mRNA Expression Of mTOR Research Articles

Dimethoxycurcumin reduces proliferation and induces apoptosis in renal tumor cells more efficiently than demethoxycurcumin and curcumin

Curcumin (Cur), is a pigment with antiproliferative activity but has some pharmacokinetic limitations, which led researchers to look for more effective structure analogs. This work investigated the effects of Cur and compared them with the two analogs, demethoxycurcumin (DeMC) and dimethoxycurcumin (DiMC), to elucidate their mechanisms of action. The cytotoxic, antiproliferative, and genotoxic effects these compounds were correlated based on gene expression analysis in the human renal adenocarcinoma cells (786-O). Cur decreased CYP2D6 expression and exhibited cytotoxic effects, such as inducing monopolar spindle formation and mitotic arrest mediated by the increase in CDKN1A (p21) mRNA. This dysregulation induced cell death through a caspase-independent pathway but was mediated by decrease in MTOR and BCL2 mRNA expression, suggesting that apoptosis occurred by autophagy. DeMC and DiMC had similar effects in that they induced monopolar spindle and mitotic arrest, were genotoxic, and activated GADD45A, an important molecule in repair mechanisms, and CDKN1A. However, the induction of apoptosis by DeMC was delayed and regulated by the decrease of antiapoptotic mRNA BCL.XL and subsequent activation of caspase 9 and caspase 3/7. DiMC treatment increased the expression of CYP1A2, CYP2C19, and CYP3A4 and exhibited higher cytotoxicity compared with other compounds. It induced apoptosis by increasing mRNA expression of BBC3, MYC, and CASP7 and activation of caspase 9 and caspase 3/7. These data revealed that different gene regulation processes are involved in cell death induced by Cur, DeMC, and DiMC. All three can be considered as promising chemotherapy candidates, with DiMC showing the greatest potency.

Epigallocatechin Gallate Protects against MNNG-Induced Precancerous Lesions of Gastric Carcinoma in Rats via PI3K/Akt/mTOR Pathway.

Objective To evaluate the therapeutic effect of epigallocatechin gallate (EGCG) on precancerous lesions of gastric carcinoma (PLGC) and to determine whether EGCG protects against PLGC by regulating PI3K/Akt/mTOR pathway. Methods Twenty-four male Wistar rats were randomly divided into 3 groups: normal control group (NC), PLGC model group (MC), and group of PLGC rats treated with EGCG (MC + EGCG). 1-Methyl-3-nitro-1-nitrosoguanidine (MNNG) and sodium salicylate were combined and used to establish the PLGC rat animal model. The therapeutic effect of EGCG on PLGC was evaluated by body weight and pathological lesions of gastric mucosa in PLGC rats. Quantitative polymerase chain reaction (qPCR) was applied to measure the mRNA expressions of PI3K, Akt, and mTOR. The protein expressions of cleaved caspase-3, PTEN, PI3K, p-PI3K, Akt, p-Akt, p-mTOR, and mTOR were determined by automated western immunoblotting. Results The body weight decreased in PLGC rats while EGCG significantly increased body weight. The gastric mucosa of PLGC rats exhibited the pathological lesions of atrophy, intestinal metaplasia, and atypical hyperplasia while EGCG could ameliorate the pathological lesions. EGCG could upregulate the expressions of cleaved caspase-3 and PTEN and reduce the expressions of PI3K, Akt, and mTOR. Conclusions EGCG ameliorated pathological lesions of PLGC and exerted the effect of apoptosis promotion in PLGC rats. The apoptotic pathway triggered by EGCG may be related to inhibition of PI3K/Akt/mTOR pathway. It provided a theoretical basis for the PLGC treatment and gastric cancer prevention.

Cloves Regulate Na+-K+-ATPase to Exert Antioxidant Effect and Inhibit UVB Light-Induced Skin Damage in Mice.

