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Objective @# To explore the mechanism of hippocampal corticotropin-releasing hormone ( CRH) receptor type 1 ( CRHR1 ) in chronic stress-induced learning and memory impairment in early aged mice．@*Methods@#C57BL /6J mice aged 12 －14 months were divided into two groups according to gender，and then divided into wild type (WT) group and hippocampal CRHR1 conditional gene knockout (KN) group according to genotype．Mice in each group were randomly divided into control group and stress group，and the stress group was subjected to chronic unpredictable stress ( CUS ) for 30 days． Genotyping of mice was performed using polymerase chain reaction ( PCR) ，agarose gel electrophoresis and real-time fluorescence quantitative PCR (RT-qPCR) ．The new object rec- ognition experiment and Morris Water maze measured learning and memory ability．Golgi-Cox staining was used to observe damage to hippocampal neuronal dendrites． The protein expressions of target protein of rapamycin (mTOR) ，p-mTOR (Ser2448) ，ribosomal protein S6 kinase ( p70S6K) and p-p70S6K ( Thr389 / Thr412 ) were detected by Western blot．Serum levels of corticotropin releasing hormone ( CRH) were measured by ELISA. @*Results @# Compared to mice without chronic stress，the cognitive coefficient of WT stress groups decreased after chron- ic stress，and the difference was statistically significant (P ＜0. 05) ，while there was no significant difference in cognitive coefficient of KN stress groups before and after chronic stress．Compared with the WT stress group，the escape latency of the WT control group was shortened (P＜0. 05) ，and the number of crossing the platform and tar- get quadrant increased (P ＜0. 01) ，and there was no significant difference in the KN groups above． Compared with the WT control group，the WT stress group had a significant reduction in the neuronal complexity in the hipp- ocampal CA1，CA3 and DG regions (P ＜0. 05) and significant reductions in the expression of p-mTOR and p- p70S6K in the hippocampus (P＜0. 05) ．There was no significant difference in the expression of p-mTOR between the KN stress group and the KN control group (P＞0. 05) ，except that the expression of p-mTOR in the hippocam- pus of the female group decreased (P＜0. 05) ．In addition，the serum level of CRH in the stress group was higher than that in the control group (P＜0. 01) ．@*Conclusion @#Hippocampal CRHR1 regulates learning and memory im- pairment and neuronal dendrite damage in early aged mice induced by chronic stress．The mechanism may be that high levels of CRH induced by chronic stress cannot bind to CRHR1 receptor，thereby enhancing the expression of down-regulated mTOR / p70S6K signaling pathway.
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Objective @#To investigate the sex difference of the effects of chronic pregnancy stress on depression-like behavior in offspring adolescent mice and whether the amygdala is involved in mediating depression-like behavior and its possible mechanism．@*Methods @#Male and female of C57BL /6J mice were put in cage together．Pregnant mice were randomly divided into normal control group ( CON group) and chronic pregnancy stress group ( CPS group) ．The day of delivery was recorded as post-natal day(PND0) ．The offspring of different groups were divided into Female group and Male groupaccording to sex，respectively．From PND35，the depressive-like behavior of off- spring was monitored in different groups．Morphological structure of basolateral amygdala (BLA) cone neurone was observed by Golgi-Cox staining，and apoptosis of BLA neurone was detected by TUNEL．Serum corticotrophin-relea- sing hormone ( CRH) was detected by ELISA．The level of protein associated with amygdala mammalian target of rapamycin (mTOR) and phosphorylated mammalian target of rapamycin ［p-mTOR ( Ser2448) ］was detected by Western blot.@*Results @#Depression-like behavior was appeared in different sexual offspring by chronic pregnancy stress，and there was an interaction between chronic pregnancy stress and gender．In the forced swimming test，the immobility time of offspring in the CPS group prominently increased(Female: P＜0. 05，Male: P＜0. 001) ．Interest- ingly，compared with female offspring ，despairing behavior of male offspring was much more clearly observed in CPS group(P＜0. 05) ．Compared with offspring of CON group，the rate of sucrose preference was significantly re- duced in the female offspring of CPS group(P＜0. 05) ，while no obvious difference was observed in the male off- spring．