Expression Levels Of Phosphatidylinositol 3-kinase Research Articles

IL-22 activates the PI3K-AKT pathway to promote colorectal cancer cell proliferation and metastasis

BackgroundColorectal cancer (CRC) is a prevalent malignancy with high morbidity and mortality rates. Previous studies have demonstrated that interleukin (IL)-22 is involved in CRC progression; however, the exact mechanism remains unclear. This study aimed to investigate the effects of IL-22 on CRC cell proliferation and metastasis.MethodsIL-22 levels in the serum and tissues of CRC patients were measured using enzyme-linked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to detect the viability of CRC (HCT116) cells treated with different IL-22 concentrations. Colony formation, Transwell invasion, and scratch assays were employed to assess the effects of IL-22 on cell proliferation, invasion, and migration. Western blotting was performed to measure the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), p-PI3K, p-AKT, E-cadherin, matrix metalloproteinase (MMP)-2, MMP-9, SNAI1, and TWIST1 in HCT116 cells treated with IL-22 or a PI3K inhibitor.ResultsELISA results showed that the expression of IL-22 was significantly increased in the serum and tissues of CRC patients compared to controls. IL-22 treatment increased cell viability and colony formation in a concentration-dependent manner and enhanced cell invasion and migration. Western blotting analysis revealed that IL-22 stimulation upregulated p-PI3K and p-AKT expression, while total PI3K and AKT levels remained unchanged. Additionally, IL-22 also decreased E-cadherin expression and increased the expression of MMP-2, MMP-9, SNAI1, and TWIST1.ConclusionsIL-22 activates the PI3K-AKT pathway and promotes HCT116 cell proliferation and metastasis. Targeting the regulation of the PI3K/AKT pathway may be a potential therapeutic strategy for CRC.

HGF ameliorates cardiomyocyte apoptosis and inflammatory response in sepsis via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway

ObjectiveThis study aimed to analyze the impact of HGF on cardiomyocyte injury, apoptosis, and inflammatory response induced by lipopolysaccharide (LPS). MethodsEnzyme-linked immunosorbent assay (ELISA) was utilized to quantify the levels of HGF, interleukin (IL)-6, IL-10, creatine phosphokinase-isoenzyme-MB (CK-MB), and cardiac troponin I (cTnI) in the samples. qPCR and Western blotting (WB) were employed to assess the mRNA and protein expressions of HGF, IL-10, IL-6, PI3K, AKT, p-PI3K, and p-AKT. ResultsThe outcomes of the in vivo experiment revealed that serum levels of IL-6, IL-10, HGF and SOFA scores in the SC group were elevated in contrast to the non-SC group. The correlation analysis indicated a substantial and positive association among serum HGF, IL-6, and IL-10 levels and SOFA scores. Relative to IL-6, IL-10 levels, and SOFA scores, serum HGF demonstrated the highest diagnostic value for SC. Following LPS administration to stimulate H9c2 cells across various periods (0, 12, 24, 48, and 72 h), the levels of myocardial injury markers (CK-MB and cTnI) in the cell supernatants, intracellular inflammatory factors (mRNA and protein levels of IL-10 and IL-6), apoptosis and ROS levels, exhibited a gradual increase followed by a subsequent decline. Following the overexpression of HGF, there was an increase in cell viability, and a decrease in apoptosis, inflammation, oxidative stress injuries, and the protein phosphorylation expressions of PI3K and AKT. After knockdown of HGF expression, the activity of LPS-induced H9c2 cells was further reduced, leading to increased cell injury, apoptosis, inflammation, oxidative stress,and the expression levels of PI3K and Akt protein phosphorylation were further elevated. ConclusionHGF was associated with decreased LPS-induced H9c2 apoptosis and inflammation in H9c2 cells, alongside an improvement in cell viability, indicating potential cytoprotective effects. The mechanism underlying these impacts may be ascribed to the suppression of the PI3K/AKT signaling pathway.

Lycium barbarum polysaccharide reverses drug resistance in oxaliplatin-resistant colon cancer cells by inhibiting PI3K/AKT-dependent phosphomannose isomerase.

