Protein Expression Levels Of PI3K Research Articles

Effects of Petasitin as Natural Extract on Proliferation and Pathological Changes of Pediatric Neuroblastoma SK-N-SH Cells

This research was developed to investigate the impact and mechanisms of petasitin as natural extract on the proliferation and pathological changes of pediatric neuroblastoma SK-N-SH cells. The sample cells were selected as experimental materials and randomly rolled into a control (Ctrl), a low-dose (LD), a medium-dose (MD), and a high-dose (HD) group, which were subjected to regular culture, 0.5, 1.5, and 5 μM/L petasitin for 12 hours, respectively. Meanwhile, the cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry (FCT) was employed to assess the cycle progression and apoptosis of SK-N-SH cells, while Hoechst 33258 fluorescent staining was applied to observe the nuclear changes. Meanwhile, the real-time quantitative PCR (RT-qPCR) was applied to measure the mRNA levels of PI3K, PTEN, AKT1, and mTOR genes in SK-N-SH cells. Western blot was conducted to determine the protein expression levels (PELs) of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR in SK-N-SH cells. The results revealed that after treatment for 48 and 72 hours, the optical density (OD) values in the petasitin treatment groups were lower and exhibited great differences to those in the Ctrl group (P < 0.05), while inhibition rates (IRs) were higher (P < 0.05). Furthermore, the petasitin treatment groups exhibited an obvious increase in the G1/G0 cell cycle ratio (CCR) and a great decrease in S and G2/M phase CCR (P < 0.05). Moreover, the apoptotic rates in the petasitin treatment groups were much higher at different time points, showing observable differences with P < 0.05. Besides, the Hoechst 33258 staining positivity rates (PRs) of SK-N-SH cell nuclei in the petasitin treatment groups were higher and presented great differences with P < 0.05 to those in the Ctrl group. In addition, the cells in petasitin treatment groups exhibited greatly downshifted mRNA levels of PI3K and AKT1 and obviously elevated PTEN (all P < 0.05). PELs of PI3K, AKT1, and mTOR exhibited no great differences (P > 0.05), but phosphorylation levels of p-PI3K, p-Akt, and p-mTOR were decreased in the petasitin treatment groups, presenting great differences with P < 0.05. Together, these results suggested that petasitin exerted a suppressive role in proliferation and promote the apoptosis of pediatric SK-N-SH cells by adjusting PI3K-Akt-mTOR signaling pathway (SPW).

Effects of catechin on the malignant biological behavior of gastric cancer cells through the PI3K/Akt signaling pathway

Catechin is a kind of flavonoids, mainly derived from the plant Camellia sinensis. It has a strong antioxidant effect, and it also has significant therapeutic effects on anti-cancer, anti-diabetes, and anti-infection. This study was intended to look at how catechin affected the malignant biological activity of gastric cancer cells. We used databases to predict the targets of catechin and the pathogenic targets of gastric cancer. Venn diagram was used to find the intersection genes, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed on intersection genes. Using the STRING database, the Protein-Protein Interaction (PPI) network was built. The top 8 genes were screened by Cytoscape 3.9.1, then their binding was verified by molecular docking. The proliferation ability, cell cycle, apoptosis and migration of gastric cancer cells were detected, as well as the protein expression levels of PI3K, p-AKT, and AKT and the mRNA expression levels of AKT1, VEGFA, EGFR, HRAS, and HSP90AA1 in gastric cancer cells. Our research revealed that different concentrations of catechin could effectively inhibit the proliferation and migration of gastric cancer cells, regulate the cell cycle, and promote the death of these cells, and it's possible that the PI3K/Akt pathway was crucial in mediating this impact. Moreover, adding the PI3K/Akt pathway agonist significantly reduced the promoting effect of catechin on the apoptosis of gastric cancer cells. This study suggested that catechin was a potential drug for the treatment of gastric cancer.

Protective Effects of Sodium Para-Aminosalicylic Acid on Lead and Cadmium Co-Exposure in SH-SY5Y Cells.

