LY294002 Group Research Articles

Inhibition of PI3K/AKT/mTOR signaling pathway promotes autophagy and relieves hyperalgesia in diabetic rats.

The purpose of this study was to explore the role and mechanism of the PI3K/AKT/mTOR signaling pathway in painful diabetic neuropathy (PDN). The diabetes mellitus (DM) model was established by intraperitoneal injection of streptozocin into SD rats. After 3 weeks of modeling, the DM + LY group was treated with PI3K inhibitor, the DM + vehicle group was treated with DMSO, and the DM group was untreated. The paw mechanical withdrawal thresholds (MWT) was measured by Von Frey filaments, and the expression of PI3K/AKT/mTOR pathway-related proteins and autophagy marker proteins were analyzed by Western blotting. We found that 3 weeks after modeling, the MWT values of diabetic rats were significantly reduced, p-PI3K, p-AKT and p-mTOR proteins expression in the spinal cord was increased, and Beclin1 and LC3-II expressions were reduced (P < 0.05). After administration of PI3K inhibitor, the MWT values in DM + LY group were improved, and the expressions of p-PI3K, p-AKT and p-mTOR proteins in the spinal cord were decreased significantly, and the expressions of Beclin1 and LC3-II were increased (P < 0.05). However, there were no significant changes in the DM + vehicle group compared with the DM group (P > 0.05). Therefore, we conclude that activation of the PI3K/AKT/mTOR pathway and impaired autophagy may be key factors that cause PDN. Inhibition of the PI3K/AKT/mTOR pathway could promote autophagy activity and alleviate PDN.

Effects of NOX4 on radiosensitivity of nasopharyngeal carcinoma cells by activating the PI3K/AKT pathway

Objective: To explore the relationship between NOX4 and radiosensitivity of nasopharyngeal carcinoma cells. Methods: Western blot was used to test the expression of NOX4 in nasopharyngeal carcinoma cells (CNE1, CNE2 and HONE1) and normal nasopharyngeal epithelial cells (NP69). The lentiviral vectors for RNA interference and overexpression of NOX4 gene were constructed and nasopharyngeal carcinoma cells were transfected. After treatment with radiation or/and PI3K/AKT inhibitor LY294002, the expressions of related proteins in cells were tested by Western blot, and the cell proliferation was detected by CCK-8 assay and the cell apoptosis was determined by flow cytometry. GraphPad Prism 5 was used for statistical analysis, and P<0.05 was statistically significant. Results: The expressions of NOX4 in nasopharyngeal carcinoma cells were higher than those in normal nasopharyngeal epithelial cells. Compared with the siNC group, the siNOX4 group of nasopharyngeal carcinoma cell had lower proliferation capacity [72 h absorbance (A) value:1.16 vs. 0.75] and higher apoptosis rate (2.9% vs. 10.0%). In contrast,compared with the vector group, the NOX4 group of nasopharyngeal carcinoma cell had higher proliferation capacity [72 h absorbance (A) value: 1.01 vs. 1.32] and lower apoptotic rate (1.7% vs. 1.1%).Treatment with LY294002 for nasopharyngeal carcinoma cells of NOX4 overexpression,compared with the NOX4 group, the proliferation ability of nasopharyngeal carcinoma cells in the NOX4+LY294002 group was reduced (72 h absorbance (A) value: 1.32 vs. 0.77), while the apoptotic rate was increased (1.1% vs. 3.1%).Treatment with radiotherapy, compared with the siNC/Vector group, the proliferation ability of nasopharyngeal carcinoma cells in the siNOX4 group was reduced (72 h absorbance (A) value: 0.72 vs. 0.33), and the apoptotic rate was increased (7.8% vs. 17.3%). However, in the NOX4 group, the proliferation of nasopharyngeal carcinoma cells was enhanced (72 h absorbance (A) value:0.65 vs. 0.78), and the apoptotic rate was reduced (8.1% vs. 3.8%). Compared with the NOX4+radiation group, the proliferation ability of nasopharyngeal carcinoma cells in the NOX4+radiation+LY294002 group was reduced (72 h absorbance (A) value: 0.79 vs. 0.56), while the apoptotic rate was increased (3.8% vs. 8.1%). Conclusion: NOX4 can inhibit radiosensitivity of nasopharyngeal carcinoma cells possibly by activating PI3K/AKT pathway.

Helix B Surface Peptide Protects Cardiomyocytes From Hypoxia/Reoxygenation-induced Autophagy Through the PI3K/Akt Pathway.

