ObjectiveTo investigate the association between mTOR pathway and pharmacoresistance of Sprague-Dawley rat epilepsy model kindled by coriaria lactone. MethodsA kindling model of pharmacoresistant temporal lobe epilepsy was developed by injecting Sprague-Dawley (SD) rats with coriaria lactone (CL) (1.75 mg/kg, every 84 h). Normal SD rats were injected with normal sodium (NS) served as control group. Rats with five or more consecutive stage 5 seizures were included in kindled group. Immunohistochemistry was used to detect the levels of P-S6 in both groups. ResultsThe expressions of P-S6 in CA1 and CA3 were significantly higher compared with control group, and were mainly in astrocytes (P < 0.001). In addition, the expression of P-S6 in DG area was significantly higher than that in control group, with more granular cell and neuron (P < 0.001). ConclusionsThe mTOR pathway may be correlated with the drug resistance of refractory lobe epilepsy kindled by coriaria lactone.
ObjectiveTo observe the expressions of p-mTOR, p-S6K1 and p-4EBP1 in the human brain with refractory epilepsy and to explore the role of mTOR signaling pathway in intractable epilepsy. MethodsCollecting the brain tissues of 24 patients with refractory epilepsy for surgical treatment from March 2010 to July 2011 as experimental group in hospitalized Epilepsy Center at the First Hospital of Jilin University, Changchun City. Collecting temporal lobe or frontal lobe brain tissue from 6 autopsy of patients who had emergency surgery for neurosurgery brain tranma during the same period. Using immunohistochemistry to observe the expression of p-mTOR, p-S6K1, p-4EBP1 in the two groups of brain tissues, and analyzed statistically. Results① p-mTOR, p-S6K1 and p-4EBP1 were expressed in both neurons and glial cells of experimental and control groups. P-mTOR, p-S6K1, p-4EBP1 positive cells of experimental group was significantly higher than the control group(P<0.01). The expression level of p-mTOR, p-S6K1, p-4EBP1 in the brain tissues of patients with different seizure frequency and with different duration:the expression level of p-mTOR, p-S6K1, p-4EBP1 in the brain tissues of patients in the group of epilepsy 10 years and more than 10 years were significantly higher than the group of epilepsy fewer than 10 years and the difference was statistically significant (P<0.05). ② The structural changes of brain tissues were observed under the optical microscope and electron microscope. Under the optical microscope:the distribution of nerve cells were uneven, the nucleus was vacuolated, the cytoplasm was less and gliosis. Under the transmission electron microscope:the number of neurons was reduced, nuclear condensation, the heterochromatin was increased, the nucleolus were dissymmetry and the nuclear membrane was breakage, also see neurons became psychotic, cell body became smaller, astrocyte cell membrane became edema, chromatin was dissymmetry, some mitochondrial were swelling and transparent, and others were vacuolated, the mitochondrial crista was in disorder. ③ p-mTOR, p-S6K1 and p-4EBP1 are expressed in the cerebral vascular of the brain in both experimental and control groups.In the experimental group, the expression is high concentration.In the control group, the expression is scattered in a small amount. Conclusionsp-mTOR, p-S6K1 and p-4EBP1 are widely expressed in neurons and glial cells with refractory epilepsy, which was significantly increased compared with control group. The expression of p-mTOR, p-S6K1and p-4EBP1 is related to frequency of epileptic seizures and course.
ObjectiveTo investigate the role of PI3K/AKT/mTOR signaling pathway in skeletal muscle atrophy in rats with chronic obstructive pulmonary diseases(COPD). MethodsPassive cigarette smoking was used to establish COPD model.The protein expression of PI3K, total mTOR, phosphorylated-mTOR, total GSK-3β, phosphorylated-GSK-3β, total 4E-BP1, phosphorylated-4E-BP1, total p70S6K1 and phosphorylated-p70S6K1 in extensor digitorum longus of rats were measured by Western blot. ResultsThe protein expression of PI3K was not significantly different between two groups(P > 0.05).Compared with the control group, the protein expression of total mTOR, phosphorylated-mTOR, total GSK-3β, and phosphorylated-GSK-3βincreased significantly in the COPD group(P < 0.05).The protein expression of total 4E-BP1 and total p70S6K1 were not significantly different between two groups(P > 0.05).While the protein expression of phosphorylated-4E-BP1 and phosphorylated-p70S6K1 significantly increased in the COPD group(P < 0.05). ConclusionThe protein expressions of PI3K/AKT/mTOR signaling pathway in extensor digitorum longus increased significantly in COPD rats, suggesting that the activity of PI3K/AKT/mTOR signaling pathway increased, which may be one of the compensatory mechanism of skeletal muscle atrophy in COPD.