The purpose of this study was to observe the effect of cloves (Syzygium aromaticum (L.) Merr. & L.M. Perry) on the mouse skin using a UVB-induced skin injury mouse model. The serum, liver, and skin indexes of mice were determined by kits, H&E tissue staining, and qPCR assay. The compound composition of cloves was determined by HPLC. The results showed that cloves increased the activity of Na+-K+-ATPase in the skin and then maintained the sodium and potassium pump in the damaged skin muscle membrane. Cloves alleviated the oxidative stress injury induced by UVB irradiation by normalizing the related oxidative stress indexes (T-SOD, CAT, AGEs, and H2O2) in serum and skin. Inhibition of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 and increased activation of anti-inflammatory cytokines IL-4 and IL-10 occurred after treatment with cloves, which ultimately reduced the inflammatory damage to the body. Further results showed that cloves upregulate SOD1, SOD2, CAT, GSH, IL-10, IκB-α, AMPK, SIRT1, LKB1, PGC-1α, APPL1, and FoxO1 and downregulate NF-κB p65, TNF-α, IL-6, and mTOR mRNA expression in the skin tissues of UVB-damaged mice. The results of composition analysis showed that the five most abundant compounds in cloves are rutin, isoquercitrin, ferulic acid, dihydroquercetin, and quercitrin. Cloves regulate the skin sarcomembrane Na+-K+-ATPase through these five compounds, and because they regulate the oxidation, inflammation, and ATP energy consumption of the body, they subsequently protect the skin from UVB damage.

Anticancer Effect of Troxerutin in Human Non-Small-Cell Lung Cancer Cell A549 and Inhibition of Tumor Formation in BALB/c Nude Mice.

This study is intended to explore the anticancer, antiproliferative, and chemopreventive action of troxerutin (TX) in human non-small-cell lung cancer cell (A549) using BALB/c nude mice. 2 × 106 A549 cells were subcutaneously injected into mice, along with 10 μM and 20 μM/kg body weight of TX orally for 19 days. On the last day, tumor weight and volume were assessed. Stress marker enzymes such as Aryl hydrocarbon hydroxylase (AHH), lactate dehydrogenase (LDH), 5'Nucleotidase (5'ND), and γ-glutamyltranspeptidase (γ-GT) were estimated in the lung tissues. Cytotoxicity of TX was assessed using MTT assay. Expression of carcinoembryonic antigen (CEA) and inflammatory cytokines were also analyzed. Histopathological examination of tissue sections and immunohistochemical examination of proliferating cell nuclear antigen (PCNA) were also performed. mRNA expression of p53, p21, cyclin D1, P13k, Akt, and mTOR were analyzed using RT-PCR. TX administered orally in a dose-dependent manner markedly reverted the level of stress marker enzymes to a significant extent. TX also exhibited significant protection against lung cancer cells, as evidenced by cytotoxicity assay and histopathological studies. It was also found to reduce the expression of PCNA, cyclin D1, P13k, Akt, and mTOR, but increase the expression of p53 and p21. TX has also been shown to reduce cancer cell inflammation, as was evidenced by reduced expression of inflammatory cytokines. Thus TX could be used as an effective chemopreventive and anticancer agent in treating cancer.

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

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

MiR-100-5p Downregulates mTOR to Suppress the Proliferation, Migration, and Invasion of Prostate Cancer Cells

BackgroundPrevious studies have shown that miR-100-5p expression is abnormal in prostate cancer. However, the role and regulatory mechanism of miR-100-5p requires further investigation. Thus, the aim of this study was to observe the effects of miR-100-5p on the proliferation, migration and invasion of prostate cancer (PCa) cells and to explore the potential related regulatory mechanism.Materials and MethodsDifferential miRNA expression analysis was performed using next-generation sequencing (NGS) in the patients with PCa and benign prostatic hyperplasia (BPH). The expression levels of miR-100-5p were detected using real-time fluorescence quantitative PCR (qRT-PCR). PCa cells were transfected with NC-mimics or miR-100-5p mimics, inhibitor by using liposome transfection. Moreover, the CCK-8 proliferation assay, colony formation assay, cell scratch assay and Transwell assay were used to detect the effects of miR-100-5p on cell proliferation, migration, and invasion. In addition, the target gene of miR-100-5p was verified by luciferase reporter gene assay, and the influence of miR-100-5p on the expression of mTOR mRNA by qRT-PCR and the expression of mammalian target of rapamycin (mTOR) protein was detected by western blot and immunohistochemical staining.ResultsDifferential expression analysis of high-throughput sequencing data showed low expression of miR-100-5p in the patients of PCa. It was further confirmed by qRT-PCR that the expression of miR-100-5p in PCa cells was significantly lower than that in RWPE-1 cells (P<0.01). miR-100-5p expression in lymph node carcinoma of prostate(LNCaP) cells was markedly upregulated after transfection with miR-100-5p mimics (P<0.01), while cell proliferation, migration and invasion capacities were clearly reduced (P<0.01). mTOR mRNA and protein expression was also substantially lowered (P<0.01) and mTOR adjusted the expression of NOX4. Finally, we further confirmed by immunohistochemical staining that miR-100-5p regulated the expression of mTOR and NOX4.ConclusionmiR-100-5p is expressed at low levels in PCa cells, and it can suppress PCa cell proliferation, migration and invasion, the mechanism of which is related to downregulating the expression of mTOR.