Helix B surface peptide (HBSP) is a newly discovered tissue-protective erythropoietin derivative that provides benefits after myocardial ischemia/reperfusion. This study explores the cardioprotective effects of HBSP in myocardial cells in response to hypoxia/reoxygenation injury and its potential mechanism. In this study, rat ventricular (H9c2) cell cultures were established and pretreated with HBSP. H9c2 cardiomyocytes were randomly assigned to the control, H/R, H/R + LY294002 (a PI3K inhibitor), HBSP + H/R, and HBSP + H/R + LY294002 groups. The pretreated cardiomyocytes underwent H/R, and the cardiomyocytes were monitored for viability through a CCK-8 assay, whereas flow cytometry was used to test cell apoptosis. Orgotein Superoxide Dismutase (SOD) and lactate dehydrogenase (LDH) expression were monitored by SOD and LDH kits, respectively. The expression of LC3 autophagosomes was determined by immunocytochemistry. The expression of LC3II/LC3I, p-Mammalian Target of Rapamycin (mTOR) mTOR, mTOR, Beclin 1, p-PI3K, PI3K p-Akt, and Akt was determined by Western blotting. HBSP increased cell viability and reduced SOD and LDH production, and it also reduced H/R-induced cell apoptosis. Moreover, the expression of the autophagy-related proteins (LC3II/LC3I) was inhibited by HBSP, whereas the expression of p-PI3K, p-Akt, and p-mTOR was enhanced. However, the PI3K inhibitor (LY294002) notably abolished these effects in H9c2 cells. HBSP inhibits excessive autophagy and apoptosis induced by H/R by activating the PI3K/Akt pathway. HBSP may potentially be a therapeutic intervention for myocardial ischemia/reperfusion injury.

Effects of live yeast on the rumen fermentation parameters and milk performance of Simmental dairy cows during the hot season

The effects of dietary supplementation with live yeast culture (LY) on the milk performance and rumen fermentation parameters of Simmental cows in the middle of lactation were examined under hot season conditions in 2019. The 28 cows, which were divided into two experimental groups of 14, were fed a controlled diet, with or without the addition of 5 g of LY per cow daily. The mean dry matter intake (DMI) and yield of milk in the LY group were greater by 0.25 kg/day and 0.42 kg/day, respectively, than for the control group. The yields of milk components and 4% fat corrected milk were also significantly greater in the LY group than in the control group, but LY supplementation did not significantly alter milk composition percentages. The acetate concentration in the rumen tended to be higher in cows fed LY but the ruminal pH, NH$_{3}$ -N, propionate and butyrate levels, and acetate/propionate ratio were similar in the two groups. It can be concluded that LY supplementation of the diet of Simmental dairy cows during the hot season improved milk production and ruminal fermentation performance to a limited degree.

Protective effects of panax notoginseng saponin on dextran sulfate sodium-induced colitis in rats through phosphoinositide-3-kinase protein kinase B signaling pathway inhibition.

BACKGROUNDIntestinal inflammation is a common digestive tract disease, which is usually treated with hormone medicines. Hormone medicines are effective to some extent, but long-term use of them may bring about many complications.AIMTo explore the protective effects of panax notoginseng saponin (PNS) against dextran sulfate sodium (DSS)-induced intestinal inflammatory injury through phosphoinositide-3-kinase protein kinase B (PI3K/AKT) signaling pathway inhibition in rats.METHODSColitis rat models were generated via DSS induction, and rats were divided into control (no modeling), DSS, DSS + PNS 50 mg/k, and DSS + PNS 100 mg/kg groups. Then, the intestinal injury, oxidative stress parameters, inflammatory indices, tight junction proteins, apoptosis, macrophage polarization, and TLR4/AKT signaling pathway in colon tissues from rats in each of the groups were detected. The PI3K/AKT signaling pathway in the colon tissue of rats was blocked using the PI3K/AKT signaling pathway inhibitor, LY294002.RESULTSCompared with rats in the control group, rats in the DSS group showed significantly shortened colon lengths, and significantly increased disease activity indices, oxidative stress reactions and inflammatory indices, as well as significantly decreased expression of tight junction-associated proteins. In addition, the DSS group showed significantly increased apoptotic cell numbers, and showed significantly increased M1 macrophages in spleen and colon tissues. They also showed significantly decreased M2 macrophages in colon tissues, as well as activation of the PI3K/AKT signaling pathway (all P < 0.05). Compared with rats in the DSS group, rats in the DSS + PNS group showed significantly lengthened colon lengths, decreased disease activity indices, and significantly alleviated oxidative stress reactions and inflammatory responses. In addition, this group showed significantly increased expression of tight junction-associated proteins, significantly decreased apoptotic cell numbers, and significantly decreased M1 macrophages in spleen and colon tissues. This group further showed significantly increased M2 macrophages in colon tissues, and significantly suppressed activation of the PI3K/AKT signaling pathway, as well as a dose dependency (all P < 0.05). When the PI3K/AKT signaling pathway was inhibited, the apoptosis rate of colon tissue cells in the DSS + LY294002 group was significantly lower than that of the DSS group (P < 0.05).CONCLUSIONPNS can protect rats against DSS-induced intestinal inflammatory injury by inhibiting the PI3K/AKT signaling pathway, and therefore may be potentially used in the future as a drug for colitis.