Combined exposure to lead and cadmium is common in occupational environments. However, the effects of co-exposure to Pb-Cd on neurotoxicity have not been fully clarified. Sodium para-aminosalicylic acid (PAS-Na) has previously been shown to protect neurons from Pb-induced toxicity. This study aimed to investigate the beneficial effect of PAS-Na against co-exposure to Pb-Cd-induced neurodegeneration in SH-SY5Y cells. The MTT assay was used to detect the effects of Pb and Cd alone, or in combination, on SH-SY5Y cell survival. The effects of Pb and Cd alone or in combination on oxidative stress were assessed by reactive oxygen species (ROS) level. Nrf2, the master switch for antioxidant responses, was detected by immunofluorescence. Protein expression levels of PI3K, Akt, p-Akt, Nrf2 and HO-1 were determined by Western blot analysis. MTT assay results established that the survival rate of SH-SY5Y cells was not significantly affected by exposure to 1 μmol/L lead, 0.25 μmol/L cadmium, and 1-fold Pb-Cd mixture (1 μmol/L Pb + 0.25 μmol/L Cd), while 10-fold Pb-Cd combined exposure (10 μmol/L Pb + 2.5 μmol/L Cd) significantly reduced the survival rate of SH-SY5Y cells. Combined Pb-Cd exposure significantly increased intracellular ROS levels, and N-Acetyl-L-cysteine (NAC) treatment in the 10 μmol/L Pb + 2.5 μmol/L Cd group significantly decreased ROS expression levels, attenuating the levels of oxidative stress. Protein expression of PI3K and p-Akt significantly decreased in the 10 μmol/L Pb + 2.5 μmol/L Cd group, while the expression of PI3K and p-Akt protein increased after PAS-Na intervention. Immunofluorescence analysis showed that levels of Nrf2 in the nucleus increased in the 10 μmol/L Pb + 2.5 μmol/L Cd group, along with Nrf2 protein levels, suggesting that Nrf2 was translocated from the cytoplasm into the nucleus upon combined Pb-Cd exposure. In addition, HO-1 protein expression level, a downstream gene product of Nrf2, was increased. In response to NAC intervention, HO-1 protein expression levels significantly decreased. PAS-Na had the same intervention effect as NAC. Combined exposure to Pb-Cd induced oxidative stress and cytotoxicity in SH-SY5Y cells. PAS-Na displayed antagonistic effects on neurodegenerative changes induced by combined Pb-Cd exposure; hence, it may afford a novel treatment modality for exposure to these metals.

The combined effect and mechanism of antiangiogenic drugs and PD-L1 inhibitor on cell apoptosis in triple negative breast cancer.

Breast cancer is the most common cancer worldwide, and triple-negative breast cancer (TNBC) has the worst prognosis. Standard systemic treatment includes chemotherapy and immunotherapy. Poly ADP-ribose polymerase (PARP) inhibitors are considered in breast cancer (BRCA) susceptibility genes mutated tumors. The role of antiangiogenic drugs is controversial. Immunotherapy with immune checkpoint inhibitor is now a standard of care for TNBC in the US, but its use in combination with anlotinib, an inhibitor of angiogenesis, on TNBC cells was never investigated. We tested the effects of anlotinib and programmed cell death-ligand 1 (PD-L1) inhibitor on the proliferation, apoptosis, migration, and invasion of MDA-MB-468 and BT-549 TNBC cells through 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cell apoptosis assay, wound healing and transwell matrix assays, and verified whether the combination of the two drugs had synergistic effect. Western blotting was used to detect the effect of anlotinib and PD-L1 inhibitor on the protein expression levels of PI3K, p-PI3K, AKT, p-AKT, Bcl-xl in MDA-MB-468 and BT-549 cells. The effects of anlotinib, PD-L1 inhibitor and the combination of the two drugs on the transplanted tumor of TNBC mice were tested by animal experiments. Anlotinib and PD-L1 inhibitor inhibited the proliferation and promote cell apoptosis of MDA-MB-468 and BT-549 cells, and the combination demonstrated the synergetic effect. Anlotinib and PD-L1 inhibitor inhibited cell migration and invasion, and the effect was strongest in the combination group. Both anlotinib and PD-L1 inhibitor reduced the expression of p-PI3K, p-AKT and Bcl-xl proteins in cells and the effects were the strongest in the combination group. Both anlotinib and PD-L1 inhibitor inhibited the growth of transplanted tumors in mice, and the combined group demonstrated the strongest growth suppression. Anlotinib and PD-L1 inhibitor can inhibit cell proliferation, migration, and invasion of TNBC and promote cell apoptosis, and the two drugs show combined anti-tumor effects in vivo and in vitro. The combination of anlotinib and PD-L1 inhibitor may promote apoptosis of TNBC cells through PI3K/AKT/Bcl-xl signaling pathways, which might offer potential clinical treatment roles for these.

Upregulation of MTHFD2 is associated with PD‑L1 activation in bladder cancer via the PI3K/AKT pathway.

Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) has been implicated in the etiology of various human malignant tumors; however, its exact role in bladder cancer (BC) remains to be explored. Through reverse transcription‑quantitative PCR, western blotting and immunohistochemistry detection of BC tissue, combined with The Cancer Genome Atlas (TCGA) database analysis, the present study demonstrated that MTHFD2 was upregulated in BC tissues. MTHFD2 expression in patients with BC was frequently associated with worse prognosis, tumor immune cell infiltration and programmed death‑ligand 1 (PD‑L1) expression. Subsequently, using short hairpin RNA, the expression levels of MTHFD2 were knocked down in BC cell lines, and the results revealed that the tumor cell proliferation and colony formation abilities of cells were greatly reduced, as determined by Cell Counting Kit 8 and colony formation assays, as was the expression of PD‑L1, as determined by western blotting. These findings were also confirmed in a xenograft nude mouse model. Simultaneously, it was revealed that abnormal expression of MTHFD2 was closely associated with the PI3K/AKT signaling pathway in both RNA‑sequencing and TCGA datasets. This observation was verified in vitro by detecting the protein expression levels of PI3K and AKT by western blotting. The activation of PI3K and AKT was enhanced in BC cells (T24) following stimulation with 740Y‑P, a PI3K activator, and cellular activities and PD‑L1 expression levels were restored. Finally, it was demonstrated that the MTHFD2 levels were correlated with chemosensitivity to traditional BC chemotherapeutic agents and various PI3K/AKT‑targeted drugs, as determined by analyzing the Genomics of Drug Sensitivity in Cancer database. Overall, the present findings revealed that upregulation of MTHFD2 was associated with PD‑L1 activation in BC via the PI3K/AKT signaling pathway, suggesting that it could be a promising marker of chemotherapy and immunotherapy for BC.