Objective: Here, we aimed to explore the effect of LBP in combination with Oxaliplatin (OXA) on reversing drug resistance in colon cancer cells through in vitro and in vivo experiments. We also aimed to explore the possible mechanism underlying this effect. Finally, we aimed to determine potential targets of Lycium barbarum polysaccharide (LBP) in colon cancer (CC) through network pharmacology and molecular docking. Methods: The invasion ability of colon cancer cells was assessed using the invasion assay. The migration ability of these cells was assessed using the migration assay and wound healing assay. Cell cycle analysis was carried out using flow cytometry. The expression levels of phosphomannose isomerase (PMI) and ATP-binding cassette transport protein of G2 (ABCG2) proteins were determined using immunofluorescence and western blotting. The expression levels of phosphatidylinositol3-kinase (PI3K), protein kinase B (AKT), B-cell lymphoma 2 (Bcl-2), and BCL2-Associated X (Bax) were determined using western blotting. Forty BALB/c nude mice purchased from Weitong Lihua, Beijing, for the in vivo analyses. The mice were randomly divided into eight groups. They were administered HCT116 and HCT116-OXR cells to prepare colon cancer xenograft models and then treated with PBS, LBP (50mg/kg), OXA (10mg/kg), or LBP + OXA (50mg/kg + 10mg/kg). The tumor weight and volume of treated model mice were measured, and organ toxicity was evaluated using hematoxylin and eosin staining. The expression levels of PMI, ABCG2, PI3K, and AKT proteins were then assessed using immunohistochemistry. Moreover, PMI and ABCG2 expression levels were analyzed using immunofluorescence and western blotting. The active components and possible targets of LBP in colon cancer were explored using in silico analysis. GeneCards was used to identify CC targets, and an online Venn analysis tool was used to determine intersection targets between these and LBP active components. The PPI network for intersection target protein interactions and the PPI network for interactions between the intersection target proteins and PMI was built using STRING and Cytoscape. To obtain putative targets of LBP in CC, we performed GO function enrichment and KEGG pathway enrichment analyses. Results: Compared with the HCT116-OXR blank treatment group, both invasion and migration abilities of HCT116-OXR cells were inhibited in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group (p < 0.05). Cells in the LBP + OXA (2.5mg/mL LBP, 10μΜ OXA) group were found to arrest in the G1 phase of the cell cycle. Knockdown of PMI was found to downregulate PI3K, AKT, and Bcl-2 (p < 0.05), while it was found to upregulate Bax (p < 0.05). After treatment with L. barbarum polysaccharide, 40 colon cancer subcutaneous tumor models showed a decrease in tumor size. There was no difference in the liver index after LBP treatment (p > 0.05). However, the spleen index decreased in the OXA and LBP + OXA groups (p < 0.05), possibly as a side effect of oxaliplatin. Immunohistochemistry, immunofluorescence, and western blotting showed that LBP + OXA treatment decreased PMI and ABCG2 expression levels (p < 0.05). Moreover, immunohistochemistry showed that LBP + OXA treatment decreased the expression levels of PI3K and AKT (p < 0.05). Network pharmacology analysis revealed 45 active LBP components, including carotenoids, phenylpropanoids, quercetin, xanthophylls, and other polyphenols. It also revealed 146 therapeutic targets of LBP, including AKT, SRC, EGFR, HRAS, STAT3, and MAPK3. KEGG pathway enrichment analysis showed that the LBP target proteins were enriched in pathways, including cancer-related signaling pathways, PI3K/AKT signaling pathway, and IL-17 signaling pathways. Finally, molecular docking experiments revealed that the active LBP components bind well with ABCG2 and PMI. conclusion: Our in vitro experiments showed that PMI knockdown downregulated PI3K, AKT, and Bcl-2 and upregulated Bax. This finding confirms that PMI plays a role in drug resistance by regulating the PI3K/AKT pathway and lays a foundation to study the mechanism underlying the reversal of colon cancer cell drug resistance by the combination of LBP and OXA. Our in vivo experiments showed that LBP combined with oxaliplatin could inhibit tumor growth. LBP showed no hepatic or splenic toxicity. LBP combined with oxaliplatin could downregulate the expression levels of PMI, ABCG2, PI3K, and AKT; it may thus have positive significance for the treatment of advanced metastatic colon cancer. Our network pharmacology analysis revealed the core targets of LBP in the treatment of CC as well as the pathways they are enriched in. It further verified the results of our in vitro and in vivo experiments, showing the involvement of multi-component, multi-target, and multi-pathway synergism in the drug-reversing effect of LBP in CC. Overall, the findings of the present study provide new avenues for the future clinical treatment of CC.

Dietary Zn Deficiency Inhibits Cell Proliferation via the GPR39-Mediated Suppression of the PI3K/AKT/mTOR Signaling Pathway in the Jejunum of Broilers.

A prior investigation revealed that a lack of Zinc (Zn) could hinder intestinal cell proliferation in broiler chickens; however, the mechanisms responsible for this effect remain unclear. We aimed to investigate the possible mechanisms of dietary Zn deficiency in inhibiting the jejunal cell proliferation of broilers. For this study, a total of 112 chickens (21 days old) were randomly divided into two treatments (seven replicate cages per treatment, eight chickens per replicate cage): the control group (CON) and the Zn deficiency group. The duration of feeding was 21 d. Chickens in the control group were provided with a basal diet containing an extra addition of 40 mg Zn/kg in the form of Zn sulfate, whereas chickens in the Zn deficiency group were given the basal diet with no Zn supplementation. The results indicated that, in comparison to the CON, Zn deficiency increased (p < 0.05) the duodenal and jejunal crypt depth (CD) of broilers on d 28 and jejunal and ileal CD on d 35, and decreased (p < 0.05) the duodenal, jejunal, and ileal villus height/crypt depth (VH/CD) on d 28 and the jejunal VH, jejunal and ileal villus surface area, and VH/CD on d 35. Furthermore, Zn deficiency decreased (p < 0.0001) the number of proliferating cell nuclear antigen-positive cells and downregulated (p < 0.01) the mRNA or protein expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, phosphorylated serine-threonine kinase (AKT), phosphorylated mechanistic target of rapamycin (mTOR), G protein-coupled receptor 39 (GPR39), and extracellular-regulated protein kinase, but upregulated (p < 0.05) the mRNA or protein expression levels of P38 mitogen-activated protein kinase, c-jun N-terminal kinase (JNK) 1 and JNK2, and phosphorylated protein kinase C in the jejunum of the broilers on d 42. It was concluded that dietary Zn deficiency inhibited cell proliferation possibly via the GPR39-mediated suppression of the PI3K/AKT/mTOR signaling pathway in the jejunum of broilers.