4-PBA Enhances Autophagy by Inhibiting Endoplasmic Reticulum Stress in Recombinant Human Beta Nerve Growth Factor–Induced PC12 cells After Mechanical Injury via PI3K/AKT/mTOR Signaling Pathway

To investigate mechanism of endoplasmic reticulum (ER) stress-mediated autophagy in spinal cord injury (SCI). An invitro model of spinal cord injury (SCI) was established by recombinant human beta nerve growth factor (NGF)-induced PC12 cells. Immunofluorescence was used to detect properties of PC12 cells induced by NGF. Western blot assay was used to detect expressions of the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)I/II, the ER stress-related protein (HSPA5/GRP78), as well as the PI3K/AKT/mTOR signaling pathway-related proteins after mechanical injury at different time points. Then the sample assigned into sham, SCI, LY294002, SCI+LY294002, 4-PBA (4-phenylbutyric acid), and SCI+4-PBA groups. The expressions of the LC3I/II and PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blot assay. NGF-induced PC12 cells have neurophysiological characteristics. After administration of the PI3K-specific inhibitor LY294002, phosphorylation levels of AKT and mTOR decreased, and the ratio of LC3II/I was higher in the inhibitor-treated injury group than the simple-injury group. After administration of the ER stress inhibitor 4-PBA, the results were similar to LY294002 group's results compared with SCI group. Our study showed that NGF-induced PC12 cells can induce autophagy and ER stress after mechanical injury. ER stress inhibitor 4-PBA obtained similar effects to PI3K inhibitor LY294002, enhanced autophagy via PI3K/AKT/mTOR signaling pathway.

Tanshinone IIA alleviates blast-induced inflammation, oxidative stress and apoptosis in mice partly by inhibiting the PI3K/Akt/FoxO1 signaling pathway

Although Tanshinone IIA (Tan IIA) has been associated with inflammation, oxidative stress and apoptosis, the effects of Tan IIA on lung blast injury remain uncertain. In this study, we explored the effects of Tan IIA on lung blast injury, studied its possible molecular mechanisms. Fifty C57BL/6 mice were randomly divided into the control, blast, blast + Tan IIA, blast + LY294002 (a PI3K inhibitor), or blast + Tan IIA + LY294002 groups. Serum and lung samples were collected 48 h after blast injury. The data showed that Tan IIA significantly inhibited blast-induced increases in the lung weight/body weight and wet/dry (W/D) weight ratios, decreased the CD44−and CD163-positive inflammatory cell infiltration in the lungs, reduced the IL-1β, TNF-α and IL-6 expression, and enhanced IL-10 expression. Tan IIA also significantly alleviated the increases in MDA5 and IRE-a and the decrease in SOD-1 and reversed the low Bcl-2 expression and the high Bax and Caspase-3 expressions. Additionally, Tan IIA significantly decreased p-PI3K and p-Akt expression and increased p-FoxO1 expression. More importantly, both LY294002 and Tan IIA pretreatment markedly protected against blast-induced inflammation, oxidative stress and apoptosis in lung blast injury. These results suggest that Tan IIA protects against lung blast injury, which may be partly mediated by inhibiting the PI3K/Akt/FoxO1 signaling pathway.

Mediated pathways of exosomes uptake by stem cells of apical papilla

To evaluate the uptake of exosomes by stem cells from apical papilla (SCAP), thus to provide experimental basis for mechanism of the exosomes endocytosis by SCAP. (1) Exosomes of dental pulp stem cells (DPSCs) were isolated by hypercentrifugation combined with ultrafiltration method. The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and western blot. (2) PKH-26 membrane labeling technology was used to mark the DPSCs derived exosomes. The labeled exosomes were co-cultured with SCAP at 37 °C as positive control group, and co-cultured with SCAP at 4 °C as the low-temperature treatment group, while the negative control group was set up. (3) Using clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ, 10 μmol /L) as CPZ group, caveolae-mediated endocytosis Genistein (200 μmol/L) as Genistein group, and macropinocytosis inhibitor LY294002 (50 μmol/L) as LY294002 group to treat the SCAP respectively. Solvent control group (DMSO group) was set. Immunofluorescence staining was used to detect the red fluorescence SCAP and flow cytometry was used to analyze the proportion of SCAP labeled with red fluorescence. (1) The bilayer membrane and cup-shaped appearance of representative exosomes were observed. The peak of the size of DPSCs-derived exosomes was at 144 nm. The exosomes expressed exosomal marker proteins TSG101 and CD63, but not GAPDH which was the cellular internal control protein. (2) Immunofluorescence staining showed that after being co-cultured at 37 °C for 6 hours, red fluorescence could be detected in SCAP but it could not be detected after being co-cultured at 4 °C for 6 hours. After endocytosis inhibition, the red fluorescence in SCAP was reduced. Flow cytometry showed that the proportion of SCAP labeled with red fluorescence in positive group was 35.0%, in negative control group was 0.5%, and in solvent control group was 29.7%, in CPZ group, Genistein group and Genistein group were reduced to 13.7%, 16.6%, and 20.9%, respectively. SCAP could uptake the DPSCs derived exosomes, and low temperature could inhibit this process. The exosomes uptake of SCAP was mediated by the clathrin endocytosis pathway, caveolae-mediated endocytosis and macropinocytosis pathway.