Effects of RFRP‑3 on an ovariectomized estrogen‑primed rat model and HEC‑1A human endometrial carcinoma cells.

The hypothalamic peptide gonadotropin inhibitory hormone (GnIH) is a relatively novel hypothalamic neuropeptide, identified in 2000. It can influence the hypothalamic-pituitary-gonadal axis and reproductive function through various neuroendocrine systems. The present study aimed to explore the effects and potential underlying molecular mechanism of RFamide-related peptide-3 (RFRP-3) injection on the uterine fluid protein profile of ovariectomized estrogen-primed (OEP) rats using proteomics. In addition, the possible effects of RFRP-3 on the viability and apoptosis of the human endometrial cancer cell line HEC-1A and associated molecular mechanism were investigated. The OEP rat model was established through injection with GnIH/RFRP-3 through the lateral ventricle. At 6 h after injection, the protein components of uterine fluid of rats in the experimental and control groups were analyzed using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Differentially expressed proteins (DEPs) were analyzed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Protein-protein interactions (PPI) were investigated using the STRING database. PPI networks were then established before hub proteins were selected using OmicsBean software. The expression of one of the hub proteins, Kras, was then detected using western blot analysis. Cell Counting Kit-8, Annexin V-FITC/PI, reverse transcription-quantitative PCR and western blotting were also performed to analyze cell viability and apoptosis. In total, 417 DEPs were obtained using LC-MS/MS, including 279 upregulated and 138 downregulated proteins. GO analysis revealed that the majority of the DEPs were secretory proteins. According to KEGG enrichment analysis, the DEPs found were generally involved in tumor-associated pathways. In particular, five hub proteins, namely G protein subunit α (Gna)13, Gnaq, Gnai3, Kras and MMP9, were obtained following PPI network analysis. Western blot analysis showed that expression of the hub protein Kras was downregulated following treatment with 10,000 ng/ml RFRP-3. RFRP-3 treatment (10,000 ng/ml) also suppressed HEC-1A cell viability, induced apoptosis, downregulated Bcl-2 and upregulated Bax protein expression, compared with those in the control group. In addition, compared with those in the control group, RFRP-3 significantly reduced the mRNA expression levels of PI3K, AKT and mTOR, while upregulating those of LC3-II. Compared with those in the control group, RFRP-3 significantly decreased the protein expression levels of PI3K, AKT, mTOR and p62, in addition to decreasing AKT phosphorylation. By contrast, RFRP-3 significantly increased the LC3-II/I ratio and G protein-coupled receptor 147 (GPR147) protein expression. In conclusion, the present data suggest that RFRP-3 can alter the protein expression profile of the uterine fluid of OEP rats by upregulating MMP9 expression whilst downregulating that of key hub proteins Gna13, GnaQ, Gnai3 and Kras. Furthermore, RFRP-3 can inhibit HEC-1A cell viability while promoting apoptosis. The underlying molecular mechanism may involve activation of GPR147 receptor by the direct binding of RFRP-3, which further downregulates the hub protein Kras to switch on the PI3K/AKT/mTOR pathway. This subsequently reduces the Bcl-2 expression and promotes Bax expression to induce autophagy.

Effect of Codonopsis Radix and Polygonati Rhizoma on the regulation of the IRS1/PI3K/AKT signaling pathway in type 2 diabetic mice.

Codonopsis Radix and Polygonati Rhizoma (CRPR) has a good hypoglycemic effect. The aims of the present study were to investigate the effect of CRPR on high-fat/high-sugar diet (HFHSD)- and streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) mice as well as to investigate the involved mechanism. A T2DM mouse model was generated by combining HFHSD and STZ. After the model was established, normal and model groups received the same volume of normal saline intragastrically, and the negative control group was treated with metformin (200 mg/kg·BW). The low, medium, and high CRPR groups received four consecutive weeks of oral gavage with CRPR doses of 2.5, 5, and 10 g/kg·BW, respectively, during the course of the study. Body weight and fasting blood glucose (FBG) were measured on a weekly basis. Enzyme-linked immunosorbent assay (ELISAs) were used to evaluate the serum and liver samples. Hematoxylin and eosin (H&E) staining was utilized to observe the pathological status of the liver and pancreas. Western blot (WB) analysis was performed to evaluate the protein expression levels of PI3K, p-PI3K, AKT, and p-AKT. Compared to model mice, each treatment group had significantly elevated levels of FBG, total cholesterol (TC), and triacylglycerol (TG) (P<0.01 and P<0.05, respectively). The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly reduced in the treatment groups compared to the model group (P<0.01). Compared to the model group, fasting insulin (FINS) levels were elevated in all groups of CRPR (P<0.05), and there were significantly higher levels of high-density lipoprotein cholesterol (HDL-C) in both the low-dose and high-dose CRPR groups (P<0.05). H&E staining indicated that CRPR treatment reduced organ enlargement, improved liver lipid accumulation, and repaired islet injury in T2DM mice. Moreover, WB analysis demonstrated that all CRPR groups significantly upregulated the protein expression of IRS1, p-GSK3β, PI3K, p-Akt and p-FOXO1(P<0.05) as well as significantly downregulated p-IRS1 and FOXO1 protein expression (P<0.05). The present study demonstrated that CRPR effectively improves the metabolic disturbance of lipids, repairs damaged liver tissues, repairs damaged pancreatic tissues, and reduces insulin resistance (IR) in T2DM mice. The mechanism of action may be associated with upregulation of the IRS1/PI3K/AKT signaling pathway and inhibition of IRS1 phosphorylation.