LARP1 knockdown inhibits cultured gastric carcinoma cell cycle progression and metastatic behavior.

This study aimed to clarify the role of la-related protein 1 (LARP1) in cell cycle progression and metastatic behavior of cultured gastric carcinoma (GC) cells. To do that, LARP1 expression was detected in clinical GC tissues and cell lines using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The cell viability, apoptosis, cell cycle, migration, invasion, and cell growth were examined using a Cell Counting Kit-8, Annexin V-FITC staining, propidium iodide staining, Transwell migration and invasion assays, and colony formation assays after LARP1 knockdown. Phosphatidyl inositol 3-kinase (PI3K) and AKT1 mRNA and protein expression levels of PI3K, p-AKT1, AKT1, p-BAD, p-mTOR, and p21 in si-LARP1 transfected GC cells were determined using qRT-PCR and western blotting. Here, we've shown that LARP1 expression was upregulated in human GC tissues and KATO III cells. LARP1 knockdown inhibited GC cell proliferation, cell cycle progression, migration, invasion, and colony formation and promoted apoptosis. In si-LARP1-transfected KATO III cells, the mRNA expression levels of PI3K and AKT1, PI3K protein expression, and the p-AKT1/AKT1 ratio were significantly suppressed. p-mTOR and p-BAD were significantly decreased, whereas p21 was significantly increased in si-LARP1-transfected KATO III cells. In conclusion LARP1 knockdown induces apoptosis and inhibits cell cycle progression and metastatic behavior via PI3K/AKT1 signaling in GC cells.

Effect of PI3K/AKT/HIF-1α signaling pathway on oxidative stress and apoptosis in nucleus pulposus cells.

The aim of the study was to explore the expression of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)/hypoxia-induced factor-1 alpha (HIF-1α) signaling pathway and the apoptosis of nucleus pulposus cells (NPCs) under different oxygen concentrations to clarify the biological characteristics of NPCs and the molecular mechanism of intervertebral disc degeneration (IVDD). Normal and degenerated human NPCs were collected. Leibovitz's medium with 100 μmol/L CoCl2 was used to establish a hypoxic culture environment, and 100 μmol/L H2O2 was used to establish an oxidative stress culture environment. Third-generation NPCs were divided into 6 groups: normal NPCs + hypoxia, normal NPCs + normoxia, normal NPCs + oxidative stress, degenerated NPCs + hypoxia, degenerated NPCs + normoxia, and degenerated NPCs + oxidative stress. Normal NPCs + hypoxia was used as the experimental control group. Cell viability and proliferation were detected by using the Cell Counting Kit-8 (CCK-8) method. Cell apoptosis rate was assessed by flow cytometry, and expression levels of PI3K, AKT, and HIF-1α were determined by Real-Time-Polymerase Chain Reaction (RT-PCR) and Western blotting. The cell proliferation rate of both normal and degenerated NPCs decreased with increasing oxygen concentration. Conversely, the apoptosis rate increased as the oxygen concentration increased (p<0.05). Compared with the control group, whether the cells degenerated had a very significant impact on the apoptosis rate (p<0.001), and oxygen concentration also had a highly significant impact on both the cell proliferation rate and apoptosis rate (both p<0.001). The interaction between cell degeneration and oxygen concentration significantly affected both cell proliferation and apoptosis rates (p<0.05). Considering the expression levels of PI3K, AKT, and HIF-1α, normal NPCs had the highest levels under low oxygen concentrations, followed by oxidative stress and normoxia. In degenerated NPCs, the expression levels also decrease as the oxygen concentration increases. The PI3K/Akt/HIF-1α signaling pathway plays a significant role in inhibiting oxidative stress, antagonizing NPC apoptosis, and consequently delaying IVDD.

Silencing of KIF2C enhances the sensitivity of hepatocellular carcinoma cells to cisplatin through regulating the PI3K/AKT/MAPK signaling pathway.