Dexamethasone on alleviating lung ischemia/reperfusion injury in rats by regulating PI3K/AKT pathway

To investigate the protective effect and mechanism of dexamethasone in lung ischemia/reperfusion injury (LIRI) rats. (1) Part one experiment: 24 Sprague-Dawley (SD) rats were divided into four groups according to the random number method (n = 6): standard ventilation group (N group), normal saline group (NS group), LIRI group, and dexamethasone+LIRI group (DEX group). The rat model of LIRI was established by clamping the left pulmonary hilum for 1 hour and reperfusing it for 2 hours. The DEX group was given dexamethasone 3 mg/kg 5 minutes before reperfusion, and NS group was injected with normal saline. Group N did not receive any treatment. The left lung tissue of the rats in each group were taken alive 2 hours after reperfusion. The lung tissue was harvested for lung wet/dry mass ratio (W/D) measurement. Hematoxylin-eosin (HE) staining and electron microscopy was used to observe the pathological changes of lung tissue and to assess the degree of injury. Ultrastructural changes of lung tissue were observed under electron microscope. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-6) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The expressions of phosphorylated protein kinase B (p-AKT) was detected by Western Blot. (2) Part two experiment: intervention with phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway inhibitor LY294002 to further explore the mechanism of dexamethasone in reducing lung injury induced by LIRI. Twenty-four SD rats were divided into four groups according to the random number method (n = 6): N group, LIRI group, DEX group, and dexamethasone+LY294002+LIRI group (LY group). All the groups except the LY group were treated with membrane and intervention according to part one experiment. The LY group was injected with LY294002 0.3 mg/kg after injection of dexamethasone. The expressions of M1 macrophage polarization markers CD11c, CD16, and M2 macrophage polarization markers CD206, Arg1 were detected by immunohistochemistry. (1) Part one experiment: compared with N group, the morphological and ultrastructural changes of lung tissue in the LIRI group were significantly changed, lung injury score, lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly increased, and p-AKT expression was significantly decreased. Compared with the LIRI group, the morphological and ultrastructural changes of the lung tissue in the DEX group were significantly improved, and the lung injury score was reduced (5.00±0.89 vs. 8.83±0.75), lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly decreased [lung W/D ratio: 6.25±0.56 vs. 8.27±0.72, TNF-α (ng/L): 93.28±16.42 vs. 205.90±25.30, IL-1β (ng/L): 130.10±10.81 vs. 209.10±19.20, IL-6 (ng/L): 195.80±21.17 vs. 310.50±20.77], p-AKT expression was significantly increased [p-AKT/AKT: (57.58±8.80)% vs. (36.62±9.25)%], and the differences were statistically significant (all P < 0.05). There was no significant difference in each index between NS group and N group. (2) Part two experiment: compared with the N group, the expression of macrophage polarization markers CD11c, CD16, CD206 and Arg1 in the LIRI group were significantly increased. Compared with the LIRI group, the expressions of CD11c and CD16 in the lung tissue of the DEX group were significantly decreased, and the expressions of CD206 and Arg1 were significantly increased. The intervention of PI3K/AKT signaling pathway inhibitor LY294002 significantly blocked the effect of dexamethasone on LIRI-mediated macrophage polarization (CD11c immunohistochemical score: 7.20±0.36 vs. 5.00±0.34, CD16 immunohistochemical score: 8.20±0.48 vs. 7.40±0.64, CD206 immunohistochemical score: 5.80±0.59 vs. 7.40±0.28, Arg1 immunohistochemical score: 7.20±0.72 vs. 8.80±0.48, all P < 0.05). Dexamethasone pretreatment can alleviate the intrapulmonary inflammatory response and lung injury caused by LIRI in rats. The mechanism of action is related to the polarization direction of pulmonary macrophagesvia activation of the PI3K/AKT pathway by dexamethasone.

The protective effects of PI3K/Akt pathway on human nucleus pulposus mesenchymal stem cells against hypoxia and nutrition deficiency