Adenosine A1 receptor agonism protection mechanism in intestinal ischemia/reperfusion injury via activation of PI3K/Akt signaling.

Intestinal ischemia/reperfusion (I/R) injury is a common clinical problem with a high mortality rate, resulting from loss of blood flow to an intestinal segment. Adenosine serves a protective role in intestinal I/R injury; however, its potential mechanism is not completely understood. The present study aimed to investigate the protective effects of adenosine A1 receptor (A1R) agonists CPA and LUF6941 and whether their mechanisms are associated with the PI3K/Akt signaling pathway. To simulate intestinal I/R injury, a cell oxygen-glucose deprivation/reoxygenation (OGD/R) model was established and the human colon cancer cell line (Caco-2) was incubated with A1R agonists before OGD/R treatment. The viability of Caco-2 cells was detected by PI and Cell Counting Kit-8 assay, apoptosis was detected using flow cytometry and western blotting was used to analyze protein expression levels of PI3K, Akt and p53 in Caco-2 cells. A1R agonist pretreatment protected Caco-2 cells against OGD/R-induced cell damage and activated PI3K/Akt signaling. Additionally, apoptosis was inhibited by downregulating phosphorylation of p53 protein, as evidenced by increased cell viability. These findings suggested that A1R agonists decreased OGD/R damage in Caco-2 cells, which may be due to their anti-apoptotic effects and activation of the PI3K/Akt/p53 signal pathway.

KIF3C Promotes the Malignant Progression of Lung Cancer Cells A549

Objective: To investigate the role of KIF3C gene in promoting the malignant phenotype of lung cancer cells and in regulating PI3K/AKT signaling pathway. Methods: CCK-8 and transwell assays were used to detect the changes in cell proliferation and cell migration ability after being transfected with siKIF3C, as well as the protein expression levels of PI3K, p-PI3K, AKT, and p-AKT following the downregulation of KIF3C by Western blot. Results: The CCK-8 assay showed that the proliferation/viability of lung cancer cells A549 significantly reduced after being transfected with siKIF3C gene (P &lt; 0.05); the migration ability of lung cancer cells A549 was significantly reduced after transfected with siKIF3C gene (P &lt; 0.05); the levels of p-PI3K and p-AKT proteins were downregulated after KIF3C protein knockdown (P &lt; 0.05); however, the detection of PI3K and AKT protein levels was not statistically significant. Conclusion: KIF3C may promote the proliferation and migration ability of lung cancer cells A549 through PI3K/AKT signaling pathway.

Resveratrol inhibits the inflammatory response and oxidative stress induced by uterine ischemia reperfusion injury by activating PI3K-AKT pathway.

Uterus transplantation is a complex surgical procedure. Uterine ischemia reperfusion (I/R) injury that occurs during this process may cause a loss of function of the uterus and the failure of transplantation. Resveratrol (RSV) is a naturally occurring polyphenol found abundantly in the skin of grapes and red wine, and it has also been used as a dietary supplement in clinical practice. RSV possesses strong anti-inflammatory and anti-oxidative effects, and exhibits a role in immune system regulation. However, the role of RSV in protecting the uterus against I/R-induced injury is yet to be fully elucidated. The aim of the present study was to investigate the effects and mechanisms underlying RSV in I/R-induced uterus injury. A total of 48 Sprague-Dawley rats were randomly divided into four groups: Control (sham operation) group, the uterus I/R group, the 20 mg/kg RSV-pre-treated I/R (I/R+RSV/20) group and the 40 mg/kg RSV-pre-treated I/R (I/R+ RSV/40) group. A regular I/R model was established to induce uterus injury in rats. RSV at 20 or 40 mg/kg was intraperitoneally administrated into rats in both I/R+ RSV groups once per day for five consecutive days prior to ischemia. The control and I/R only groups received an intraperitoneal injection of the vehicle (ethanol) for the same period prior to ischemia. Samples from blood and uterine horns were collected 3 h after reperfusion. Changes in the levels of malondialdehyde, interleukin (IL)-6, tumor necrosis factor-α, IL-10 and IL-37 were determined using ELISA, the activity levels of myeloperoxidase, catalase, and superoxidase dismutase were also determined using ELISA, the protein expression levels of PI3K, phosphorylated (p)-PI3K, AKT and p-AKT were determined using western blot analysis, and the uterine histology was investigated using H&E staining. Results of the present study demonstrated that treatment with RSV increased the capacity of antioxidants and suppressed uterine oxidative injury induced by I/R. Moreover, treatment with RSV decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines. In addition, RSV promoted the phosphorylation of PI3K and AKT, thus activating the PI3K-AKT signaling pathway. Therefore, administration of RSV prior to uterine I/R effectively alleviated inflammatory response and oxidative stress via activation of the PI3K-AKT pathway, suggesting that RSV may play a protective role in I/R-induced uterus injury.