In the treatment of unresectable advanced hepatocellular carcinoma (HCC), cisplatin is administered transhepatic arterially for local treatment, but the clinical application of cisplatin drugs is frequently hindered by the emergence of drug resistance. Kinesin family member 2C( KIF2C ) has been shown as oncogene in a variety of tumors. Nevertheless, its effect on cisplatin sensitivity has yet to be ascertained. Herein, we aim to investigate the impact of the KIF2C gene on cisplatin sensitivity within HCC and the plausible underlying molecular mechanism. We examined the expression level of the KIF2C gene in HCC cells by real-time quantitative reverse transcription PCR and Western blot analysis, and analyzed bioinformatically by The Gene Expression Omnibus database and The Cancer Genome Atlas database. The KIF2C gene was silenced using the small interfering RNA technology, and its effect on cisplatin drug sensitivity in HCC cells was evaluated by flow cytometry, cell proliferation, cell migration, and invasion assays. Our results indicated that KIF2C was highly expressed in HCC cells. KIF2C silencing inhibits HCC cell proliferation, migration and invasion, promotes apoptosis, and keeps the cell cycle in G2 phase. In addition, KIF2C silencing enhanced the sensitivity of HCC cells to cisplatin. KIF2C silencing down-regulates the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and mitogen-activated protein kinase 3 (MAPK3) proteins. In conclusion, KIF2C silencing amplifies the sensitivity of HCC cells to cisplatin by regulating the PI3K/AKT/MAPK signaling pathway. Consequently, targeting KIF2C shows great application potential as a strategy for enhancing the effectiveness of HCC treatment.

Mechanism of protective effect of resveratrol on poor ovarian response in mice

This study aims to investigate the therapeutic effects and potential mechanisms of resveratrol(Res) on poor ovarian response(POR) in mice. The common target genes shared by Res and POR were predicted by network pharmacology, used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment, and then validated by animal experiments. The mice with regular estrous cycle after screening were randomized into normal, POR, and low-and high-dose(20 and 40 mg·kg~(-1), respectively) Res groups. The normal group was administrated with an equal volume of 0.9% sodium chloride solution by gavage, and the mice in other groups with tripterygium glycosides suspension(50 mg·kg~(-1)) by gavage for 2 weeks. After the modeling, the mice in low-and high-dose Res groups were treated with Res by gavage for 2 weeks, and the mice in normal and POR groups with an equal volume of 0.9% sodium chloride solution by gavage. Ovulation induction and sample collection were carried out on the day following the end of treatment. Vaginal smears were collected for observation of the changes in the estrous cycle, the counting of retrieved oocytes, and the measurement of ovarian wet weight and ovarian index. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of anti-mullerian hormone(AMH), follicle-stimulating hormone(FSH), estradiol(E_2), and luteinizing hormone(LH) in the serum. The ovarian tissue morphology and granulosa cell apoptosis were observed by hematoxylin-eosin(HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL), respectively. Western blot was employed to determine the protein levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), forkhead box O(FOXO) 3a, hypoxia-inducible factor(HIF)-1α, B-cell lymphoma-2(Bcl-2), and Bcl-2-associated X protein(Bax). A total of 222 common targets shared by Res and POR were collected. GO annotation indicated that these targets were mainly involved in oxidative stress response. KEGG enrichment analysis revealed that Res can intervene in POR via PI3K/AKT, HIF-1, and FOXO signaling pathways. Animal experiments showed that the model group had higher rate of estrous cycle disorders, lower number and poorer morphology of normally developed follicles at all levels, more atretic follicles, higher apoptosis of ovarian granulosa cells, lower number of retrieved oocytes, lower ovarian wet weight and ovarian index, higher serum levels of FSH and LH, lower levels of AMH and E_2, higher expression levels of HIF-1α, FOXO3a and Bax, and lower expression levels of PI3K, AKT, and Bcl-2 in the ovarian tissue than the normal group. Compared with the POR group, low-and high-dose Res decreased the rate of estrous cycle disorders, improved the follicle number and morphology, reduced atretic follicles, promoted the apoptosis of ovarian granulosa cells, increased retrieved oocytes, ovarian wet weight and ovarian index, and lowered serum FSH and LH levels. Moreover, Res down-regulated the expression levels of HIF-1α, FOXO3a and Bax, and up-regulated the expression levels of PI3K, AKT and Bcl-2 in the ovarian tissue. In summary, Res can inhibit apoptosis and mitigate poor ovarian response in mice by regulating the PI3K/AKT/FOXO3a and HIF-1α pathways.