ObjectiveTo investigate the effect of PI3K/Akt/mTOR signaling pathway on liver injury induced by severe acute pancreatitis (SAP). MethodsForty healthy adult male Sprague-Dawley (SD) rats were randomly divided into 4 groups: Sham operation group (SO group), SAP group, PI3K inhibitor LY294002 group (LY294002 group), and mTOR kinase inhibitor rapamycin group (rapamycin group). The rat model with SAP was made by injection with 5% sodium deoxycholate through retrogradely bilio pancreatic duct. Serum levels of amylase (AMY), alanine aminotransferase (ALT), and aspartate transaminase (AST) were detected through the inferior vena at 6 h after modeling. Pathologic change of the liver was observed under the light microscope. TUNEL analysis was used to detect apoptotic index (AI) of the heptocyte. Expressions of Akt, phosphated-Akt (p-Akt), mTOR, phosphated-mTOR (p-mTOR) protein were evaluated by Western blot. Results①Compared with the SO group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the serum levels of AMY, ALT, AST, and the hepatocyte AI were significantly decreased in the LY294002 group and rapamycin group (P < 0.05).②Compared with the SO group, the damages of the liver tissues were aggravated among the other three groups. The pathologies of the liver tissues were ameliorated in the LY294002 group and rapamycin group as compared with the SAP group.③Compared with the SO group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly increased among the other three groups (P < 0.05). Compared with the SAP group, the levels of p-Akt/Akt, p-mTOR/mTOR were significantly decreased in the LY294002 group (P < 0.05), but in the rapamycin group, only the p-mTOR/mTOR level was significantly decreased (P < 0.05). ConclusionThe activation of PI3K/Akt/mTOR signaling pathway might be one of the reasons for the liver injury induced by SAP and blocking this signaling pathway might be a potential target of preventing progress of SAP and alleviating liver injury induced by SAP.
ObjectiveTo systematically review the correlation between mTOR protein expression and different clinical pathological features as well as the response to radiotherapy and chemotherapy of cervical cancer. MethodsWe electronically searched databases including The Cochrane Library (Issue 1, 2015), PubMed, EMbase, CNKI, CBM, VIP and WanFang Data from inception to April 2015 to collect case-control studies investigating the correlation between mTOR protein expression and different clinical pathological features as well as the response to radiotherapy and chemotherapy of cervical cancer. Two reviewers independently screened literature, extracted data and assessed the risk bias of the included studies. Then meta-analysis was performed using RevMan 5.2 software. ResultsA total of 8 case-control studies involving 591 patients were included. Among these cases, 365 cases were in the cervical cancer group, 135 cases were in the cervical intraepithelial neoplasia (CIN) group, and 91 cases were in the normal cervix tissue group. The results of meta-analysis showed that:(1) Compared with the normal cervix tissue group, mTOR protein was overexpressed in the cervical cancer group (OR=24.14, 95%CI 4.47 to 130.35, P=0.000 2) and the CIN group (OR=4.71, 95%CI 2.15 to 10.33, P=0.000 1); Compared with the CIN group, mTOR protein was overexpressed in the cervical cancer group (OR=5.12, 95%CI 2.96 to 8.86, P<0.000 01). (2) Compared with the non-lymphnode-metastasis group, mTOR protein was overexpressed in the lymph node metastasis group (OR=3.29, 95%CI 1.61 to 6.69, P=0.001); Compared with the FIGO I group, mTOR protein was overexpressed in the FIGO Ⅱ group (OR=3.00, 95%CI 1.49 to 6.04, P=0.002); Compared with the radiotherapy and chemotherapy responsive group, mTOR protein was overexpressed in the non-response group (OR=15.64, 95%CI 3.17 to 77.15, P=0.000 7). In addition, there was no significant difference between the medium/high differentiation group and low differentiation group (OR=1.70, 95%CI 0.75 to 3.81, P=0.20). ConclusionmTOR protein expression is associated with cervical cancer, and mTOR protein overexpression was associated with lymph node metastasis, higher FIGO and non-response to radiotherapy and chemotherapy. Due to the limited quantity and quality of the included studies, the above conclusion needs to be further verified by more high quality studies.
