Objective To investigate the cell growth inhibition and apoptosis induced by rapamycin on human hepatocellular carcinoma Bel-7402 cells and to study the role of mitochondrium membrane potential in the process of apoptosis. Methods Bel-7402 cells in vitro were given 5, 10, 20, 30, 40 and 50 nmol/L different concentrations of rapamycin, and the cell growth inhibiting ratio of Bel-7402 was assessed by MTT assay. The changes of morphology of Bel-7402 were observed by Hoechst 33258 staining and flow cytometry (FCM), respectively; The cell mitochondrial membrane potential was detected by using JC-1 staining method. Results Rapamycin could inhibit the growth of Bel-7402 cells significantly by inducing apoptosis, and the growth suppression and the cell apoptosis both presented time-effect relationship and were also dose-dependent. The rates of inhibiting and cell apoptosis after 72 h exposure to 50 nmol/L rapamycin were significantly higher that those of other groups (P＜0.01). Typical morphological changes of cell apoptosis were observed very clearly after the Bel-7402 cells had been exposed to rapamycin for 48 hours using Hoechst 33258 staining method, and it was also observed that the mitochondrial membrane potential decreased when apoptosis occured (P＜0.01). Conclusion Rapamycin could inhibit the growth of Bel-7402 cells by inducing cell apoptosis, and the descent of mitochondrial membrane potential may play an important role in the process of cell apoptosis.
ObjectiveTo investigate the mechanism of mTOR signaling pathway in bleomycin (BLM)-induced pulmonary fibrosis in mice.MethodsSixty C57BL/6 mice were randomly divided into a control group and a BLM group. Pulmonary fibrosis model was induced by single intratracheal instillation of bleomycin (2.5 mg/kg) in the BLM group. Similarly, 0.9% saline was instilled directly into the trachea in the control group. Then all mice were sacrificed at 21 days. The lungs were collected for morphometric analysis with HE and Masson staining. The degree of pulmonary fibrosis was evaluated with Ashcroft score. The activity of mTOR signaling pathway was measured by Western blot. The level of collagen1, collagen3 mRNA was assessed with quantitative real time PCR.ResultsThe thickening alveolar septa, accumulation of inflammatory cells, and fibrous obliteration in the BLM group were exhibited predominantly compared with the control group. There was a significant difference in Ashcroft score between the BLM group and the control (P<0.05). Also, the activity of mTOR signaling pathway was up-regulated and the expression of collagen1 mRNA and collagen3 mRNA was increased in the BLM group.ConclusionAberrant activation of mTOR signaling pathway aggravates the pulmonary fibrogenesis.
ObjectiveTo explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs). MethodsThe hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 hof co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. ResultsAt 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group (F=95.900, P＜0.05). Compared with the PBS group, The mTOR, HIF-1 α protein (t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression (t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant (P＜0.05). The relative expression level of VEGF-Aprotein (t=3.337, 7.380) and mRNA (t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant (P＜0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant (t=4.664, 6.136, 6.247; P＜0.05). ConclusionsmiR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
ObjectiveTo investigate the relationships between the onset age, genotype, clinical phenotype and the efficacy of Rapamycin in patients with tuberous sclerosis complex.MethodsRetrospectively analyze the clinical data of patients with tuberous sclerosis complex (TSC) who were diagnosed with epilepsy in Guangdong Sanjiu Brain Hospital from October 2013 to December 2018. Meanwhile, the relationships between the onset age of epilepsy and genotype, clinical phenotype and Rapamycin efficacy were analyzed comprehensively.ResultsTSC gene was detected in 104 patients with tuberous sclerosis complex, of which 85 (81.7%) were positive and 44 (51.8%) were males as well as 41 (48.2%) were females, with an average age of (4.0±4.9) years old. And there were 34 (40.0%) TSC1 mutations and 51 (60.0%) TSC2 mutations. The patients were divided into 3 groups according to their ages: ≤1 year old, 1 ~ 6 years old and ≥6 years old. Among them, 31 cases (36.5%) were in the ≤1 year old group, 31 cases (36.5%) in the 1 ~ 6 years old group and 23 cases (27.0%) in the ≥6 years old group. Through statistical analysis, we found that the onset age of epilepsy in patients with TSC1 and TSC2 gene mutations was statistically different (χ2=9.030, P=0.011). Further analysis of the relationship between the onset age of epilepsy and other clinical phenotypes showed that there were statistical differences in the probability of mental retardation and spasm seizure in different onset age groups of epilepsy (P<0.05). In addition, patients with epilepsy onset age ≤1 year old are more likely to have renal disease and patients with epilepsy onset age ≥6 years old are more likely to have SEGAs. There was no significant difference between the onset age of epilepsy and the efficacy of Rapamycin (P>0.05).ConclusionTSC2 mutation, mental retardation and spasm seizure are more likely to occur in patients with epilepsy onset age ≤1 year old. The study on multiple factors of epilepsy onset age may have a certain guiding role in judging the development and prognosis of TSC with epilepsy.
Objective To review the role of mTOR signal pathway in chemo-resistance of gastric cancer. Methods Domestic and international publications related mTOR signal pathway in chemo-resistance of gastric cancer in recent years were collected and reviewed. Results mTOR was a central signaling molecule of mTOR signal pathway， which regulated key cellular processes such as cell growth， cell proliferation， cell metabolism， and angiogenesis. Signaling molecules of mTOR signal pathway were overexpressed in gastric cancer. Moreover， mTOR signal pathway might play an important role in chemo-resistance of gastric cancer， and tumor stem cells were involved in it too. Conclusion As mTOR signal pathway plays an important role in chemo-resistance of gastric cancer， the combination of mTOR inhibitors and chemotherapy drugs may overcome the chemo-resistance of gastric cancer.
ObjectiveTo analyze the clinical efficacy and safety of rapamycin in the treatment of Tuberous sclerosis complex ( TSC ) complicated with refractory epilepsy, and to provide scientific basis for the clinical treatment of this disease.MethodsRetrospective analysis was performed on 22 children with TSC complicated with refractory epilepsy admitted to Henan People's Hospital from 2017 to 2019, including 11 males and 11 females who met the inclusion criteria, with an average age of (27.91±36.92) months. They were treated with antiepileptic drugs and rapamycin at the same time, and followed up for at least 1 year.To observe the change of seizure frequency before and after treatment with rapamycin.ResultsThe mean reduction rate of seizure frequency in children with tuberous sclerosis complicated with refractory epilepsy was 52.1% 6 months after the addition of rapamycin, and 51.2% 12 months after the addition of rapamycin. The number of seizure-free days could be maintained. The difference before and after the addition of rapamycin was statistically significant (P<0.05).ConclusionThe addition of rapamycin in the treatment of TSC complicated with refractory epilepsy can reduce the frequency of seizure and increase the number of days without seizure, and the adverse reactions are mild/moderate. Rapamycin has certain safety in children with regular follow-up.
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 review the possible mechanisms of the mammal ian target of rapamycin (mTOR) in theneuronal restoration process after nervous system injury. Methods The related l iterature on mTOR in the restoration ofnervous system injury was extensively reviewed and comprehensively analyzed. Results mTOR can integrate signals fromextracellular stress and then plays a critical role in the regulation of various cell biological processes, thus contributes to therestoration of nervous system injury. Conclusion Regulating the activity of mTOR signaling pathway in different aspects cancontribute to the restoration of nervous system injury via different mechanisms, especially in the stress-induced brain injury.mTOR may be a potential target for neuronal restoration mechanism after nervous system injury.