BackgroundTo study the effects of hypoxia and nutrition deficiency mimicking degenerated intervertebral disc on the biological behavior of human nucleus-derived pulposus mesenchymal stem cells (hNP-MSCs) and the role of PI3K/Akt pathway in the process in vitro.MethodshP-MSCs were isolated from lumbar disc and were further identified by their immunophenotypes and multilineage differentiation. Then, cells were divided into the control group, hypoxia and nutrition deficiency group, the LY294002 group, and insulin-like growth factor 1 (IGF-1) group. Then cell apoptosis, the cell viability, the caspase 3 activity, and the expression of PI3K, Akt, and functional genes (aggrecan, collagen I, and collagen II) were evaluated.ResultOur work showed that isolated cells met the criteria of International Society for cellular Therapy. Therefore, cells obtained from degenerated nucleus pulposus were definitely hNP-MSCs. Our results showed that hypoxia and nutrition deficiency could significantly increase cell apoptosis, the caspase 3 activity, and inhibit cell viability. Gene expression results demonstrated that hypoxia and nutrition deficiency could increase the relative expression of PI3K and Akt gene and inhibit the expression of functional genes. However, when the PI3K/Akt pathway was inhibited by LY294002, the cell apoptosis and caspase 3 activity significantly increased while the cell viability was obviously inhibited. Quantitative real-time PCR results showed that the expression of functional genes was more significantly inhibited. Our study further verified that the above-mentioned biological activities of hNP-MSCs could be significantly improved by IGF1.ConclusionsPI3K/Akt signal pathway may have protective effects on human nucleus pulposus-derived mesenchymal stem cells against hypoxia and nutrition deficiency.

MiR‑21 promotes osteoclastogenesis through activation of PI3K/Akt signaling by targeting Pten in RAW264.7 cells.

The present study aimed to investigate the effects of microRNA (miR)-21 on osteoclastogenesis and its underlying molecular mechanisms. The expression levels of tartrate-resistant acid phosphatase (TRAP) and miR-21 were detected during osteoclastogenesis in receptor activator of NF-κB ligand (RANKL)-induced RAW264.7 cells via reverse transcription-quantitative PCR. Bioinformatics and dual luciferase reporter assays were performed to analyze the association between miR-21 and Pten. RANKL-induced RAW264.7 cells were divided into the following groups: MiR-negative control (NC), miR-21 mimic, miR-21 inhibitor and miR-21 mimic + LY294002. The effects of miR-21 on osteoclastogenesis and bone resorption were then detected using TRAP staining and a bone resorption assay. Pten, phosphorylated-Akt and nuclear factor of activated T cell (NFATc1) expression levels were measured by western blotting to analyze the effects of miR-21 on the PI3K/Akt signaling pathway. The present study revealed that miR-21 was upregulated during osteoclastogenesis in RANKL-induced RAW264.7 cells. Furthermore, miR-21 negatively regulated Pten. Compared with the miR-negative control (NC) group, the number of osteoclasts and the percentage of bone resorption were increased in the miR-21 mimic group, whereas they were decreased in the miR-21 inhibitor group. The number of osteoclasts and the percentage of bone resorption in the miR-21 mimic + LY294002 group were lower than in the miR-21 mimic group. Compared with the miR-NC group, the protein expression levels of Pten were decreased, whereas p-Akt and NFATc1 were increased in the miR-21 mimic group. Conversely, Pten protein expression was increased, whereas p-Akt and NFATc1 were decreased in the miR-21 inhibitor group. In the miR-21 mimic + LY294002 group, Pten protein expression was higher, and p-Akt and NFATc1 were lower than in the miR-21 mimic group. In conclusion, miR-21 is upregulated during osteoclastogenesis, and may promote osteoclastogenesis and bone resorption through activating the PI3K/Akt signaling pathway via targeting Pten.

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroine kinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEM medium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the following groups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylation inhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342 and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD) for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevated levels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, the H9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group, while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group was lower than the hypoxia group (12.34 ± 2.00% <i>vs</i>. 21.73 ± 2.58%; <i>p</i> < 0.05), while the LY group was significantly higher (22.54 ± 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (<i>p</i> < 0.05), but decreased in CM group (<i>p</i> < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secrete multiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which the activation of Akt phosphorylation is involved to achieve the protective effect.

Exercise Preconditioning Plays a Protective Role in Exhaustive Rats by Activating the PI3K-Akt Signaling Pathway.

Objective To investigate whether exercise preconditioning (EP) protects the rat heart from exhaustive exercise- (EE-) induced injury by inducing the PI3K-Akt signaling pathway. Methods 84 male Sprague-Dawley rats were randomly divided into 6 groups (n = 14 rats per group): control group (Con), exhaustive exercise group (EE), exercise preconditioning group (EP), exercise preconditioning + exhaustive exercise group (EP + EE), LY294002 (PI3K inhibitor) + exercise preconditioning + exhaustive exercise group (LY + EP + EE), and LY294002 group (LY). The Con and LY did not exercise. The remaining groups were subjected to treadmill running. The structure of myocardial tissue and serum biomarkers of myocardial injury were observed. Hemodynamic parameters were recorded with a pressure-volume catheter. TUNEL assay was used to detect the apoptosis of cardiac myocytes, and the level of mitochondrial membrane permeability transforming pore (mPTP) in myocardium was evaluated using ELISA. Pathway and apoptosis-related proteins in myocardium were assessed using western blotting. Results Compared to the Con group, the EE group showed remarkable myocardial injury, such as cardiac dysfunction and myocardial apoptosis. Compared to the EE group, the injuries in the EP + EE group were improved. EP increased the PI3K-Akt signaling pathway and regulated Bcl-2 family to decrease the mPTP openness level. However, the cardioprotective effects of EP were attenuated when pretreated with the LY294002. Conclusions EP protected the heart from EE-induced injury, and it may improve the cardiac function and reduce the cardiomyocyte apoptosis by activating the PI3K-Akt signaling pathway.