ObjectiveTo observe the effect of metformin on airway remodeling in asthma and its possible mechanism.MethodsTwenty-eight B/N rats were randomly divided into control group, asthma group, metformin intervention group and rapamycin intervention group. After that, the asthma model was established and intervened with metformin and rapamycin. The airway resistance and airway reactivity were measured 48 hours after the last challenge, and then the lung tissue samples were collected. Histopathological examination was used to observe airway inflammatory cell infiltration, goblet cell proliferation, airway wall fibrosis and remodeling, as well as airway smooth muscle proliferation. The expression of AMPK/mTOR pathway related proteins was detected by Western blot.ResultsCompared with the asthma group, metformin and rapamycin significantly reduced the airway responsiveness induced by high concentration of acetylcholine (P<0.05), reduced the infiltration of inflammatory cells in lung tissue and the changes of airway wall structure (P<0.05), reduced goblet cell proliferation in airway epithelium, collagen fiber deposition in lung tissue and bronchial smooth muscle hyperplasia (P<0.05). Further studies showed that the effects of metformin and rapamycin were related to AMPK/mTOR pathway. Compared with the asthma group, metformin and rapamycin could significantly reduce the expression of p-mTOR, p-p70s6k1 and SKP2, while p21 protein expression was significantly increased (P<0.05). In addition, metformin and rapamycin had similar effects (P>0.05).ConclusionMetformin can alleviate airway hyperresponsiveness and airway remodeling by activating AMPK and then inhibiting mTOR pathway, which may be a potential drug for treating asthma and preventing airway remodeling.
Objective To explore the protective effect of rapamycin on brain tissues injury in severe acute pancreatitis (SAP) and its possible mechanism in experimental rats. Methods Ninety SPF males SD rats were randomly divided into 3 groups by random envelope opening method: sham operation group (SO group), SAP group, and rapamycin group (RAPA group), then the rats of each group were divided into 24 h, 36 h, and 48 h 3 subgroups by random number table method. Rats in each group underwent laparotomy, the model was prepared by retrograde injection of solutions into biliopancreatic duct, rat of the SO group was injected with 0.9% normal saline (2 mL/kg), rats of the SAP group and the RAPA group were injected with 5% sodium taurocholate solution (2 mL/kg), but rat of the RAPA group was injected with rapamycin (1 mg/kg) at 30 min before narcosis. All survival rats in each subgroup were killed at 24 h, 36 h, and 48 h respectively, then the pancreas and brain tissues of rats were collected, pancreas and brain tissues were stained by hematoxylin-eosin staining, brain tissues were stained by Luxol fast blue additionally, pathological changes of brain tissues were scored under light microscope. The protective effect of rapamycin on brain tissues injury was determined by comparing the differences in the degree of brain tissues among 3 groups. The phosphorylated mammaliantarget of rapamycin (p-mTOR) and phosphorylated ribosomal 40S small subunitS6 protein kinase (p-S6K1) expression levels in brain tissues were detected by Western blot. In addition, the correlations between the expression levels of p-mTOR and p-S6K1 in brain tissues and the degree of brain tissues injury were analyzed to further explore the possible mechanism of rapamycin’s protective effect on brain tissues injury in SAP. Results① At the point of 24 h, 36 h, and 48 h, the order of the relative expression levels of p-mTOR and p-S6K1 in brain tissues of three groups were all as follows: the SO group < the RAPA group < the SAP group (P<0.05). ② At the point of 24 h, 36 h, and 48 h, the order of brain histological score in three groups were all as follows: the SO group < the RAPA group < the SAP group (P<0.05). ③ The relative expression levels of p-mTOR and p-S6K1 in brain tissues were positively correlated with pathological scores of brain tissues (r=0.99, P<0.01; r=0.97, P<0.01). ConclusionRapamycin plays a protective role in pancreatic brain tissues injure by down-regulating the expression levels of p-mTOR and p-S6K1 in mTOR signaling pathway.
