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.
ObjectiveTo investigate the effect of dexamethasone on mammalian target of rapamycin (mTOR) expression of astrocytes in hippocampus of rats with sepsis associated encephalopathy (SAE). MethodsTotally, 90 cases of 30-day-old male Wistar rats were randomly divided into sham-operation group (n=10) and cecal ligation and puncture (CLP) group (n=80). Models of rats with sepsis were established by CLP. At 12 hours after CLP, if rats appeared lower neurobehavioral scores, abnormal electroencephalogram (EEG) and somatosensory evoked potential (SEP), they were diagnosed with SAE. And then, they were randomly divided into non-treated group and dexamethasone group. Rats in the dexamethasone group were injected with dexamethasone (1 mg/kg) via tail vein every other day for a total of 3 times. The same dose of saline was used in the non-treated group. The neurobehavioral score was measured, SEP and EEG were examined in the age of 40 days, and then the rats were killed and the hippocampus was taken. Expressions of mTOR protein were measured by Western blot. The glial fibrillary acidic protein (GFAP) and mTOR were detected by immunofluorescence assay, and the number of positive cells was calculated by image analysis system software. ResultsSix of 80 CLP rats died in 12 hours after operation, and 28 of 74 rats were diagnosed as SAE because they appeared lower neurobehavioral scores, abnormal EEG and SEP at 12 hours after CLP. The incidence of SAE was 37.84% (28/74). In the age of 40 days, compared with non-treated group, neurobehavioral score of rats in the dexamethasone group was low, the amount of alpha waves in EEG reduced, delta waves increased, the amplitude of P1 waves in SEP was decreased, and the latencies of P1 and N1 waves were prolonged (P＜0.05). GFAP immunofluorescence staining showed astrocytic body and processes were small in the sham operation group. However, astrocytes in the non-treated group had large body and hypertrophic processes, and compared with the sham operation group, the number of these cells increased significantly (P＜0.05). Astrocytic body and processes were small in the dexamethasone group compared with the non-treated group, and the number of cells also decreased (P＜0.05). The mTOR positive astrocytes in the non-treated group were more than those in the sham operation group (P＜0.05). But mTOR positive astrocytes in the dexamethasone group were fewer than those in the non-treated group (P＜0.05). ConclusionsAstrocytes are activated in the hippocampus of rats with SAE. They show features of reactive hyperplasia, and the expression of mTOR is up-regulated, while dexamethasone can inhibit effects on these.
Objective To observe efficacy of rapamycin combined with sorafenib in hepatocellular carcinoma (HCC) patients with tumor recurrence after liver transplantation beyond Milan criteria. Methods Forty-one beyond Milan criteria HCC patients who underwent the classic orthotopic liver transplantation without bypass and the tumor postoperatively recurred in the Tianjin First Center Hospital from February 1, 2012 to August 31, 2015 were collected retrospectively, then were divided into a local treatment group (n=21) and a comprehensive treatment group (n=20). The local treatment included the surgical resection, radiofrequency ablation, transcatheter arterial chemoembolization, radioactive seed implantation, etc.. The comprehensive treatment was on the basis of the local treatment plus rapamycin in combination with sorafenib. Results There were 12 patients with stable disease and 9 patients with progressive disease in the local treatment group. There were 12 patients with partial response, 10 patients with stable disease and 8 patients with progressive disease in the comprehensive group. In the local treatment group and the comprehensive treatment group, the median survival time were 9 months and 12 months, and the 1-year and 2-year survival rates after the recurrence were 14% versus 55%, 0 versus 15%, respectively. The survival of the comprehensive treatment group was significantly better than that of the local treatment group (P<0.01). Conclusion Combination of rapamycin and sorafenib in HCC patients with tumor recurrence after liver transplantation beyond Milan criteria can significantly improve survival time of patient with recurrence.
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.