Effect of ginsenoside Rg_1 in delaying premature ovarian failure induced by D-gal in mice through PI3K/Akt/mTOR autophagy pathway

The aim of this paper was to study the role of phosphoinositide 3-kinase(PI3 K), protein kinase B(Akt) and mamma-lian target of rapamycin(mTOR) in the inhibition of premature ovarian failure induced by D-galactose(D-gal) in mice model by ginsenoside Rg_1(Rg_1). Fifty-four female SPF BALB/c mice were randomly divided into PBS group, D-gal group, and Rg_1 group. In the D-gal group, D-galactose(200 mg·kg~(-1)·d~(-1)) was injected subcutaneously into the neck and back for 42 days. In the PBS group, an equal amount of phosphate buffered saline(PBS) was injected into the neck and back for 42 days. In addition to the therapy of D-gal group, Rg_1 group was given Rg_1(20 mg·kg~(-1)·d~(-1)) through intraperitoneal injection since the 15 th day for 28 days, at the same time, the D-gal group and the PBS group were also given an equal amount of PBS through intraperitoneal injection since the 15 th day for 28 days. After the treatment, the estrous cycle changes of the mice were detected, and the ovarian SA-β-Gal staining was used to detect the changes of ovarian aging. Western blot was used to detect the changes in protein expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). Fluorescence quantitative PCR was used to detect the changes in mRNA expressions of PI3 K, Akt, mTOR, S6 k, LC3-Ⅱ and P16~(INK4 a). According to the findings, compared with the PBS group, the D-gal group began to show estrous cycle disorder in the 3 rd week,the ovarian SA-β-Gal staining positive granulosa cells increased in the D-gal group, the expression of senescence marker P16~(INK4 a) increased, while the expression of autophagy signaling molecule LC3-Ⅱ decreased. After treatment with Rg_1, the positive rate of ovarian SA-β-Gal staining in Rg_1 group decreased, the expression level of autophagy signaling molecule LC3-Ⅱ in Rg_1 group was higher than that in D-gal group, while the expression level of senescence marker P16~(INK4 a) was lower than that in D-gal group. Compared with the PBS group, the protein and mRNA expressions of PI3 K, Akt, mTOR and S6 k in the D-gal group were up-regulated, the protein expressions of Akt, mTOR and S6 k in the Rg_1 group were up-regulated, and the mRNA expressions of PI3 K and mTOR were up-regulated. After treatment with Rg_1, the protein expressions of PI3 K, Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group, while the mRNA expressions of Akt, mTOR and S6 k in the Rg_1 group were lower than those in the D-gal group. The finding ssuggested that Rg_1 has the effect in delaying ovarian premature failure in D-gal-induced mouse models, and PI3 K/Akt/mTOR autophagy signaling pathways play an important role.

MiR-100 inhibits cell proliferation in mantle cell lymphoma by targeting mTOR

BackgroundmiR-100 is reported to be associated with cell proliferation and apoptosis. However, the function of miR-100 in mantle cell lymphoma (MCL) is unknown. The purpose of this study is to analyze the abnormal expression of miR-100 and mTOR in MCL together with their potential biological function and pathogenesis.MethodEighteen MCL tissue samples and 3 cell lines (Jeko-1, Mino, Granta-519) were investigated in this research study, while eighteen samples of proliferative lymphadenitis from patients and peripheral lymphocyte cells from healthy volunteers served as controls. The expression and alteration of miR-100 and mTOR mRNA were detected by RT-PCR. The expression and alteration of mTOR protein were explored by Western blot. LV-miR-100-up and LV-mTOR-RNAi were constructed and transfected by lentivirus transfection. Cell proliferation, cell apoptosis and the cell cycle were detected using CCK-8 and flow cytometry. Bioinformatics prediction software was used to predict the miR-100 target gene of mTOR. A double luciferase experiment was used to verify miR-100 targeting at the mTOR-3′-UTR. The interaction between miR-100 and mTOR was further studied using recovery experiments. GraphPad Prism 7 software (version 7.2) was used for statistical analysis, and a P value < 0.05 was considered statistically significant.ResultsWe found that the expression of miR-100 mRNA in MCL tissues and cell lines was lower, while that of the mTOR protein was higher. There was a negative correlation between miR-100 and mTOR in both MCL tissues and cell lines. Promoting miR-100 and inhibiting mTOR could inhibit cell proliferation, induce cell apoptosis and block the cell cycle in the G1 phase. A double luciferase reporter assay showed that mTOR was one of the target genes of miR-100. The recovery experiment demonstrated that PV-mTOR-up partially set off the effect of LV-miR-100-up on decreasing mTOR expression, inhibiting proliferation, inducing apoptosis and blocking the cell cycle in G1 phase in both Jeko-1 and Mino cells.ConclusionsAbnormal expression of miR-100 and mTOR was found in MCL, which included downregulation of miR-100 and upregulation of mTOR. The expression of mTOR is negatively correlated with miR-100. It may play an important role in MCL pathogenesis. miR-100 up-regulation can inhibit cell proliferation, promote cell apoptosis, and inhibit cell cycle in G1 phase by targeting the mTOR gene. miR-100 may potentially be an anti-mantle cell lymphoma gene.