Objective To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1β group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 μmol/L EGCG+10 ng/mL IL-1β group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1β group (group B), EGCG+10 ng/mL IL-1β group (group C), and EGCG+10 ng/mL IL-1β+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by β-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1β group significantly decreased (P<0.05). Compared with the 10 ng/mL IL-1β group, the cell activity of EGCG+10 ng/mL IL-1β groups increased, and the 50.0, 100.0, and 200.0 μmol/L EGCG significantly promoted the activity of chondrocytes (P<0.05). The 100.0 μmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant (P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend (P<0.05). ConclusionEGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.
ObjectiveTo study the effect of Tangeretin on non-small cell lung cancer (NSCLC) and the tumor stemness, and to find the molecular mechanism of its effect. MethodsWe used cell counting and cell cloning experiments to study the effect of Tangeretin on the proliferation of NSCLC cells in vitro. The effect of Tangeretin on the invasion of NSCLC cells was detected by transwell assay. We detected the effect of Tangeretin on the proliferation of NSCLC cells in vivo by nude mouse tumor-bearing experiment. The effect of Tangeretin on tumor stemness of NSCLC cells was detected by self-renew assay, and CD133 and Nanog protein expressions. The expressions of PI3K/AKT/mTOR signaling pathway-related proteins were detected by Western blotting (WB). ResultsTangeretin had a good inhibitory effect on the proliferation of NSCLC cells in vivo and in vitro. Cell counting experiment, clonal formation experiment and nude mouse tumor-bearing experiment showed that Tangeretin could inhibit the proliferation activity, clonal formation ability, and tumor size of NSCLC cells in vivo. Self-renew experiments showed that Tangeretin could inhibit the self-renew ability of NSCLC cells. WB experiments showed that Tangeretin inhibited the expressions of tumor stemness markers CD133 and Nanog in NSCLC cells. Tangeretin could inhibit the activation of PI3K/AKT/mTOR signaling pathway-related proteins in NSCLC cells, and the activation of PI3K/AKT/mTOR signaling pathway could partially remit the inhibitory effect of Tangeretin on tumor stemness of NSCLC cells. ConclusionTangeretin can inhibit the tumor stemness of NSCLC cells, which may be related to the regulation of PI3K/AKT/mTOR signaling pathway.
Objective To investigate the effects and mechanisms of hydroxychloroquine sulfate (HCQ) on pulmonary fibrosis through the PI3K/AKt/mTOR signalling pathway. Methods Paraquat intraperitoneal injection was used to establish a mouse model of pulmonary fibrosis. Thirty-six SPF C57BL/6J female mice were randomly divided into a blank group, a paraquat group (20 mg/kg) and a HCQ intervention group. The HCQ intervention group was divided into two subgroups (10 mg/kg and 30 mg/kg) according to different doses. The general condition and body weight changes of mice were observed. twenty-one days later, lung tissues were stained with hematoxylin-eosin and Masson’s pathological staining, and the content of inflammatory factors (IL-1β, IL-6, TNF-α) and hydroxyproline (HYP) were detected by ELISA. Alpha-smooth muscle actin (α-SMA), E-cadherin (E-cad), the expression levels of PI3K/Akt/mTOR pathway-related proteins, phosphatidylinositol 3 kinase (PI3K) and protein kinase B (AKt), and mammalian target of rapamycin (mTOR) were detected by Western blot. The gene expression levels of α-SMA and E-cad were detected by q-PCR. Results Compared with the blank group, the mice in the paraquat group had lower body weight, worse general condition, higher serum levels of inflammatory factors, increased lung structure destruction and collagen deposition, significantly increased HYP content, and higher expression level of PI3K/AKt/mTOR signaling pathway related proteins (all P<0.05). The expression levels of E-cad protein and gene decreased, α-SMA protein and gene increased (all P<0.05). While the HCQ intervention group improved the degree of pulmonary fibrosis in different degrees, and the relevant indexes of PI3K/AKt/mTOR signaling pathway decreased compared with the paraquat group (all P<0.05). Conclusion HCQ can ameliorate paraquat-induced pulmonary fibrosis by inhibiting the PI3K/AKt/mTOR signaling pathway.