A rat study model of depression-driven chronic prostatitis by modulating the PI3K/Akt/mTOR network.

Depression and chronic prostatitis (CP) are two common diseases that affect the human population worldwide. Clinically, it has been demonstrated that andrological patients often simultaneously suffer from depression and CP. Prior investigations have established that depression acts as an independent risk factor for CP. Herein, we explored the correlation between depression and CP using bioinformatics tools and through animal experiments. The potential targets and signalling pathways involved in depression and CP were predicted using bioinformatics tool, while depression in the rat model was established through chronic restraint stress. The expression of the related proteins and mRNA was assessed by Western blotting and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Relative to those in the control rats, the protein contents of PI3K, p-Akt, and p-mTOR were lower in the model rats (p < 0.05). Similarly, the transcript levels of PI3K, Akt, and mTOR was also relatively lower in the model rats (p < 0.05). And PI3K/Akt agonists reduced inflammation in rat prostate tissue, accompanied by significant increases in the transcript and protein expression levels of PI3K, Akt, and mTOR. Thus, we proposed that depression model rats may induce CP as a result of mediation by the negative regulation of the PI3K/Akt/mTOR signalling network.

Study on the Mechanism of Shenjing Guben Prescription Regulating PI3K and NRF2 Signaling Pathway in the Treatment of Immune Infertility.

Objective To explore the mechanism of Shenjing Guben prescription (SP) in the treatment of immune infertility by regulating PI3K-NRF2/p38 signal pathway. Methods 60 adult male SD rats were randomly divided into control group (NC group), ACN group, low concentration AP intervention group (low group), middle concentration SP intervention group (middle group), and high concentration SP intervention group (high group). 12 rats in each group were administered by gavage once a day, 6 days/w, and the rats were killed after 28 days. Bilateral testis and epididymis were removed and weighed and organ coefficients were calculated, and testicular histopathological sections were prepared to evaluate the changes of testicular tissue structure. The relative expression levels of PI3K, MKK7, JNK, p38 mRNA, and protein in testis were measured by QRT-PCR and western blot. Results (1) Compared with the control group, the proportion of grade A and B sperms in ACN group increased significantly, and the proportion of grade D sperm decreased significantly (P < 0.05). After SP intervention, compared with ACN group, there was no significant difference in the proportion of sperm at all levels in low, medium, and high SP intervention groups (P > 0.05). (2) Compared with the control group, the sperm VCL, VSL, VAP, and mad in ACN group increased significantly, and the BCF decreased significantly (P < 0.05). After SP intervention, compared with ACN group, there was no significant difference in sperm motility parameters among low, medium, and high SP intervention groups (P > 0.05). (3) Compared with the control group, the activities of AKP and SDH in testicular tissue of rats in ACN group decreased significantly (P < 0.05). After SP intervention, compared with ACN group, AKP activity increased significantly and LDH activity decreased significantly in low, medium, and high SP intervention groups (P < 0.05). (4) Compared with the control group, the expression levels of PI3K, p-PI3K, MKK7, p-MKK7, JNK, p-JNK, p38, and p-p38 proteins and the ratios of p-JNK/JNK and p-p38/p38 increased in the testis of ACN group (P < 0.05). After SP intervention, compared with ACN group, the protein expression levels of PI3K, p-PI3K, MKK7, p-MKK7, JNK, p-JNK, p38, and p-p38 in testicular tissue of SP intervention group decreased, and the ratio of p-JNK/JNK and p-p38/p38 decreased (P < 0.05). Conclusion SP can reduce the oxidative stress of testis induced by ACN and inhibit the activation of PI3K-NRF2/p38 signal pathway.

Fengreqing Oral Liquid Exerts Anti-Inflammatory Effects by Promoting Apoptosis and Inhibiting PI3K/AKT and NF-κB Signaling Pathways.

Fengreqing oral liquid (FOL), a Chinese patent drug frequently used in clinical practice in China, is effective in treating inflammatory diseases of the upper respiratory tract such as colds and flu. However, its anti-inflammatory effects and mechanisms remain to be elucidated. In this study, the anti-inflammatory effects of FOL and its mechanisms on PI3K/AKT and NF-κB signaling pathways in LPS-induced RAW264.7 cells were explored, as well as the regulatory effect of FOL on apoptosis. In addition, the potential of FOL for the treatment of acute lung injury was explored in LPS-induced ALI mice. The results showed that treatment with FOL significantly reduced the levels of interleukin 1β (IL-1β), interleukin 6 (IL-6), nitric oxide (NO), and tumor necrosis factor α (TNF-α) in the supernatant of LPS-induced RAW264.7 cells, and also significantly reduced the phosphorylated protein levels of PI3K and AKT in the PI3K/AKT signaling pathway and also protein levels of NF-κB p50, phosphorylated NF-κB p65, and IκBα in the NF-κB signaling pathway. In addition, the results showed that FOL induced apoptosis in LPS-induced RAW264.7 cells at the level of 80%–90%, and significantly increased the protein expression levels of the pro-apoptotic Bax and cleaved-caspase-3. In LPS-induced ALI mice, FOL administration showed inhibition of IL-1β, IL-6, and TNF-α in Bronchoalveolar lavage fluid (BALF) and decreased protein expression levels of PI3K, AKT, NF-κB p50, and NF-κB p65, and elevated protein expression levels of Bax and cleaved-caspase-3 significantly. These results suggest that FOL may exert anti-inflammatory effects by inhibiting the PI3K/AKT signaling pathway to promote apoptosis and leading to attenuated activation of the NF-κB signaling pathway.