Discovery of Turn-On Fluorescent Probes for Detecting PDEδ Protein in Living Cells and Tumor Slices.

The first small-molecule fluorescent turn-on probes for detecting PDEδ protein were rationally designed, showing reasonable fluorescent properties and the fluorescent ability has been applied for visualization of the PDEδ protein in living cells and at tissue levels. The qPCR results showed that the mRNA expression of KRAS, PDEδ, AKT1, MAPK1, MEK7, RAF1, and mTOR were downregulated by probes 1-3 through PI3K/AKT/mTOR and MAPK signal pathways. The probes also can downregulate the protein level of pErk and tErk. Therefore, these small-molecule fluorescent probes are expected to be used in the screening of antipancreatic cancer drugs targeting the PDEδ protein, as well as in obtaining a better understanding of the pathological and physiological roles of PDEδ protein.

HMGB1/PI3K/Akt/mTOR Signaling Participates in the Pathological Process of Acute Lung Injury by Regulating the Maturation and Function of Dendritic Cells.

Background: High-mobility group box 1 protein (HMGB1) was identified as a highly conserved DNA binding nuclear protein, which participates in the processes of acute lung injury (ALI). HMGB1 binds to its specific receptors not only to activate the nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways but also to regulate the activation of the phosphatidylinositol 3′-kinase/protein kinase B/mammalian target of the rapamycin (PI3K/AKT/mTOR) pathway. Mature dendritic cells (DCs) regulate acute lung inflammation and pathological injury in ALI. In addition, studies have shown that the activation of the PI3K/AKT/mTOR signaling pathway may regulate the function and maturation of DCs.Objective: Therefore, we speculate that HMGB1/PI3K/Akt/mTOR signaling participates in regulating the pathological process of ALI by regulating the maturation and function of DCs.Methods: Anti-HMGB1 antibody, rHMGB1, or LY294002 (PI3K inhibitor) was administered in a murine model of lipopolysaccharide (LPS)-induced ALI. For in vitro studies, generated bone marrow-derived dendritic cells (BMDCs) primed by LPS were stimulated with the same reagents. The effects of these different treatments were observed on the expression of PI3K, AKT, and mTOR and on the function of DCs.Results: HMGB1 upregulated the expression of PI3K, Akt, and mTOR mRNA and phosphorylated proteins in BMDCs. The HMGB1/PI3K/Akt/mTOR signaling pathway induced the maturation and antigen-presenting ability of lung DCs, mediated the percentage of myeloid DCs (mDCs), and enhanced the adhesion and chemotactic ability of lung DCs.Conclusions: HMGB1/PI3K/Akt/mTOR signaling participates in the pathological process of ALI by regulating the maturation and functions of DCs.

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

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

Dingkun Pill replenishes diminished ovarian reserve through the PI3K/AKT/mTOR signaling pathway in TWP-induced mice