Short term exposure to polystyrene nanoplastics in mice evokes self-regulation of glycolipid metabolism

With the detection of nano-plastics (NPs) in daily essentials and drinking water, the potential harm of NPs to human health has become the focus of global attention. Studies have shown that long term exposure to NPs can lead to disorders of glucose and lipid metabolism in organisms, while the effects of short term exposure are rarely reported. Moreover, environmental factors cause the aging of NPs, and it is unclear whether this has an effect on their toxicity. In this study, we use 100 nm polystyrene (PS) NPs and ultraviolet (UV) aging PS (aPS) NPs to gavage mice for 7 days at an exposure dose of 50 mg/kg/day. To evaluate the effects of exposure on mice hepatic glucose lipid metabolism, we performed blood biochemical, pathological and metabolomic analyses. The results showed that exposure to PS NPs and aPS NPs increased serum glucose, disrupted serum lipoprotein levels, and up-regulated the expression levels of phosphatidylinositol 3-kinase (PI3K)/ phosphoprotein kinase B (p-AKT)/Glucose transporter 4 (GLUT4) proteins in the glucose metabolism pathway. The expression levels of key proteins sterol regulatory element binding protein-1 (SREBP-1)/peroxisome proliferator-activated receptor-γ (PPARγ)/adipose triglyceride lipase (ATGL) in the lipid metabolism signaling pathway were significantly increased. These findings suggest that short term exposure to PS NPs and aPS NPs induces glycolipid metabolism disturbance in mice, which may subsequently awaken the mice to self-regulate the serum levels of various lipoproteins and the expression of related key proteins. Compared with PS NPs, the aPS NPs interfered more strongly with glucose metabolism, and the corresponding self-regulation in mice was also more obvious. These findings not only provide a basis for environmental factors to increase the health risk of NPs but also provided a reference for the selection of test substances for further studies on the toxicity of NPs.

PI3K/Akt/CncC signaling pathway mediates the response to EPN-Bt infection in Holotrichia parallela larvae.

Combining the entomopathogenic nematode (EPN), Heterorhabditis beicherriana LF strain, and Bacillus thuringiensis (Bt) HBF-18 strain is a practical strategy to manage the larvae of Holotrichia parallela Motschulsky (white grubs). However, the mechanisms underlying the larval defense response to this combined biocontrol strategy are unknown. The activities of some antioxidant enzymes (SOD, POD, CAT) and some detoxifying enzymes (AChE, P-450, CarE, GST) in grubs showed an activation-inhibition trend throughout the EPN-Bt exposure time course. Eight potentially key antioxidant and detoxifying enzyme genes in response to EPN-Bt infection were identified from the midgut of grubs through RNA sequencing. After silencing CAT, CarE18, and GSTs1, the enzyme activities were significantly decreased by 30.29%, 68.80%, and 34.63%, respectively. Meanwhile, the mortality of grubs was increased by 18.40%, 46.30%, and 42.59% after exposure to EPN-Bt for 1day. Interestingly, the PI3K/Akt signaling pathway was significantly enriched in KEGG enrichment analysis, and the expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), cap 'n' collar isoform-C (CncC), kelch-like ECH-associated protein 1 (Keap1), and CarE18 were all up-regulated when exposed to EPN-Bt for 1day. Furthermore, RNAi-mediated PI3K silencing showed a similar down-regulated trend between PI3K/Akt/CncC and CarE18. Moreover, silencing PI3K rendered grubs more susceptible to EPN-Bt and accelerated symbiotic bacteria multiplication in grubs. These results suggest that the PI3K/Akt/CncC pathway mediates the expression of CarE18 and participates in the defense response of H. parallela larvae against EPN-Bt infection. Our data provide valuable insights into the design of appropriate management strategies for this well-known agricultural pest. © 2022 Society of Chemical Industry.

Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway

To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 μmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 μmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 μmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Compared with the control group, MYR at 50, 100 and 200 μmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

ADAM33 Silencing Inhibits Vascular Smooth Muscle Cell Migration and Regulates Cytokine Secretion in Airway Vascular Remodeling via the PI3K/AKT/mTOR Pathway.

Introduction Vascular smooth muscle cells (VSMCs) are highly involved in airway vascular remodeling in asthma. Objectives This study aimed to investigate the mechanisms underlying the effects of a disintegrin and metalloproteinase-33 (ADAM33) gene on the migration capacity and inflammatory cytokine secretion of VSMCs. Methods Human aortic smooth muscle cells (HASMCs) were transfected with lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 or negative control vectors. The migration capacity of HASMCs was evaluated by a transwell assay. The levels of secreted inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative polymerase chain reaction and Western blot assays were performed to detect mRNA and protein expression levels. Results Silencing of ADAM33 significantly inhibited the migration of HASMCs. The expression of tumor necrosis factor alpha (TNF-α) in the supernatant of HASMCs was decreased, while that of interferon gamma (IFN-γ) was increased after the transfection of shRNA targeting ADAM33. Insufficient ADAM33 expression also suppressed the expression levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTOR), Rho-associated protein kinases, phospho-forkhead box protein O1 (FOXO1), and cyclin D1, but it did not affect the levels of AKT, mTOR, or Rho. Conclusion Silencing of the ADAM33 gene inhibited HASMC migration and regulated inflammatory cytokine secretion via targeting the PI3K/AKT/mTOR pathway and its downstream signaling. These data contribute to a better understanding of the regulatory mechanisms of airway vascular remodeling in asthma.

Explore the effects of pulmonary fibrosis on sperm quality and the role of the PI3K/Akt pathway based on rat model.