Neuroprotective effect of AGGF1 against isoflurane-induced cognitive dysfunction in aged rats through activating the PI3K/AKT signaling pathways.

This study aimed to evaluate and identify the value and explore the mechanisms of Angiogenic Factor with G-patch and FHA domains 1 (AGGF1) in postoperative cognitive dysfunction (POCD). Rats were separated into four different groups, namely sham, isoflurane, isoflurane + recombinant human Aggf1 (rh-Aggf1) (5 μg kg-1), and isoflurane + rh-Aggf1 (10 μg kg-1). qPCR and western blot assays were applied to detect the correlation between the expression of AGGF1 and isoflurane administration. Then, the Morris water maze (MWM) test was applied to evaluate the effect of AGGF1 on improving the POCD rats. Subsequently, TUNEL assay was applied and the cell apoptosis-related proteins were tested to reveal the anti-apoptotic effect of AGGF1 in POCD rats. Furthermore, the mRNA and protein levels of TNF-α, IL-6, and IL-1β were also detected by qPCR and ELISA to verify the anti-inflammatory effects of AGGF1 on POCD rats. Besides, the protein expression levels of PI3K, Akt, and NF-κB in each group were examined by western blot. In this study, the results revealed that isoflurane induced a decrease in AGGF1 expression in the hippocampus of aged rats. In addition, exogenous AGGF1 attenuated POCD in aged rats. Meanwhile, exogenous AGGF1 had anti-apoptotic and anti-inflammatory effects in POCD rats. Further research indicated that AGGF1 activated the PI3K/Akt pathway. AGGF1 has neuroprotective effect against isoflurane-induced cognitive dysfunction in aged rats via activating the PI3K/AKT signaling pathways.

Neuroprotective Effect of Chrysophanol as a PI3K/AKT/mTOR Signaling Inhibitor in an Experimental Model of Autologous Blood-induced Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) refers to predominant, sporadic, and non-traumatic bleeding in the brain parenchyma. The PI3K/AKT/mTOR signaling pathway is an important signal transduction pathway regulated by enzyme-linked receptors and has many biological functions in mammals. It plays a key role in neuronal metabolism, gene expression regulation, and tissue homeostasis in the healthy and diseased brain. In the present study, the role of the PI3K/AKT/mTOR pathway inhibitor chrysophanol (CPH) (10 mg/kg and 20 mg/kg, orally) in the improvement of ICH-associated neurological defects in rats was investigated. Autologous blood (20 µL/5 min/unilateral/intracerebroventricular) mimics ICH-like defects involving cellular and molecular dysfunction and neurotransmitter imbalance. The current study also included various behavioral assessments to examine cognition, memory, and motor and neuromuscular coordination. The protein expression levels of PI3K, AKT, and mTOR as well as myelin basic protein and apoptotic markers, such as Bax, Bcl-2, and caspase-3, were examined using ELISA kits. Furthermore, the levels of various neuroinflammatory cytokines and oxidative stress markers were assessed. Additionally, the neurological severity score, brain water content, gross brain pathology, and hematoma size were used to indicate neurological function and brain edema. CPH was found to be neuroprotective by restoring neurobehavioral alterations and significantly reducing the elevated PI3K, AKT, and mTOR protein levels, and modulating the apoptotic markers such as Bax, Bcl-2, and caspase-3 in rat brain homogenate. CPH substantially reduced the inflammatory cytokines like interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. CPH administration restored the neurotransmitters GABA, glutamate, acetylcholine, dopamine, and various oxidative stress markers. Our results show that CPH may be a promising therapeutic approach for overcoming neuronal damage caused by the overexpression of the PI3K/AKT/mTOR signaling pathway in ICH-induced neurological dysfunctions in rats.