Compared with the CON group，the density of neuronal dendrite branches in the BLA of offspring mice in the CPS group decreased(Female: P＜0. 01，Male : P＜0. 01) and the degree of neuronal apoptosis increased( Fe- male: P＜0. 001，Male : P ＜0. 001) ，the expression level of p-mTOR in amygdala of offspring mice in CPS group significantly decreased(Female: P＜0. 001，Male: P＜0. 001) ．Chronic pregnancy stress increased the serum CRH level of offspring mice(P＜0. 001) ，and the gender had significant influence on serum CRH level，the serum CRH level of female in CPS group was higher than that of male(P＜0. 05) ．@*Conclusion @#Chronic pregnancy stress leads to depression-like behavior in offspring adolescent mice，and the depression-like behavior has gender differences. In addition，chronic pregnancy stress leads to dendrite atrophy and apoptosis of BLA neurons in offspring mice，and the mechanism may be that the activation of mTOR in the amygdala of offspring mice is inhibited．CRH may be in- volved in mediating sex differences in depression-like behavior and BLA neuron damage in offspring induced by chronic pregnancy stress.
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Aim To observe the effect of mineralocorticoid receptor blocker eplerenone on autophagy in obstructive nephropathy and its mechanism. Methods Totally 36 male Wistar rats were randomly divided into sham-operation group, model group and eplerenone group. The animal models were established with unilarteral urteral obstruction ( UUO). The rats in eplerenone group were treated with eplerenone (100 mg • kg"1 • d"1). The obstructed kidneys were collected lOd after UUO. The expression of NR3C2 was detected by laser confocal microscopy, the expression of serum and glucocorticoid-induced protein kinase 1 ( SGK-1), phosphorylated mammal target of rapamycin (p-mTOR) , autophagy associated gene 5 (Atg5) , Be-clin-1 and microtubular-associated protein 1 light chain 3 (LC3) were detected by immunohistochemistry and Western blot. Results The expression of NR3C2 was detected in cytoplasm of renal tubular distal epithelial cells, but not in nucleus in sham-operation group with laser confocal microscopy. The expression of NR3C2 was enhanced significantly in model group, mainly in nucleus but significantly inhibited in eplerenone group. The immunohistochemistry and Western blot showed that the expressions of SGK-1, Atg5, Beclin-1 and the ratio of LC3 11/I in model group were up-regulated and down-regulated by eplerenone treated group. The expression of p-mTOR was down-regulated in model group compared with sham-operation group and up-regulated in eplerenone group. Conclusions Eplerenone plays a role in reducing autophagy in obstructive nephropathy via inhibiting the activation of mineralocorti-coid receptor.
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@#Objective: To investigate the effect of sulforaphane (SFN) on CD8+ T cells differentiation, phenotype and the secretion of intracellular cytokines, as well as to study the underlying molecular mechanism. Methods: In the in vitro culture experiment, the cells were categorized into control group, SNF 10 mmol/L group and SNF 20 mmol/L group according to the SNF concentration. The effect of SFN treatment on CD8+ T cells differentiation, phenotype and cytokine secretion were detected by flow cytometry, and the effect of mTOR siRNA on the expression of CD127 and LKRG1 in CD8+T cells was also detected by flow cytometry. Expression of Bcl-2 and Bcl-6 were analyzed by qRT-PCR. The effect of SFN on apoptosis of CD8+T cells was examined byAnnexin-V/PI staining. The protein expressions of p-mTOR, p-S6 and b-actin were detected by western blotting. Results: SFN significantly promoted the formation of memory precursor CD8+ T cells and decreased the expression level of PD-1 and Tim-3 in CD8+T cells(P<0.01); meanwhile, after the treatment of SFN, the expressions of anti-apoptosis genes Bcl-2 and Bcl-6 were significantly increased while the apoptosis of CD8+ T cells was significantly inhibited and the protein expressions of p-mTOR and p-S6 were also significantly inhibited(P<0.05 or P<0.01). Moreover, mTOR siRNA could significantly increasethe expression of CD127 and decrease the expression of LKRG1 (all P<0.01). Conclusion: Sulforaphone promotes the formation of memory precursor CD8+T cells possibly by inhibiting the p-mTOR signaling pathway, and this could obtain more T cells to provide new thoughts for clinical immunotherapy.