Researches were reported that respiratory diseases can lead to male infertility; however, it is unclear whether there is a relationship between pulmonary fibrosis (PF) and male infertility. This study examined the influence of PF on sperm quality and its mechanisms. The key signalling pathway of male infertility caused by PF was predicted based on bioinformatics research. After modelling, we evaluated semen quality. Real-time quantitative polymerase chain reaction and Western blotting were used to measure the protein and mRNA expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylation-protein kinase B (p-Akt) and B-cell lymphoma 2 (Bcl2) in rat testicular cells. Compared with group A (48.77±4.67; 59.77±4.79), the sperm concentration and total sperm viability of group B (8.44±1.71; 15.39±3.48) showed a downward trend (p<0.05). Western blotting showed that the protein expressions of PI3K, p-Akt and Bcl2 in the testes of group B (0.30±0.06; 0.27±0.05; 0.15±0.03) was significantly lower than those of group A (0.71±0.07; 0.72±0.06; 0.50±0.06) (p<0.05). The hypoxic environment induced by PF can inhibit the expression of PI3K, p-Akt and Bcl2 protein and eventually cause dysfunctional spermatogenesis.

Effect of Inonotus obliquus (Fr.) Pilat extract on the regulation of glycolipid metabolism via PI3K/Akt and AMPK/ACC pathways in mice

Ethnopharmacological relevanceInonotus obliquus (Fr.) Pilat is a mushroom belonging to the family Hymenochaetaceae. It is popularly called the Chaga mushroom in Russian folk medicine and has been used as a traditional medicine to treat diabetes mellitus in Eastern European and Asian countries. However, its effects on glycolipid metabolism disorders and underlying molecular mechanism of action remain unclear. Aim of the studyI. obliquus contains abundant functional components, which provide potential medicinal value. The purpose of this study was to investigate compositions of I. obliquus extract with a high-pressure water extraction method, and investigate the anti-type 2 diabetic effects of I. obliquus extract and the possible underlying mechanisms involved. Materials and methodsThe I. obliquus was extracted by a high-pressure water extraction method, and tested its main components by special assay kit and instrumental analysis. Type 2 diabetic C57BL/6 mice were induced by high-fat diet with low-dose STZ injection, and were daily gavaged with different doses of I. obliquus extract for 8 weeks. Glycemic, blood lipid profile, and histopathology of liver and pancreas were assessed. Underlying mechanisms related to glycemic control in liver were further performed. ResultsThe I. obliquus extract main compounds were β-Glucans, triterpenoids and polyphenol by determination. Oral administration of 250 mg/kg and 500 mg/kg I. obliquus extract significantly alleviated blood glucose and insulin resistance. Moreover, 250 mg/kg and 500 mg/kg of I. obliquus extract increased liver glycogen content and high-density lipoprotein cholesterol (HDL-C) levels while decreased total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels. Furthermore, the protein expression levels of phosphatidylinositol-3 kinase (PI3K), p-protein kinase B (Akt), p-adenosine monophosphate activated protein kinase (AMPK), and p-acetyl-CoA carboxylase (ACC) were upregulated, whereas sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) were downregulated after supplement with 250 mg/kg and 500 mg/kg of I. obliquus extract. Interestingly, I. obliquus extract was a dose-effect relationship within a certain range. 250 mg/kg had obvious anti-diabetes effect, and the effect of 500 mg/kg dose was the same as that of metformin. ConclusionI. obliquus extract ameliorated insulin resistance and lipid metabolism disorders in diabetic mice. The hypoglycemic and hypolipidemic properties of I. obliquus extract were supposedly exerted via the regulation of the PI3K/Akt and AMPK/ACC signaling pathways.

MiR-200b-5p inhibits proliferation of ovarian cancer cells by targeting ATAD2 and regulating PI3K/AKT signaling pathway.

The purpose of this study was to investigate the influences of micro ribonucleic acid (miR)-200b-5p on proliferation and apoptosis of ovarian cancer (OC) cells, and to explore its correlations with the target gene ATPase family, AAA domain containing 2 (ATAD2), and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Human ovarian fibroblasts (HOFs) or human OC cell lines (A2780) were cultured in vitro, and then, A2780 cells were separately transfected with miR-200b mimics or miR-NC or cultured with ATAD2-specific inhibitor BAY-850. Thereafter, the expression levels of miR-200b and ATAD2 messenger RNA (mRNA) were measured via qRT-PCR, and the proliferative capacity of cells was detected by CCK-8 assay. Next, the cell apoptosis was determined by means of flow cytometry and one-step TUNEL assay. Finally, the targeted regulatory relationship between miR-200b and ATAD2 was examined using a Luciferase reporter assay system, and the protein expressions were detected through Western blot (WB) assay. It was found that the expression level of miR-200b was remarkably lower (p<0.05), while the mRNA expression level of ATAD2 was notably higher (p<0.05) in A2780 cells than those in HOFs. The transfection with miR-200b mimics markedly reduced the mRNA expression level of ATAD2 (p<0.05) and the proliferative capacity (p<0.05) and increased the apoptosis rate (p<0.05) of A2780 cells. Besides, it was detected via the Luciferase reporter assay system that miR-200b inhibited ATAD2. BAY-850 significantly decreased the expression level of ATAD2 protein (p<0.05) and the proliferative capacity (p<0.05) but improved the apoptosis rate (p<0.05) of cells. Moreover, both miR-200b mimics and BAY-850 could distinctly repress the protein expression levels of PI3K and p-Akt of the PI3K/Akt signaling pathway (p<0.05) and enhance the expression of suppressor gene p53 (p<0.05). MiR-200b-5p can inhibit the proliferation and promote the apoptosis of OC cells through targeted inhibition of ATAD2 expression and regulation of the PI3K/Akt signaling pathway.