SULF2 promotes tumorigenesis and inhibits apoptosis of cervical cancer cells through the ERK/AKT signaling pathway.

The objective of this study was to explore the role of the SULF2-mediated ERK/AKT signaling pathway in cervical cancer. SULF2 expression was detected in tumor tissues and tumor-adjacent normal tissues from cervical cancer patients. HeLa cells were divided into six groups: control group, NC group, SULF2 siRNA group, SULF2 group, SULF2 + LY294002 group, and SULF2 + U0125 group. In each group, HeLa cells received the corresponding treatment, followed by measurement of the cellular biological characteristics and expression of the ERK/AKT signaling pathway. We also confirmed the effect of SULF2 in vivo using a xenograft model in nude mice. SULF2 was upregulated in cervical cancer tissues, which was specifically associated with the clinical stage, histological differentiation, and lymphatic metastasis. Compared to the control group, the SULF2 siRNA group displayed decreased expression of SULF2, concomitant with reduced proliferation, migration, and invasion, but there was an increase in the apoptosis rate of HeLa cells, as well as downregulation of the p-Akt/Akt, p-ERK/ERK, and Bax/Bcl-2 ratios and cyclin D1. Additionally, tumor growth was significantly inhibited in the xenograft model of nude mice. The results in the SULF2 group were quite the opposite in which SULF2 facilitated the growth of cervical cancer cells, which was reversed by LY294002 or U0126. SULF2 is highly expressed in cervical cancer, and thus, downregulation of SULF2 can inhibit the ERK1/2 and AKT signaling pathways to suppress the proliferation, invasion, and migration of cervical cancer cells while facilitating apoptosis.

Herbal complex 'Buyang Huanwu Tang' improves motor endplate function of denervated-dependent skeletal muscle atrophy in rat.

Denervated-dependent skeletal muscle atrophy is a disease induced by skeletal muscle associated peripheral neuro-disconnection. Its specific molecular mechanisms remain unknown. The treating for denervated-dependent skeletal muscle atrophy is applied with an herbal complex Buyang Huanwu Tang used in traditional Chinese medicine and subjected to the established denervated-dependent skeletal muscle atrophy in rat models, and the therapeutic effects and associated mechanisms were evaluated in the pathogenesis of denervated-dependent skeletal muscle atrophy. Denervated-dependent skeletal muscle atrophy in rats was established and randomly divided into eight groups, including Normal control, Model, Positive control, Model + Buyang Huanwu Tang, Model + astragalus extracts, Model + Buyang Huanwu Tang-astragalus, Buyang Huanwu Tang + LY294002, and astragalus extract + LY294002 group. Hematoxylin-eosin staining and quantitative RT-PCR (qRT-PCR) assay were used to examine the inflammatory response of muscle tissues. Quantitative RT-PCR and Western blotting assay were utilized to analyze mRNA and protein expression. Immunohistochemistry assay was used to detect molecule expression in anterior cervical muscle tissues. Motor endplate activity was examined using the wholemount acetylcholinesterase staining method. The wet mass ratio of anterior cervical muscle was measured. The results indicated that Buyang Huanwu Tang treatment significantly alleviated inflammatory response, enhanced acetylcholinesterase activity, and motor endplate functions, and promoted wet mass of anterior cervical muscle compared to denervated-dependent skeletal muscle atrophy rat models (P < 0.05). Buyang Huanwu Tang regulated molecules of PI3K/PKB/GSK3β/FOXO1 signaling pathway. Buyang Huanwu Tang significantly reduced muscle atrophy F-box protein, MuFR-1, Bax and caspase 9 expression, significantly enhanced Bcl-2 expression, and remarkably increased element-binding protein and vascular endothelial growth factor levels, compared to Model group (P < 0.05). Buyang Huanwu Tang suppressed caspase 9 and caspase 3 activity and associated apoptosis. Moreover, PI3K specific blocker, LY294002, significantly inhibited the effects of Buyang Huanwu Tang on the above molecule expression (P < 0.05). In conclusion, Buyang Huanwu Tang improved motor endplate functions of denervated-dependent skeletal muscle atrophy rat model through suppressing mitochondria-mediated apoptosis and activating PI3K/PKB/FOXO1 signaling pathway.

Beneficial effect of Rhodopseudomonas palustris on in vitro rumen digestion and fermentation.