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Aim To study the effect of resveratrol on proliferation of liver cancer SMMC-7721 cells and the relevant mechanism.Methods SMMC-7721 cells were treated with different concentrations of resveratrol.Cell proliferation was tested by cell proliferation assay and colony-forming experiment.The effect of resveratrol on cell cycle of SMMC-7721 cells was detected by flow cytometry.The activation of mTOR/p-mTOR proteins was detected by Western blot.Results Compared with control group, resveratrol inhibited SMMC-7721 cell proliferation in a dose-dependent manner(P<0.01);cell cycle detection showed that SMMC-7721 cells were arrested at G0/G1 phase by resveratrol;Westren blot assay showed that resveratrol down-regulated p-mTOR expression.Conslusion Resveratrol inhibits human SMMC-7721 cell proliferation and arrests the cell cycle at G0/G1 phase, while the inhibition mechanism may be related to activation of mTOR signaling pathway by resveratrol.
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Objective To investigate the effect of propofol on autophagy in SD rat heart during myocar-dial ischemia-reperfusion (I/R) injury. Methods Twenty-one male SD rats were randomly divided into three groups as follows (n = 7): the sham operation group, in which rats underwent sham operation without tightening of the coronary artery sutures; the myocardial ischemia-reperfusion group , in which rats were induced by occlud-ing the left anterior descending coronary artery for 30 min , followed by 120 min reperfusion and 0.9% NaCl in-fusion at 3 mL/(kg·h) at 10 min before occluding the left anterior descending coronary artery; the myocardial ischemia- reperfusion- propofol group, in which rats underwent I/R and propofol infusion at 6 mg/(kg·h) at 10 min before occluding the left anterior descending coronary artery. Before tightening of the coronary artery, at 30 min post-tightening of the coronary artery and at 120 min post-reperfusion, HR、 LVSP and ± dp/dtmax of rats were recordedin each group. Atter 120 min post-reperfusion, the serum concentrations of cTnT was measured. The in-jured cardiac tissue was collected to investigate the ultrastructure change under the TEM and to determine the levels of mTOR and p-mTOR. Results No signifcant differences in HR, LVSP and ± dp/dtmax before tighten-ing of the coronary artery. But, at 30 min post- tightening of the coronary artery, compared with groupⅠ, the HR, LVSP and ±dp/dtmax were significantly decresed in groupⅡ and Ⅲ(P < 0.05). Then, at 2 h post-reper-fusion, compared with groupⅠ, the HR, LVSP, ±dp/dtmax and the level of p-mTOR were significantly de-creased, but the serum concentration of cTnT was significantly increased in groupⅡ(P < 0.05); but, compared with groupⅡ, the HR, LVSP, ± dp/dtmax and the level of p-mTOR were significantly increased, the serum concentration of cTnT and the level of mTOR were significantly decreased in group Ⅲ(P < 0.05). Conclusions These data suggest that propofol could heighten the level of p-mTOR, and attenuate the expression of mTOR dur-ing the myocardial ischemia-reperfusion injury in SD rats.