Ethnopharmacological relevanceDiminished ovarian reserve (DOR) can lead to poor fertility and shorten the reproductive lifespan of females. The Dingkun Pill (DKP), a traditional Chinese-patented medication, has been an integral part of traditional Chinese medicinal treatment for the management of gynecological diseases for centuries. Relevant clinical studies have shown that DKP is able to protect against DOR, however, its mechanism of action is not yet fully elucidated. Study goalsThis study was conducted with the aim of exploring the impact of tripterygium wilfordii polyglycosidium (TWP) on the PI3K/AKT/mTOR pathway in the context of the pathophysiology of DOR and the mechanism of action of DKP. Materials and methodsEighty female balb/c mice with regular estrous cycles were assigned to Blank, Model, DKP and hormone replacement therapy (HRT) groups in a random manner. With the exception of the Blank group, mice in the other groups were exposed to 40 mg/kg/d TWP suspension for 30 days to DOR induction. Following this, either DKP or hormones were orally administrated to determine their effect on disease progression. During the experiment, changes in body weight and the estrous cycles of the mice were observed. Post treatment, serum sample anti-mullerian hormone (AMH), estradiol (E2), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were quantified using enzyme-linked immunosorbent assay (ELISA). The mice were then sacrificed in order to harvest their ovaries for hematoxylin and eosin (HE) staining. This process allowed for the assessment of ovarian morphology and follicular quantification. Apoptotic ovarian cells of the ovary were assessed using TUNEL technique, while Caspase-3 and Cytochrome C (Cyt C) expressions of the ovary were examined through immunohistochemistry (IHC). Western blotting analysis was used to quantify levels of Bax, Bcl-2, Caspase-3, Cyt C, mTOR, P-mTOR, AKT, P-AKT, P-PI3K and PI3K proteins, while mRNA levels of Bax, Bcl-2, PI3K, AKT and mTOR were measured in ovarian tissue using RT-PCR. ResultsThe findings revealed that DKP was able to improve levels of serum hormones and promote the recovery of the estrous cycle. DKP augmented the total amount of primordial follicles while reducing the number of follicles that were atretic follicles. The apoptosis index of growing follicles and Bax, Cyt C and Caspase-3 expressions decreased, while the Bcl-2: Bax ratio increased. DKP suppressed levels of phosphorylation and the mRNA expressions of mTOR, AKT and PI3K. ConclusionsIt was demonstrated that DKP was able to increase ovarian reserves through inhibition of the PI3K/AKT/mTOR signaling pathway, which lead to the suppression of primordial follicle activity and a reduction in levels of apoptosis of early growing follicles. This highlights its potentially beneficial role for the treatment of DOR.

Gut microbiota mediates the protective role of Lactobacillus plantarum in ameliorating deoxynivalenol-induced apoptosis and intestinal inflammation of broiler chickens

The protection of Lactobacillus plantarum JM113 against deoxynivalenol (DON)-induced apoptosis and intestinal inflammation on the jejunum of broiler chickens and the potential roles of gut microbiota were determined. A total of 144 one-day-old male broilers (Arbor Acres) were randomly divided into 3 treatment groups consisting of 6 replicates with 8 birds per replicate, including the CON (basal diet), the DON (basal diet + 10 mg/kg DON), and the DL (basal diet + 10 mg/kg DON + 1 × 109 CFU/kg L. plantarum JM113). The DON-diet decreased (P < 0.05) the mRNA expression of mucosal defense proteins and mechanistic target of rapamycin pathway genes. Meanwhile, DON challenge significantly increased Bcl-2-associated X gene/B-cell lymphoma 2 gene (Bcl-2) in the jejunum (P < 0.05) and demonstrated proapoptosis status. In contrast, the DL group showed normal immunity-related gene expression of jejunal mucosa and manifested a superior antiapoptosis status. Adding L. plantarum JM113 significantly raised (P < 0.05) propionic acid, n-butyric acid, and total short-chain fatty acids concentrations in cecal contents of birds fed with DON diet. In addition, DON exposure altered bacterial community structure and disturbed the abundance of several bacterial phyla, families, and genera, leading to dysbiosis. Supplementation with JM113 shifted the gut microbiota composition to that of the CON group. Finally, Spearman correlation analysis suggested that most positive correlations with the mRNA expression of immunity-related and apoptosis-regulatory gene were observed within the phylum Bacteroidetes, and most negative correlations with the indicators were observed within the phylum Firmicutes. The mRNA expression of Bcl-2, TLR2, mTOR, Raptor, and RPS6KB1 (P < 0.05), which are regarded as important cell proliferation and antiapoptosis parameters, were significantly negatively associated with the relative abundances of norank_f__Erysipelotrichaceae, Subdoligranulum, and Anaeroplasma, whereas they had a strong positive correlation with Ruminococcaceae_UCG-004, Alistipes, and Ruminococcaceae_NK4A214_group. These results implied that L. plantarum JM113 supplementation could ameliorate DON-induced apoptosis and intestinal inflammation via manipulating the bacterial community composition and could be used as a potential candidate to attenuate intestinal impairments.