As a member of photosynthetic bacteria, Rhodopseudomonas palustris, which has extraordinary metabolic versatility, has been applied as one of potential probiotics in feed industry. To explore whether R. palustris can increase rumen microbial viability and thus improve microbial fermentation, a 2×5 factorial experiment was conducted to evaluate the effect of R. palustris at dose rates of 0, 1.3, 2.6, 3.9, 5.2×106 cfu/ml on ruminal fermentation of two representative total mixed rations (HY, a ration for high-yield (>32 kg/d) lactating cows; LY, a ration for low-yield (<25 kg/d) lactating cows). After a 48 h in vitro rumen incubation, both rations resulted in different fermentation characteristics. The HY in comparison with LY group presented greater in vitro dry matter disappearance (IVDMD), cumulative gas production (GP48) and total volatile fatty acids (VFA, P<0.01). Increasing R. palustris addition linearly increased IVDMD (P<0.01) and GP48 (P<0.05), and the IVDMD increment in response to R. palustris addition was greater in LY than HY group (6.4% vs 1.4%). Meanwhile, increasing R. palustris addition also linearly enhanced microbial protein synthesis and increased total VFA production (P<0.01), especially in LY group (up to 21.5% and 24.5% respectively). Unchanged acetate and declined propionate in molar percentage were observed in response to the R. palustris addition. Furthermore, increasing R. palustris addition altered fermentation gas composition in which molar O2 proportion in headspace of fermentation system was linearly reduced by 46.1% in LY and 32.9% in HY group, respectively (P<0.01), and methane production in both ration groups was enhanced by 1.9-4.1% (P=0.02). In summary, the R. palustris addition exhibited high potential for promoting the growth of rumen microorganism and enhancing microbial fermentation towards non-glucogenic energy supply by maintaining an anaerobic environment to microbe equilibrium.

Mitochondrial Coenzyme Q Protects Sepsis-Induced Acute Lung Injury by Activating PI3K/Akt/GSK-3β/mTOR Pathway in Rats.

The aim of our study was to assess the effects of mitochondrial coenzyme Q (MitoQ) on sepsis-induced acute lung injury (ALI) and investigate its possible mechanisms. The cecal ligation and puncture (CLP) method was used to establish a septic ALI model. Rats were randomly divided into Con group, CLP group, MitoQ group, and MitoQ + LY294002 group. The survival rate of the rats was recorded, and the survival rate curve was plotted. Moreover, the ratio of wet/dry weight (W/D) in lung tissue was measured. The activity of myeloperoxidase (MPO) was measured by using the MPO colorimetric activity assay kit. The levels of high-mobility group box 1 (HMGB1) and interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP2), and keratinocyte chemoattractant (KC) were analyzed by ELISA. The histopathological changes were measured by HE staining, and the lung injury was scored. TUNEL assay was applied to detect the apoptotic cells in lung tissue. The protein expressions were detected by western blot. MitoQ increased the survival rate and alleviated pulmonary edema in septic ALI rats. In addition, MitoQ inhibited the MPO activity and decreased the levels of HMGB1 and IL-6. After treatment with MitoQ, alveolar wall edema, inflammatory cell infiltration, and red blood cell exudation were relieved. MitoQ inhibited cell apoptosis in lung tissue of septic ALI rats. Meanwhile, MitoQ treatment remarkedly increased the expression of p-Akt, p-GSK-3β, and p-mTOR but decreased Bax, caspase-3, caspase-9, Beclin-1, and LC-3II/LC-3I. The effects of MitoQ were significantly reversed by the PI3K inhibitor (LY294002). Our study demonstrated that MitoQ could protect sepsis-induced acute lung injury by activating the PI3K/Akt/GSK-3β/mTOR pathway in rats.

Silencing of Tenascin-C Inhibited Inflammation and Apoptosis Via PI3K/Akt/NF-κB Signaling Pathway in Subarachnoid Hemorrhage Cell Model

ObjectivesTenascin-C (TNC) is upregulated in serum and cerebrospinal fluid after subarachnoid hemorrhage (SAH) and the deficiency of TNC could alleviate neuronal apoptosis and neuroinflammation after SAH. However, the specific mechanism of TNC regulating neuronal apoptosis and neuroinflammation after SAH is not well recognized. The aim of this study was to investigate whether PI3K/Akt/ NF-κB signaling pathway is involved in the regulation of TNC on early brain injury after SAH. MethodsOxygen hemoglobin (OxyHb) was used to induce SAH models in PC12 cells, and classified into control, SAH, LY294002, SAH+TNC-siRNA and SAH+TNC-siRNA+LY groups. Western blotting was applied to examine the protein expression of TNC, Caspase-3, Bax, Bcl-2, PI3K, p-Akt, and p-NF-κB. Reverse transcription quantitative polymerase chain reaction was applied to examine the TNC mRNA expression. Cholecystokinin (CCK)-8 and flow cytometry were used to examine cell proliferation and apoptosis, respectively. ELISA was applied to examine the content of interleukin 6, interleukin 1β, and tumor necrosis factor α. We showed that the TNC protein was highly expressed in SAH cell model. ResultsOxyHb inhibited cell proliferation, promoted cell apoptosis and the expression of proapoptotic protein, and promoted proinflammatory cytokine secretion in PC12 cells, which were restored following TNC gene silencing. Moreover, OxyHb decreased the expression of PI3K and p-Akt and increased the expression of p-NF-κB p65 in PC12 cells, which were activated following TNC gene silencing. The LY294002 weakened the effect of TNC gene silencing. ConclusionsThe TNC gene silencing relieved neuronal apoptosis and neuroinflammation by activating the PI3K/Akt/ NF-κB signaling pathway. TNC-induced neuroinflammation would be a new target to improve outcome after SAH.