Neuroprotective Effect of Chrysophanol as a PI3K/AKT/mTOR Signaling Inhibitor in an Experimental Model of Autologous Blood-induced Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) refers to predominant, sporadic, and non-traumatic bleeding in the brain parenchyma. The PI3K/AKT/mTOR signaling pathway is an important signal transduction pathway regulated by enzyme-linked receptors and has many biological functions in mammals. It plays a key role in neuronal metabolism, gene expression regulation, and tissue homeostasis in the healthy and diseased brain. In the present study, the role of the PI3K/AKT/mTOR pathway inhibitor chrysophanol (CPH) (10 mg/kg and 20 mg/kg, orally) in the improvement of ICH-associated neurological defects in rats was investigated. Autologous blood (20 µL/5 min/unilateral/intracerebroventricular) mimics ICH-like defects involving cellular and molecular dysfunction and neurotransmitter imbalance. The current study also included various behavioral assessments to examine cognition, memory, and motor and neuromuscular coordination. The protein expression levels of PI3K, AKT, and mTOR as well as myelin basic protein and apoptotic markers, such as Bax, Bcl-2, and caspase-3, were examined using ELISA kits. Furthermore, the levels of various neuroinflammatory cytokines and oxidative stress markers were assessed. Additionally, the neurological severity score, brain water content, gross brain pathology, and hematoma size were used to indicate neurological function and brain edema. CPH was found to be neuroprotective by restoring neurobehavioral alterations and significantly reducing the elevated PI3K, AKT, and mTOR protein levels, and modulating the apoptotic markers such as Bax, Bcl-2, and caspase-3 in rat brain homogenate. CPH substantially reduced the inflammatory cytokines like interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. CPH administration restored the neurotransmitters GABA, glutamate, acetylcholine, dopamine, and various oxidative stress markers. Our results show that CPH may be a promising therapeutic approach for overcoming neuronal damage caused by the overexpression of the PI3K/AKT/mTOR signaling pathway in ICH-induced neurological dysfunctions in rats.

The anti-fatigue activity of corn peptides and their effect on gut bacteria.

Corn peptides (CPs) are rich in branched-chain amino acids such as leucine and have a variety of biological activities such as antioxidant and improved lipid distribution. In this article, we prepared CPs by enzymatic digestion of corn proteins and evaluated their anti-fatigue activity. We evaluated the anti-fatigue effect of CPs through an exhaustive swimming experiment. The results showed that CPs were able to significantly reduce the rate of body weight gain and prolong the duration of exhaustive swimming. Besides, CPs reduced blood urea nitrogen (BUN) levels after exercise, while they significantly increased muscle glycogen and liver glycogen stores. They reduced muscle cell damage from exercise. In addition, CPs were effective in increasing AMPK, PGC-1α and PI3K protein expression levels and promoting Akt phosphorylation. Correlation analysis showed that CPs increased the abundance of probiotics such as Lactobacillus and Akkermansia in the gut microflora. CPs, which enhanced exercise performance in mice and could modulate gut microbial composition, had significant anti-fatigue activity. © 2021 Society of Chemical Industry.

Bai-Hu-Tang regulates endothelin-1 and its signalling pathway in vascular endothelial cells

Ethnopharmacological relevanceBai-Hu-Tang (BHT) is traditionally used to treat human and animal fever syndrome with four symptoms: large and vigorous pulse, large thirst, high sweat, and high heat. Aim of the studyTo investigate the mechanism of vasodilation regulation of Bai-Hu-Tang in primary vascular endothelial cells stimulated by lipopolysaccharide (LPS). Materials and methodsA hydrophilic concentrate of BHT was prepared, and the main components of mangiferin and timosaponin BⅡ were determined by HLPC analysis. The rabbit fever model was constructed by intravenous injection of LPS (15 μg/kg body weight), and BHT was gavaged to treat febrile rabbits. After treatment for 6 h, animal peripheral blood was collected, and serum was isolated for endothelin-1 (ET-1) and nitric oxide (NO) assays. Rabbit vascular endothelial cells (RVECs) were isolated and stimulated with 1 μg/mL LPS, and then inflammatory cells were treated with 125 or 250 μg/mL BHT for 24 h. The supernatant cytokines TNF-ɑ, IL-1β, IL-6, and ET-1 were detected by ELISA kits. Gene expression levels of endothelin receptor type B (ETB receptor) were analysed by real-time polymerase chain reaction (RT-PCR), and protein expression levels of PI3K and Akt were detected by Western blot. A nitrite assay was used to measure intracellular nitric oxide (NO) production, and nitric oxide synthase (NOS) was measured by the T-NOS colorimetric method. ResultsAnimal experiments demonstrated that BHT significantly restored ET-1 and NO in animal peripheral blood, which were disordered in LPS-induced fever rabbits. Moreover, a cytotoxicity assay demonstrated that BHT ≤700 μg/mL is innoxious to RVECs. BHT significantly repressed cellular TNF-α, IL-1β, and ET-1, which were originally elevated by LPS in RVECs. Meanwhile, BHT elevated the gene expression level of the ETB receptor and promoted NOS and NO production in RVECs induced by LPS. ConclusionBHT can inhibit excessive ET-1 secretion induced by LPS in vascular endothelial cells and activate the classic ET-1 signalling pathway to promote NO production, which may facilitate vasodilation of smooth muscle cells.