Total flavones of Dracocephalum moldavica L. protect astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway

BackgroundThe active components of Dracocephalum moldavica L. (TFDM) can inhibit myocardial ischemia by inhibiting oxidative stress. However, the effects of TFDM on astrocytes have not been investigated in vitro. The current study aimed to explore whether TFDM protects astrocytes against H2O2-induced apoptosis through a mitochondria-dependent pathway.MethodsThe human glioma cell line U87 was used to investigate the ability of TFDM to protect astrocytes against H2O2-induced apoptosis. The cell counting kit-8 assay and flow cytometry were used to detect cell viability, apoptosis, MMP, Ca2+ influx and reactive oxygen species (ROS). Lactate dehydrogenase (LDH) and malonic dialdehyde (MDA) levels were measured by ELISA. In addition, protein and mRNA expression changes were detected by Western blotting and qRT-PCR.ResultsTFDM (0.78~200 μg/ml) had limited cytotoxic effects on the viability of U87 cells. Compared with the model group (treated with H2O2 only), cells treated with medium- and high-dose TFDM exhibited reduced MDA concentrations (P < 0.05) and ROS production (P < 0.05) and decreased MMP (P < 0.05) and reduced apoptosis (P < 0.05). The percentage of annexin V-FITC-stained cells was markedly suppressed by TFDM, confirming its anti-apoptotic properties. WB results showed that protein expression of Bcl-2-associated X protein (BAX), Caspase-3, Caspase-9, Caspase-12, and B-cell leukemia/lymphoma 2 (Bcl2) was reduced in the TFDM group compared with that in the model group (P < 0.05) and that expression of these proteins was normalized by TFDM treatment in a dose-dependent manner. According to RT-qPCR results, TFDM pretreatment resulted in reduced mRNA expression of BAX, Caspase-9, Caspase-12, p38MAPK, and CaMKII and increased mRNA expression of mTOR compared with the model group.ConclusionsThe current study revealed the protective effects of TFDM on U87 cells under oxidative stress conditions through the inhibition of a mitochondria-dependent pathway that is associated with the CaMKII/P38MAPK/ERK1/2 and PI3K/AKT/mTOR pathways.

Long-term caloric restriction activates the myocardial SIRT1/AMPK/PGC-1α pathway in C57BL/6J male mice.

BackgroundCaloric restriction (CR) can help in improving heart function. There is as yet no consensus on the mechanism of the effect of CR. Silent mating-type information regulation 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK), and mTOR are key players in metabolic stress management. We aimed to explore the effect of CR on the myocardial SIRT1/AMPK/mTOR pathway in mice.MethodsThirty-six 6-week-old male C57BL/6J mice were randomly divided into three groups: normal control group (NC group, n = 12), high-energy group (HE group, n = 12) and CR group (n = 12) according to different diets. After 11 months, western blot was used to examine proteins such as p-AMPK, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), SIRT1, and p-mTOR, whereas real-time PCR was used to examine the expression of AMPK, PGC-1α, and SIRT1 transcripts.ResultsCompared to the HE group, the CR group displayed increased expression of myocardial p-AMPK protein, SIRT1 protein and mRNA, and PGC-1a mRNA. However, no difference was observed in the expression of p-mTOR protein and mTOR mRNA in the myocardium among the three groups.ConclusionsCR improves the SIRT1/AMPK/PGC-1α pathway in mice myocardium with no effect on the mTOR pathway.

Antitumor Effects of Trimethylellagic Acid Isolated From Sanguisorba officinalis L. on Colorectal Cancer via Angiogenesis Inhibition and Apoptosis Induction.

Previous studies have demonstrated that tannin could inhibit the proliferation and angiogenesis of cancer cells. However, the mechanism(s) associated with its antitumor effect remains unclear. Here, we investigated the effects of 3,3',4'-trimethylellagic acid (TMEA), a tannin compound isolated from Sanguisorba officinalis L., on the proliferation, angiogenesis, and apoptosis in cancer cells, as well as the underlying mechanism(s) related to its antitumor activity. TMEA was isolated from Sanguisorba officinalis L. by silica gel column chromatography. Molecular docking was carried out to assess active pocket binding between TMEA and vascular endothelial growth factor receptor 2 (VEGFR2). The antiangiogenic effect of TMEA on the migration and tube formation was detected in HUVECs by wound healing and tube formation assays, respectively. The antitumor effects of TMEA on the cell proliferation were determined in HepG2, A549, and SW620 cells by MTS assay in vitro and on the tumor growth of SW620 xenografts bearing in nude mice in vivo. The mRNA expression of Bcl-2, Bax, caspase-3, VEGF, PI3K, and mTOR were measured by qRT-PCR and protein expression of Bcl-2, Bax, caspase-3, VEGF, PI3K, and mTOR by Western blotting, and the protein expression of Bcl-2, Bax, caspase-3 and CD31 were detected by immunohistochemical analysis in vivo, respectively. The results showed that TMEA combined with VEGFR2 in the functional pockets of Asn223A, Gly922A, and Leu840A and inhibited the proliferation, migration, tube formation, and expression of VEGF and its downstream signaling mediators in HUVECs. TMEA also significantly inhibited the proliferation of HepG2, A549, and SW620 cancer cells in vitro, and suppressed the growth of SW620 tumors in vivo. Moreover, TMEA upregulated the expression of proapoptotic factors Bax and caspase-3 and downregulated the expression of antiapoptotic factors CD31 and Bcl-2 in cancer cells and/or tumor tissues. The data indicate that TMEA executes its anticancer activity by inducing apoptosis and inhibiting angiogenesis in cancer cells in vitro and tumor growth in vivo. The underlying anticancer mechanism is associated with the apoptotic and VEGF/PI3K/AKT/mTOR pathways.