Effect of electroacupuncture on levels of serum sex hormones and expression of ovarian auto-phagy related factors in rats with polycystic ovary syndrome

To observe the effect of electroacupuncture (EA) on the expression of autophagy mediated PI3K/AKT pathway and sex hormones in polycystic ovary syndrome (PCOS) rats, so as to explore the mechanisms underlying improvement of PCOS anovulatory infertility. Six-week-old female Sprague-Dawley rats were randomly divided into control, model and EA groups (n=10 per group). The PCOS model was established by gavage of Letrozole solution (1.0 mg/kg), once daily for 21 consecutive days. Then, EA (2 Hz, 1 mA) was applied to the "Sanyinjiao"(SP6) and "Taichong"(LR3) for 20 min, once a day for 14 successive days. The morphological changes of the ovary were observed after H.E. staining. The contents of serum androgen (T), luteinizing hormone (LH) and anti-mullerian hormone (AMH) were detected by ELISA. The expression levels of phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT) and microtubule-associated protein 1 light chain 3 (LC3) in the ovary tissues were detected by Western blot. Compared with the control group, the levels of serum T, LH and AMH contents, and ovarian LC3 Ⅱ expression and the ratio of LC3 Ⅱ/Ⅰwere significantly increased (P<0.05), and the expression levels of PI3K and AKT proteins were considerably decreased in the model group (P<0.05). After EA intervention, the levels of serum T, LH, AMH, and the ratio of LC3 Ⅱ/Ⅰwere considerably down-regulated (P<0.05), and those of PI3K and AKT were obviously up-regulated in the EA group (P<0.05). EA intervention can reduce serum T, LH and AMH contents, and the ratio of ovarian LC3 Ⅱ/Ⅰ and up-regulate ovarian PI3K and AKT in PCOS rats, which may contribute to its effect in improving anovulatory infertility by suppressing autophagy.

Interleukin-17A Promotes Human Disc Degeneration by Inhibiting Autophagy Through the Activation of the Phosphatidylinositol 3-Kinase/Akt/Bcl2 Signaling Pathway

Previous studies have suggested that interleukin (IL)-17A is a key factor that contributes to intervertebral disc degeneration (IDD), whereas autophagy has been shown to be a protective mechanism in IDD. However, the relationship between IL-17A and autophagy in IDD remains to be fully elucidated. This study sought to evaluate the association between IL-17 and autophagy and the potential mechanism through which IL-17A affects autophagy in IDD. Intervertebral disc specimens were collected from 10 patients with lumbar disc herniation. Human degenerated nucleus pulposus (NP) cells were cultured in the presence or absence of IL-17A treatment. Western blot and monodansylcadaverine staining were used to measure autophagy levels in human degenerated NP cells. Subsequently, phosphatidylinositol 3-kinase (PI3K)/Akt/Bcl-2 pathway inhibitors were used to reveal the potential mechanism. IL-17A treatment inhibited the autophagic activity in human NP cells in a time- and dose-dependent manner. Moreover, monodansylcadaverine staining showed that cells treated with IL-17A had significantly fewer changes in their autophagic vacuoles compared with control-treated cells. After IL-17A treatment, expression levels of PI3K, p-Akt, and Bcl-2 in NP cells were significantly increased. Further assays with PI3K/Akt/Bcl-2 inhibitors revealed that IL-17A suppressed autophagy in NP cells by activating the PI3K/Akt/Bcl-2 signaling pathway. These data suggest that IL-17A promotes IDD by inhibiting autophagy through activation of the PI3K/Akt/Bcl-2 signaling pathway and may offer new insights for targeted therapy of this disease.