Resveratrol increase myocardial Nrf2 expression in type 2 diabetic rats and alleviate myocardial ischemia/reperfusion injury (MIRI).

Oxidative stress is one of the main factors that increases reperfusion injury in the diabetic heart. Resveratrol could decrease oxidation and promote antioxidant factor expression. Nrf2 is an important endogenous antioxidant. making the heart more resistant to ischemic injury. As Nrf2 expression is considered to be reduced in diabetic heart, we therefore hypothesized that up-regulation of Nrf2 in the diabetic heart may overcome its increased susceptibility to ischemic injury. A total of 50 diabetic rats were randomly divided into five groups: the sham operation group (DM + sham), the MIRI group (DM + MI/R), the resveratrol treatment group (DM + MI/R + RSV), the resveratrol + ex527 (SIRT1 inhibitor) group (DM + MI/R + RSV + ex527), and the resveratrol + LY294002 (Akt inhibitor) group (MD + MI/R + RSV + LY294002). Another 20 normal rats were randomly divided into two groups: the sham-operated group (CON + sham) and the myocardial ischemia-reperfusion group (CON + MI/R). A type 2 diabetes model was induced by a high-fat diet-fed and intraperitoneal injection of streptozotocin (STZ). All rats were subjected to 30 min myocardial ischemia followed by 120 min reperfusion except sham groups. Plasma were collected to measure the creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), malondialdehyde (MDA) level, glutathione (GSH) level, and superoxide dismutase (SOD) activity. Pathologic changes in myocardial tissues were observed by Hematoxylin and eosin (HE) straining. SIRT1, p-GSK3β, Nrf2, HO-1 protein expressions were measured by western blot. Compared with the control (CON) group, the diabetic (DM) group had more severe myocardial injury, higher oxidative stress index increase, and a more reduced expression of Nrf2 in the myocardium. After supplementing with resveratrol, the myocardial damage was reduced and the oxidative stress index decreased in the DM group, while the Nrf2 in the myocardium was increased. It also was found that inhibition of SIRT1 also partially inhibited the expression of Nrf2 and the corresponding antioxidant factor HO-1. Decreased expression of p-GSK3β by Akt inhibitors also partially inhibited Nrf2 and HO-1 expression. Resveratrol can enhance the expression of Nrf2 in a diabetic heart by stimulating SIRT1 or inhibiting GSK3β, alleviating myocardial oxidative stress, and improving ischemia-reperfusion injury.

Dexmedetomidine prevents septic myocardial dysfunction in rats via activation of α7nAChR and PI3K/Akt- mediated autophagy

Background and purposeDexmedetomidine (Dex) has been shown to elicit cardio-protective effects in sepsis. The aim of this study was to investigate the role of autophagy in the protective effects of Dex and its possible mechanism in vivo and vitro. Experimental approach6-8-week-old male Wistar rats were performed cecal ligation puncture (CLP) and administered 0.9% saline (CLP group), 50 μg/kg Dex (Dex group), Dex plus chloroquine (20 mg/kg; Dex + CQ group), or 40 μg/kg methyllycaconitin (Dex + MLA group), or 25 μM LY294002 (Dex + LY294002 group). After study, cardiac histology, cardiac function, level of autophagy, cardiomyocytes apoptosis and inflammatory mediators including protein IL-1β, IL-6, and TNF-α were measured. The LPS induced-H9C2 cardiomyocytes were treated with Dex, Dex + CQ and detected for cell apoptosis, autophagy level and cell cycle. Key resultsCLP-induced sepsis resulted in cardiac dysfunction, apoptosis, and inflammatory response. Dex exhibited protective effects on the myocardium by the induction of myocardial autophagy and ameliorated the LPS-induced blockade of autophagic flux in H9C2 cells. CQ was found to significantly inhibit Dex-mediated protection of myocardial apoptosis and inflammation. CLP rats treated with Dex in combination with MLA, an antagonist of α7 nicotinic acetylcholine receptor (α7nAChR), exhibited decreased autophagy and increased inflammation and cell death, identifying α7nAchR was involved in the Dex-mediated pathway. In addition, we found that the PI3K/Akt pathway is involved in Dex-mediated autophagy and convergent with α7nAChR-mediated stimulation of autophagy response. Conclusions and implicationsFor the first time, these data indicate that autophagy is central in Dex-mediated cardio-protection in sepsis. These observations provide the foundation for further study, and may serve as the basis for innovative therapeutic strategies against septic myocardial dysfunction.