Objective To evaluate the short and long term effectiveness and safety of rapamycin-based immunosuppression regimes with CsA preserving versus CsA withdrawal. Methods We searched MEDLINE, EMBASE, The Cochrane Library and CNKI from Jan. 1995 to Dec. 2005. We identified randomized controlled trials of rapamycin-hased immunosuppression regimes with CsA preserving versus CsA withdrawal for renal transplantation patients. The quality of included trials was evaluated by two reviewers. Meta-analysis was conducted on homogeneous studies. Results Ten studies （1 121 patients） undergoing renal transplantation were included. All included studies were graded in term of randomization, allocation concealment and bhnding. Six studies were graded A and the other 4 were graded B. Meta-analysis results showed CsA withdrawal in sirolimus-based therapy in renal transplantation patients survival rate OR.（95% CI ） values were 0,77（0.17, 3.52）, 1.24（0.48, 3.16）, 1.32（0.57, 3.08）, 1.21（0.60, 2.41） at the end of 6, 12, 24, 36 months respectively; renal allografts survival rate OR. （95% CI） values were 1.79 （0.63, 5.06）, 1.15 （ 0.56, 2.36） , 1.39 （0.68, 2.85）, 1.80（0.99, 3.29）, 2. 13（1.16, 3.89）, 2.01（1.15, 3.51） at the end of 6, 12, 24, 36, 48, 54 months respectively; and acute rejection OP,（95% CI） values were 0.92（0.48, 1.78）, 1.90（1.25, 2.89）, 2. 01 （0.94,4.27）, 1.93（0.93, 4.00）, 1.52（0.77, 3.02） at the end of6, 12, 24, 36, 48 months respectively. Conclusions Available evidence shows that compared with CsA preserving, CsA withdrawal in rapamycin-based immunosuppression regimes can lead to higher incidence rates of acute rejection at the end of one year while there is no statistical difference to survival rate of patients/renal allograft in cases with stabilized renal function post-transplantation. And CsA withdrawal is of benefit to allografts for long term survival rate and is helpful to recovery of renal function. Owing to high possibility of selection bias and measurement bias in included studies, there must be a negative impact on evidence intensity of our results. We expect best evidence from with high quality double blind randomized control trials.
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.
ObjectiveTo explore the protective effect of rapamycin on pancreatic damage in severe acute pancreatitis (SAP) and further to explain its protective mechanism.MethodsNinety selected SPF males SD rats were randomly divided into 3 groups: sham-operated group (SO group), SAP group, and rapamycin group (RAPA group), with 30 rats in each group. Then each group of rats were randomly divided into 3 subgroups of 24 h, 36 h, and 48 h, 10 rats in each subgroup. Rats in each group underwent laparotomy, the model was prepared by retrograde injection of solutions into biliopancreatic duct, rats of the SO group were injected with 0.9% normal saline, rats of the SAP group and RAPA group were injected with 5% sodium taurocholate solution, but rats of the RAPA group were injected with rapamycin at 30 min before the injection of 5% sodium taurocholate. All the survival rats in corresponding subgroup were killed at 24 h,36 h, and 48 h after operation respectively, then serum and pancreas tissues of rats were collected, serum inflammatory factors content of IL-1β, IL-6, and TNF-α were detected by ELISA method, expression levels of p-mTOR and p-S6K1 in pancreas were detected by Western blot, pancreas tissues were stained by Hematoxylin-Eosin Staining and pathological changes of pancreas were scored under light microscope.Results① At the timepoint of 24 h, 36 h, and 48 h, the order of the expression levels of p-mTOR and p-S6K1 in pancreatic tissues of 3 groups were all as follows: SO group<RAPA group<SAP group, there were significant difference among any 2 groups (P<0.05). ② IL-1β: at the timepoint of 48 h, the order of the content of IL-1β in 3 groups were as follows: SO group<RAPA group<SAP group, there were significant differences among any 2 groups (P<0.05); IL-6: at the timepoint of 36 h and 48 h, the order of the content of IL-6 in3 groups were as follows: SO group<RAPA group<SAP group, there were significant differences among any 2 groups (P<0.05); TNF-α: at the timepoint of 48 h, the order of the content of TNF-α in 3 groups was as follows: SO/RAPA group<SAP group (P<0.05), but there was no significant difference between the SO group and RAPA group (P>0.05). ③ Pancreatic histological score: at the timepoint of 24 h, 36 h, and 48 h, the order of the pancreatic histological score in3 groups was all as follows: SO group<RAPA group <SAP group, there were significant differences among any 2 groups (P<0.05). ④ The expression levels of p-mTOR and p-S6K1 in pancreatic tissue were positively correlated with the pathological scores of pancreatic tissue (r=0.97, P<0.01; r=0.89, P<0.01).ConclusionRapamycin can reduce the degree of pancreatic damage in SAP and has protective effect on pancreatic tissue.
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.