85 Regulation of mammalian target of rapamycin signaling post-fertilisation is essential for efficient development of bovine pre-implantation embryos

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is considered a key regulator of various cellular responses related to growth, proliferation, survival, apoptosis, and autophagy. The expression of mTOR in cumulus cells and all oocyte stages highlights a role in embryonic development, but its function in the context of embryo development is not completely clarified. In the current study, we investigated the effect of mTOR activation on the total cleavage and blastocyst development rates in bovine. Oocytes collected from bovine ovaries were invitro matured (around 50 oocytes per group) and fertilised before incubation with the mTOR activator MHY1485 (1 µM) for 72h at 38.5°C and 5% CO2. The total cleavage rate at Day 4 post-fertilisation and the blastocyst development rate at Day 8 post-fertilisation were recorded. The expression levels of the mTOR and PI3K in Day 8 blastocysts were investigated using immunofluorescence and imaged using confocal laser-scanning microscope, whereas the fluorescence intensity was analysed using ImageJ software. The difference between groups was analysed using Student's t-test (GraphPad Prism). Results of microscopic investigations showed higher rates of Day 4 cleavage (83.7±1.3 vs. 72±0.8% for control) and Day 8 blastocyst development (40.4±1.7 vs. 33±1.8% for control) upon addition of MHY1485 (P&amp;lt;0.05). The immunofluorescence showed higher mTOR and PI3K expression levels in MHY1485-treated group than the untreated control (P&amp;lt;0.05). The total number of cells per blastocyst, using 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei, was higher in the mTOR-activated group (194.9±4.8 vs. 171.3±3.5 for control). In conclusion, our data reflect the essential role of the mTOR signaling for bovine pre-implantation embryonic development.

Downregulation of osteopontin inhibits browning of white adipose tissues through PI3K-AKT pathway in C57BL / 6 mice

Brown adipose tissue (BAT) plays important roles in regulating energy homeostasis and combating obesity. Accordingly, increasing the abundance and/or activating BAT would be effective and promising approaches to combat obesity and obesity-relative diseases. Our previous data in vitro have shown that osteopontin (OPN) induces the brown adipogenesis in 3T3-L1 cells via a phosphatidylinositol 3 kinase (PI3K)-AKT pathway. However, it is currently unknown whether OPN exerts such an effect on animals in vivo. Therefore, in the study we sought to investigate the pro-browning effects of OPN and to explore its underlying mechanisms by transfecting with Ad-GFP-aP2-OPN-shRNA to specifically down-regulate the OPN of white adipose tissue (WAT) in mice. Our present results show that downregulation of OPN in WAT exacerbates obesity and inhibits WAT-browning. Moreover, immunohistochemical results also exhibit that the downregulation of OPN significantly diminishes the expression and sub-cellular localization of UCP-1, PRDM16 and PGC-1α. Besides, the western blotting results reveal that the expression levels of PI3K, AKT-pS473 and PPARγ markedly reduce. Consequently, we conclude that the downregulation of OPN inhibits the browning of WAT through inhibiting the expression of PPARγ mediated by the PI3K-AKT pathway. The findings suggest that OPN is involved in regulation of WAT-browning and regulating its expression would become a potential strategy to combat obesity and obesity-relative metabolic diseases.

Protective effect of nicorandil on collapse‑induced lung injury in rabbits by inhibiting apoptosis.

The one‑lung ventilation (OLV) technique is vital in thoracic surgery. However, it can result in severe lung injury, which is difficult to manage. The main solution at present is the use of ventilation strategies, including continuous positive oxygen pressure, low tidal volume and high frequency ventilation, and the administering of drugs, including phenylephrine, dexmedetomidine and morphine. However, the protective effect of these methods on the lungs is not sufficient to improve the prognosis of patients. Therefore, how to develop a novel protective drug remains an open question. Nicorandil, a mitochondrial (mito)KATP‑specific opener, serves an important role in cardioprotection, although its effect on lung injury remains unclear. The present study examined the protective role of nicorandil against collapse‑induced lung injury in rabbits undergoing OLV. Changes in arterial oxygen saturation (SaO2), arterial partial pressure for oxygen (PaO2), wet/dry weight ratio, and the microstructure of tissues and cells were observed. Enzyme‑linked immunosorbent assays were used to determine the concentrations of malondialdehyde(MDA) and tumor necrosis factor (TNF)‑α, and the activity of superoxide dismutase (SOD) in rabbits treated with nicorandil. Terminal deoxynucleotidyl transferase transfer‑mediated dUTP nick end‑labeling was used to detect apoptosis and western blotting was used to analyze the relative proteins involved in apoptosis. Western blotting and reverse transcription‑quantitative polymerase chain reaction analysis were used to examine the expression of hypoxia inducible factor1α(HIF‑1α), phosphatidylinositol‑3‑kinase (PI3K), protein kinaseB (Akt) and nuclear factor (NF)‑κB in the lungs of rabbits treated with nicorandil. The SaO2 and PaO2 in the high‑dose group were significantly higher than those in the control group in the process of OLV. The wet/dry weight ratio, and the concentrations of MDA and TNF‑α in the collapsed lung of the high‑dose group were significantly lower than those in the control group. The activity of SOD in the high‑dose group was significantly higher than that in the control group. The lung had improved microstructure and less apoptosis, which was determined by the Bax/Bcl2 ratio in the high‑dose group. The expression levels of PI3K, phosphorylated Akt and HIF‑1α were upregulated, whereas the expression of NF‑κB was downregulated. In conclusion, nicorandil had a protective effect via inhibiting apoptosis in non‑ventilated lung collapsed and re‑expansion during OLV in the rabbit. It acted on mitoKATP through the PI3K/Akt signaling pathway.