A network pharmacology-based approach to explore the active ingredients and molecular mechanism of Lei-gong-gen formula granule on a spontaneously hypertensive rat model

BackgroundLei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, the largest ethnic minority among 56 nationalities in China. It consists of three herbs, namely Eclipta prostrata (L.) L., Smilax glabra Roxb, and Centella asiatica (L.) Urb. It has been widely used as health protection tea for hundreds of years to prevent hypertension in Guangxi Zhuang Autonomous Region. The purpose of this study is to validate the antihypertensive effect of LFG on the spontaneously hypertensive rat (SHR) model, and to further identify the effective components and anti-hypertension mechanism of LFG.MethodsThe effects of LFG on blood pressure, body weight, and heart rate were investigated in vivo using the SHR model. The levels of NO, ANG II, and ET-1 in the serum were measured, and pathological changes in the heart were examined by H&E staining. The main active components of LFG, their corresponding targets, and hypertension associated pathways were discerned through network pharmacology analysis based on the Traditional Chinese Medicine Systems Pharmacology (TCMSP), Traditional Chinese Medicine Integrated Database (TCMID), and the Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM). Then the predicted results were further verified by molecular biology experiments such as RT-qPCR and western blot. Additionally, the potential active compounds were predicted by molecular docking technology, and the chemical constituents of LFG were analyzed and identified by UPLC-QTOF/MS technology. Finally, an in vitro assay was performed to investigate the protective effects of potential active compounds against hydrogen peroxide (H2O2) induced oxidative damage in human umbilical vein endothelial cells (HUVEC).ResultsLFG could effectively reduce blood pressure and increase serum NO content in SHR model. Histological results showed that LFG could ameliorate pathological changes such as cardiac hypertrophy and interstitial inflammation. From network pharmacology analysis, 53 candidate active compounds of LFG were collected, which linked to 765 potential targets, and 828 hypertension associated targets were retrieved, from which 12 overlapped targets both related to candidate active compounds from LFG and hypertension were screened and used as the potential targets of LFG on antihypertensive effect. The molecular biology experiments of the 12 overlapped targets showed that LFG could upregulate the mRNA and protein expressions of NOS3 and proto-oncogene tyrosine-protein kinase SRC (SRC) in the thoracic aorta. Pathway enrichment analysis showed that the PI3K-AKT signaling pathway was closely related to the expression of NOS3 and SRC. Moreover, western blot results showed that LFG significantly increased the protein expression levels of PI3K and phosphorylated AKT in SHR model, suggesting that LFG may active the PI3K-AKT signaling pathway to decrease hypertension. Molecular docking study further supported that p-hydroxybenzoic acid, cedar acid, shikimic acid, salicylic acid, nicotinic acid, linalool, and histidine can be well binding with NOS3, SRC, PI3K, and AKT. UPLC-QTOF/MS analysis confirmed that p-hydroxybenzoic acid, shikimic acid, salicylic acid, and nicotinic acid existed in LFG. Pre-treatment of HUVEC with nicotinic acid could alleviate the effect on cell viability induced by H2O2 and increase the NO level in cell supernatants.ConclusionsLFG can reduce the blood pressure in SHR model, which might be attributed to increasing the NO level in serum for promoting vasodilation via upregulating SRC expression level and activating the PI3K-AKT-NOS3 signaling pathway. Nicotinic acid might be the potential compound for LFG antihypertensive effect.

Tacrolimus inhibits insulin release and promotes apoptosis of Min6 cells through the inhibition of the PI3K/Akt/mTOR pathway.

As a calcineurin inhibitor, tacrolimus is commonly used as a first‑line immunosuppressant in organ transplant recipients. Post‑transplantation diabetes mellitus (PTDM) is a common complication following kidney transplantation and is associated with immunosuppressant drugs, such as tacrolimus. PTDM caused by tacrolimus may be related to its influence on insulin secretion and insulin resistance. However, the specific mechanism has not been fully elucidated. The aim of the present study was to investigate whether the PI3K/Akt/mTOR signaling pathway served an important role in the pathogenesis of PTDM induced by tacrolimus. In the present study, the Cell Counting Kit‑8 assay was used to measure the effect of tacrolimus on the viability of Min6 mouse insulinoma cells. The effects of tacrolimus on the insulin secretion and the activity of caspase‑3 of Min6 cells stimulated by glucose exposure were measured by ELISA. Superoxide dismutase (SOD) and malondialdehyde(MDA) levels were measured using WST‑8 and thiobarbituric acid assays, respectively. The effects of tacrolimus on the mRNA expression levels of PI3K, Akt and mTOR were detected by reverse transcription‑quantitative PCR (RT‑qPCR), whereas the protein expression levels of PI3K, Akt, mTOR, phosphorylated (p)‑AKT and p‑mTOR in Min6 cells were assessed using western blotting. The present data indicated that, compared with the control group, 5, 25and50ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50ng/ml tacrolimus markedly increased the activity of caspase‑3 by 175.1% (P<0.05), it also decreased the SOD activity (P<0.01) and increased MDA levels (P<0.05). The RT‑qPCR results demonstrated that the mRNA expression levels of PI3K, Akt and mTOR were downregulated by 25and50ng/ml tacrolimus (P<0.01). Furthermore, the western blotting results suggested that tacrolimus had no significant effects on the expression levels of total PI3K, Akt and mTOR proteins (P>0.05), but 25and50ng/ml tacrolimus could significantly inhibit the expression levels of p‑Akt and p‑mTOR (P<0.01). In conclusion, tacrolimus decreased the activity and insulin secretion of pancreatic β cells and induced the apoptosis of islet β cells by inhibiting the mRNA expression levels of PI3K, Akt and mTOR and reducing the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway, which may ultimately lead to the occurrence of diabetes mellitus, and may be considered as one of the specific mechanisms of PTDM caused by tacrolimus.