the protein expressions of PI3K, Akt and phosphorylated Akt (p-Akt) were detected by Western Blot. (1) Experiment one: the survival rate of the blank control group was 100%. Cell activity gradually decreased with the increase of H2O2 concentration, and the survival rate of the H2O2 action 20 minutes cells of 2.50 μmol/L was reduced to about 50%, so a cell injury model was established at this concentration. With the increase of hydrogen-rich water pretreatment concentration, and the duration of action, the cell survival rate increased first and then decreased. The cell survival rate was highest when 50 μmol/L hydrogen-rich water was pretreated with 9 hours, so a hydrogen-rich water pre-protection model was established. After 200 nmol/L or 400 nmol/L wortmannin was cultured together with hydrogen-rich water, cell activity was inhibited, and the cell survival rate of 200 nmol/L wortmannin group was no significantly different compared with that of H2O2 injury group, so the astrocyte suppression model was established. (2) Experiment two: compared with the blank control group, the mRNA expressions of PI3K and Akt and the protein expressions of PI3K, Akt and p-Akt were significantly decreased in the H2O2 injury group. Compared with the H2O2 injury group, the PI3K, Akt mRNA expressions and PI3K, Akt, p-Akt protein expressions were significantly increased in the HW+H2O2 group [PI3K mRNA (2-ΔΔCT): 0.843±0.019 vs. 0.631±0.038, Akt mRNA (2-ΔΔCT): 0.591±0.025 vs. 0.558±0.037, PI3K/β-actin: 1.277±0.008 vs. 0.757±0.004, Akt/β-actin: 1.308±0.015 vs. 0.682±0.006, p-Akt/β-actin: 1.210±0.005 vs. 0.614±0.005, all P < 0.05]. The mRNA expressions of PI3K, Akt in the HW+WM+H2O2 group was 0.784±0.159 and 0.556±0.037, respectively, and the protein expressions of PI3K, Akt, p-Akt was 0.715±0.006, 0.686±0.005, and 0.606±0.004, respectively, both were significantly lower than those in HW+H2O2 group (all P < 0.05), and there was no significant difference with H2O2 injury group (all P > 0.05). Hydrogen-rich water activates the PI3K/Akt pathway, thereby mediates mice astrocytes to exert the biological function of antioxidant.

Influence of laser needle-knife on PI-3K, AKT and VEGF mRNA expression in cervical spondylotic arteriopathy model rabbits.

ObjectiveTo determine the effect of laser needle-knife on PI-3K, AKT and VEGF mRNA expression of vertebral arteries in a rabbit model of cervical spondylotic arteriopathy (CSA) and the mechanism of action involved.MethodsForty healthy general-grade rabbits were divided into a normal control group, model group, acupuncture group, and laser needle-knife group (n = 10 rabbits per group), and the CSA rabbit model was established in all but groups but the normal control group. CSA model rabbits in the acupuncture group were treated by acupuncture at the Fengchi (GB 20) and Cervical Jiaji (EX-B 2) points, whereas rabbits in the laser needle-knife group were treated with laser needle-knife targeting the Jiaji points near the C5 spinous process. Rabbits in the normal control and model groups were fixed using similar methods. Behavioral characteristics of all rabbits were evaluated before and after treatment. Peak systolic velocity (PSV) of the right carotid and vertebral arteries in each group were examined using beside B ultrasound, and PI-3K, AKT, VEGF mRNA expression in vertebral arteries were determined by real-time PCR.ResultsThe behavioral signs of rabbits were improved after treatment in both the acupuncture and laser needle-knife groups. In comparison with control group, PSV of right carotid arteries in acupuncture group and laser needle-knife group were enhanced significantly (P < 0.05 and P < 0.01), PSV of right vertebral arteries in acupuncture group and laser needle-knife group were enhanced significantly too (P < 0.01 and P < 0.05). PI-3K mRNA expression in laser needle-knife and acupuncture group was significantly higher than that in control group (P < 0.01, P < 0.05). AKT mRNA expression in laser needle-knife and acupuncture group was significantly higher than that in control group (P < 0.01). VEGF mRNA expression in laser needle-knife and acupuncture group was significantly higher than that in control group too (P < 0.01, P < 0.05). No significant differences were found in PI-3K, AKT and VEGF mRNA expression levels among acupuncture and laser needle-knife groups (P > 0.05).ConclusionLaser needle-knife could effectively intervene the mRNA expression of PI-3K, AKT and VEGF, this may be one of the mechanisms of the effect of laser needle-knife in treating CSA in rabbits.

Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

RETRACTED: Effects of Artesunate on chondrocyte proliferation, apoptosis and autophagy through the PI3K/AKT/mTOR signaling pathway in rat models with rheumatoid arthritis

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figure 5D and 5F, which appear to have a similar phenotype as contained within another publication, as detailed here: https://pubpeer.com/publications/CD4DF7B6DCA28182EC6809846F3653; and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. Concerns were also raised about the provenance of the flow cytometry data in Figure 7A. In addition, suspected duplications were detected in Western blots contained within Figure 5E and 5F. The journal requested the corresponding author comment on these concerns and provide raw data. The corresponding author communicated that the raw data was not available and some of the results were not repeatable and therefore not solid enough to support the conclusions. The authors requested retraction of this article. The Editor-in-Chief assessed this case and decided to retract the article.

The Antitumor Effect of Xihuang Pill on Treg Cells Decreased in Tumor Microenvironment of 4T1 Breast Tumor-Bearing Mice by PI3K/AKT~AP-1 Signaling Pathway.

To study the antitumor effect of Xihuang pill (XHP) on the number of Treg cells in the tumor microenvironment of 4T1 breast tumor-bearing mice by PI3K/AKT/AP-1 pathway, a mouse model was established. Flow cytometry (FCM) and immunohistochemistry (IHC) were used to detect the number of Treg cells in the tumor microenvironment; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect the apoptosis of Treg cells in tumor microenvironment. Quantitative real-time PCR (RT-qPCR) was used to detect the mRNA expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment; immunofluorescence (IF) and Western Blot (WB) were used to detect the protein expression of PI3K, AKT, and AP-1 in Treg cells in tumor microenvironment. Compared with the naive control group, the tumor weight in XHP groups decreased significantly (P < 0.05); FCM and IHC results showed that the number of Treg cells in the tumor microenvironment decreased with the dose of XHP groups (P < 0.05); TUNEL staining showed that the number of Treg cells in tumor microenvironment increased with the dose of XHP groups (P < 0.05); RT-qPCR results showed that the mRNA expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups, while RNA expression of AP-1 increased with the dose of XHP groups (P < 0.05); IF and WB results showed that the protein expression of PI3K and AKT in Treg cells decreased with the dose of XHP groups and the protein expression of AP-1 increased with the dose of XHP groups (P < 0.05). The results suggested that XHP decreased the number of Treg cells via inhibiting PI3K and AKT expression and upregulating AP-1 expression in Treg cells and then promoting the apoptosis of Treg cells. Thus, XHP could improve the immunosuppressive state of tumor microenvironment and reverse the immune escape to inhibit tumor growth.

Aloperine induces apoptosis and inhibits invasion in MG-63 and U2OS human osteosarcoma cells

Aloperine (ALO) is a novel type of alkaloid drug that is extracted from S. alopecuroide, and exert an anti-inflammatory, anti-allergenic, antitumor and antiviral effects. In our study, we evaluated the effects and underlying mechanisms of ALO on MG-63 and U2OS osteosarcoma (OS) cells. ALO suppressed the proliferation and clonogenecity of both cell lines in a dose- and time-dependent manner as observed by CCK-8 and clonogenic survival assays. Data of morphologic changes, DAPI assays and flow cytometry showed that ALO induced apoptosis of OS cells, and the results of western blotting and qRT-PCR indicated that ALO upregulated protein and mRNA of Bax and cleaved caspase-3, while downregulated Bcl-2. Besides, ALO inhibited the invasion of MG-63 and U2OS cells as shown by transwell invasion assay. The protein and mRNA of MMP-2 and MMP-9 were decreased with ALO treatment. ALO also downregulated the protein and mRNA expression of PI3K and p-AKT1. In conclusion, ALO induced apoptosis and inhibited invasion in MG-63 and U2OS cells, which maybe through suppression of PI3K/AKT signaling pathway.

Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary

The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.