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Objective To investigate the autophagy capacity in clear cell renal cell carcinoma tissue compared with adjacent normal tissue.Methods Sixty-nine pairs of samples from human clear cell renal cell carcinoma tissues and relatively healthy renal tissues adjacent to the tumor were collected during surgical resection.The expressions of proteins that were participating in the regulation and execution of autophagy were detected by immunohistochemisty.Electron-microscopy was also carried out for the morphometrical analysis.Results The protein expression of p-mTOR (P =0.004),P62 (P =0.000) and ULK1 (P =0.000) were up-regulated in the carcinoma tissue,while the expression of Beclin1 (P=0.000),LC3 (P =0.000) and ATG7 (P =0.000) were down-regulated,and the expression of ATG5 (P =0.349) had no signif-icant difference compared with adjacent normal tissue.Morphometrical analysis showed that the basal autophagic activity (measured by the presence of autophagy vacuole compartment) was remarkably down-regulated in carcinoma tissue,compared with adjacent normal tissue.The expression level of mTOR was correlated with P62,LC3 and ATG7,but results showed no correlation between mTOR and Beclin 1.Conclusion Our studies show a relatively reduced autophagy capacity in clear cell renal cell carcinoma,which is regulated by multiple autophagy-related proteins such as mTOR,Beclin 1 and LC3.
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Objective: To observe the expression of phosphorylated mammalian target of rapamycin(p-mTOR), epidermal growth factor receptor(EGFR), growth factor receptor-bound protein 2(Grb2) and vascular endothelial growth factor(VEGF) in human colorectal cancer tissues and to explore their roles in the carcinogenesis of colorectal cancer. Methods: Tissue microarray containing 185 colorectal cancer tissues was constructed and the expression of EGFR, Grb2, p-mTOR and VEGF in the colorectal cancer tissues and the corresponding adjacent tissues was examined by immunohistochemistry methods. The relationship between their expression with the clinicopathological characteristics such as age, sex, invasion depth, lymphatic metastasis, clinical stage and differentiation degree was analyzed. Results: EGFR, Grb2, p-mTOR and VEGF were scarcely expressed or absent in the corresponding adjacent tissues; their positive rates in the colorectal caner tissues were 21.1%, 44.9%, 42.2% and 54.1%, respectively, which were significantly higher than those in the corresponding adjacent tissues (P<0.05). The expression of EGFR, Grb2, p-mTOR and VEGF was not correlated with the patients' sex, age and differentiation degree of cancer. Overexpression of EGFR was found significantly associated with the invasion depth and clinical stage of cancer(P<0.05); and overexpression of p-mTOR and VEGF was significantly associated with lymphatic metastasis, invasion depth and clinical stage(P< 0.05). There was a correlation between every two of the four proteins (r=0.245-0.567, P<0.05). Conclusion: Over-expression of EGFR, Grb2, p-mTOR and VEGF is closely associated with the development and progresssion of colorectal cancer, and they may be worth further studying as new targets for the molecular target therapy of colorectal cancer.
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Objective: To explore the roles of mammalian target of rapamycin (mTOR) and the activated mTOR (phosphorylated mTOR, p-mTOR) in the development and progression of colorectal cancer, and to discuss the clinical significance. Methods: The expression of mTOR and p-mTOR in 185 coLorectal cancer specimens and the corresponding adjacent tissues were evaluated by tissue microarray and immunohistochemistry,and the relationship between the expression and the age, sex, invasion depth( T stage) ,lymph metastasis, TNM stage and differentiation degree was analyzed. Results1 Diffused expression of mTOR and hardly any expression of p-mTOR were found in the adjacent tissues. The expression of mTOR and p-mTOR was obviously stronger in the colorectal cancer tissues compared with that in the adjacent tissues. The over-expression rates of mTOR and p-mTOR in colorectal cancer were 45. 9% and 42. 2%, respectively. There was no significant correlation of mTOR and p-mTOR over-expression with age, sex( P> 0. 05); the over-expression of mTOR was correlated with the differentiation degree (P0. 05). The correlation of p-mTOR over-expression with the invasion depth (T stage) ,lymph metastasis and TNM stage was significant (P0. 05). Conclusion:Over-expression of p-mTOR is closely associated with the malignant phenotype of colorectal cancer. It is also indicated that p-mTOR may be involved in the development and progression of colorectal cancer.