MicroRNA files in the prevention of intestinal ischemia/reperfusion injury by hydrogen rich saline.

Background: Hydrogen-rich saline (HRS) has been proven effective against ischemia/reperfusion (I/R) injury. However, knowledge on the underlying signaling events remain poor. Having recent highlight of microRNAs (miRNAs) in mediating intestinal I/R injury, we hypothesized that HRS may protect intestine against I/R injury by regulating miRNAs.Method: Mice were given intraperitoneal injection of saline or HRS once daily for five consecutive days before undergoing intestinal I/R that was induced by 60-min ischemia followed by 180-min reperfusion of superior mesenteric artery. The intestine was collected for histopathological assay, miRNA microarray profiling, Real-Time PCR, and Western blotting. Next, miR-199a-3p mimics or inhibitors were transfected into IEC-6 cells to explore the relationship between HRS treatment and miR-199a-3p.Results: I/R-induced mucosal injury and epithelial cells apoptosis were attenuated by HRS pretreatment. A total of 64 intestinal I/R-responsive miRNAs were altered significantly by HRS pretreatment, in which we validated four novel miRNAs with top significance by Real-Time PCR, namely miR-199a-3p, miR-296-5p, miR-5126, and miR-6538. Particularly, miR-199a-3p was drastically increased by I/R but reduced by HRS. Computational analysis predicts insulin-like growth factor (IGF)-1, mammalian target of rapamycin (mTOR), and phosphoinositide-3-kinase (PI3K) regulatory subunit 1 as targets of miR-199a-3p, suggesting involvement of the pro-survival pathway, IGF- 1/PI3K/Akt/mTOR. In in vitro experiment, HRS treatment reduced miR-199a-3p level, increase IGF-1, PI3K and mTOR mRNA expression, restore IEC-6 cells viability, and this protective effects were reversed under miR-199a-3p mimics treatment.Conclusion: Collectively, miR-199a-3p may serve a key role in the anti-apoptotic mechanism of HRS that contributes to its protection of the intestine against I/R injury.

MiR-18a Inhibitor Suppresses Leukemia Cell Proliferation by Upregulation of PTEN Expression.

BackgroundLeukemia is common in aging adults and has very high mortality worldwide. The present study was designed to investigate the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP-1 leukemia cells.Material/MethodsThe changes in miR-18a inhibitor-transfected WEHI-3 and THP-1 cell proliferative potential was measured by use of the Cell Counting Kit-8 assay. Apoptotic changes were analyzed by electron microscopy, and evaluation of PI3K, AKT, mTOR, and PTEN expression was assessed by RT-qPCR assay.ResultsTransfection of miR-18a inhibitor significantly (P<0.05) suppressed the proliferative potential of WEHI-3 and THP4 cells. The WEHI-3 cells showed the presence of characteristic apoptotic bodies on transfection with miR-18a inhibitor at 48 h. Flow cytometry showed that miR-18a inhibitor transfection significantly (P<0.05) increased the WEHI-3 cell percentage in G1 phase. The transfection of miR-18a inhibitor significantly (P<0.05) promoted apoptosis in WEHI-3 cells. In WEHI-3 cells, miR-18a inhibitor transfection markedly suppressed the expression of PI3K, AKT, and mTOR mRNA. The expression of PTEN mRNA was significantly (P<0.05) upregulated by miR-18a inhibitor transfection in WEHI-3 cells.ConclusionsThe present study investigated the therapeutic efficacy of miR-18a inhibitor against WEHI-3 and THP1 leukemia cells. The study demonstrated that miR-18a inhibitor suppressed the proliferative potential of WEHI-3 and THP1 cells and activated apoptotic process through upregulation of PTEN mRNA expression. Therefore, miR-18a inhibitor can be of therapeutic importance for the treatment of leukemia.

Combined Extracts of Epimedii Folium and Ligustri Lucidi Fructus with Budesonide Attenuate Airway Remodeling in the Asthmatic Rats by Regulating Apoptosis and Autophagy.

This study aimed to investigate the effects of the coadministration of budesonide (Bud) and the extracts of Epimedii Folium and Ligustri Lucidi Fructus (EEL) on regulating apoptosis and autophagy in asthmatic rats. Forty Sprague-Dawley rats were divided randomly into five groups (8 rats in each group): normal control (control), asthma model (asthma), Bud (1 mg Bud suspension in 50 ml sterile physiological saline for 30 min), EEL (100 mg/kg EEL), and group of coadministration of Bud and EEL (Bud&EEL, 100 mg/kg EEL plus Bud by nebulized inhalation for 30 min). Rats were sensitized and challenged with ovalbumin for 7 weeks and treated with corresponding drug for 4 weeks. We anesthetized all rats with 25% ethyl carbamate (4 ml/kg) and took lung tissues and BALF after final ovalbumin challenge to observe the lung histopathology and morphometry; apoptosis in BALF and lung tissue; protein expressions of Ki-67, α-SMA, cleaved Caspase-3, p-mTOR, and LC3; and protein and mRNA expressions of Bax, Bcl-2, Caspase-3, P53, mTOR, and Beclin-1. Results showed that Bud&EEL could alleviate airway remodeling, inhibit cell proliferation and autophagy in lung tissue, and promote apoptosis in BALF and lung tissue in ovalbumin-induced asthma rats through downregulating the protein expressions of α-SMA and Ki-67, the protein ratio of LC3-II/LC3-I and Bcl-2/Bax, and the protein and mRNA expressions of Bcl-2 and Beclin-1, while upregulating the protein expressions of cleaved Caspase-3 and p-mTOR, and the protein and mRNA expressions of Bax, Caspase-3, P53, and mTOR. Bud&EEL had better effects than single-use Bud on improving airway remodeling, promoting apoptosis, and regulating the expressions of autophagy- and apoptosis-related proteins. This study suggested that the effects of coadministration of EEL and Bud on regulating apoptosis and autophagy were better than those of single-use Bud treatment, and that might be the mechanism of attenuating airway remodeling, providing an alternative therapy for asthma.

Study on myelin injury of AD mice treated with Shenzhiling oral liquid in the PI3K/Akt–mTOR pathway

Shenzhiling oral liquid (SZL) is a Traditional Chinese Medicine (TCM) compound to be approved by the China Food and Drug Administration (CFDA) (Z20120010) for the treatment of mild-to-moderate Alzheimer’s disease (AD). However, its mechanism in early AD is not clear. We studied its mechanism in protecting myelin. Three-month-old APPswe/PS1dE9double transgenic mice were used as AD model and wild-type C57BL/6 mice were used as control. After 3-month intervention, the Morris water maze was used to detect behavioural changes. Myelin mTOR pathway (PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR), myelin basic protein (MBP) and postsynaptic density protein 95 (PSD95) were detected by immunohistochemistry and western blot and reverse transcriptase polymerase chain reaction (RT-PCR). After 3 months of SZL treatment, compared with the model group (M), SZL medium-dose (SM) and SZL low-dose groups (SL) exhibited increased staying and crossing results in Morris water maze (P < 0.05). Compared with M, PI3K-positive cells in SM and SL groups were increased (P < 0.01), p-PI3K expression increased in the Donepezil group (D), SZL high-dose group (SH) and SM (P < 0.05); number of Akt-positive cells and Akt expression in D, SM and SL were increased (P < 0.01, P < 0.05); number of p-Akt- and mTOR-positive cells and mTOR expression in all drug-treated groups were significantly increased (P < 0.01); p-Akt and p-mTOR expression increased in all drug-treated groups (P < 0.05, P < 0.01); MBP expression in D and SH increased (P < 0.05), while in SM and SL it increased more significantly (P < 0.01); and PSD95 expression in D, SM and SL was increased (P < 0.05). RT-PCR results showed that compared with M, PI3K mRNA and Akt mRNA expression in all drug-treated groups increased, but there was no statistical difference (P > 0.05), mTOR mRNA expression in all the drug-treated groups increased significantly (P < 0.01) and MBP mRNA and PSD95 mRNA expression in D and SH increased (P < 0.05). SZL oral liquid could play a role in myelin protection in early AD.