1.Isolation of differently expressed cDNAs from the hippcampus of rats with vascular dementia by RNA fingerprinting
Chinese Journal of Geriatrics 2001;0(03):-
Objective To isolate differently expressed cDNAs associated with vascular dementia from hippocampus of rats. Methods The creation of vascular dementia model of rat was by 4 vessel occlusion method. The learning and memory of vascular dementia(VaD) rats were examined by Morris water maze. RNA fingerprinting was used to analyse the differently expressed cDNAs in the hippocampus of normal aging and VaD rats. At the same time, cDNAs expressed differently during the progression of VaD were isolated. Results Thirty two candidate cDNA fragments were isolated by RNA fingerprinting. The two cDNA fragments specifically expressed in VaD rats were analysed by Northern blot. Homology analysis through BLAST revealed that these two were novel genes and were given numbers of BG937392 and BG937393 in the gene bank. Conclusions RNA fingerprinting is a simple and quick method for isolating differently expressed genes. These two new gene fragments may contribute significantly to the process of VaD as pathogenic genes or protective genes.
2.Predictive value of preoperative imaging and postoperative pathology on clinical complete response after neoadjuvant chemoradiation for locally advanced rectal cancer.
Yi XIAO ; Huadan XUE ; Guangxi ZHONG ; Weixun ZHOU ; Lai XU ; Xuezhao DU ; Guannan ZHANG ; Bin WU ; Guole LIN ; Huizhong QIU
Chinese Journal of Gastrointestinal Surgery 2015;18(5):474-477
OBJECTIVETo evaluate the accuracy and clinical significance of clinical complete response (cCR) after neoadjuvant themoradiotherapy for locally advanced rectal cancer.
METHODSLocally advanced rectal cancer patients who received neoadjuvant chemoradiotherapy following radical resection were retrospectively assessed for tumor response during 2005 to 2014 from the database of colorectal cancer. The concomitant preoperative chemoradiation consisted of 50 Gy radiation, fractionated within 5 weeks and 5-FU combined with oxaliplatin. Endorectal ultrasound and MRI were applied to preoperative staging, and postoperative gross pathologic inspection was retrospectively employed to evaluate the status of clinical complete response(cCR).
RESULTSA total of 227 patients undergoing radical surgery were enrolled in the study. Complete pathological response (ypT0N0, pCR) was found in 40 patients(17.6%) by postoperative pathologic examination while the rate of node involved in ypT0 patients was 11.1%. The preoperative rectal MRI was more sensitive to correlate ypT0 than endorectal ultrasound and gross pathologic inspection(60.0% vs 19.4% and 17.8%), but the accuracy and specificity showed no significant differences among these three tests. Multivariate Logistic regression analysis revealed preoperative MRI evaluation of cT0 might predict ypT0 independently(OR=4.975, 95% CI: 1.073 to 23.067, P=0.040).
CONCLUSIONIt is difficult to diagnose the primary tumor to be a cCR status based on preoperative MRI, EUS, or ulceration of rectal mucosa, and further to predict pCR. Preoperative MRI is more sensitive. The strategy of "wait and see" for cCR patients after neoadjuvant chemoradiation should be seriously considered in the decision-making before surgery.
Chemoradiotherapy ; Fluorouracil ; Humans ; Magnetic Resonance Imaging ; Neoadjuvant Therapy ; Neoplasm Staging ; Postoperative Period ; Preoperative Period ; Rectal Neoplasms ; Remission Induction ; Retrospective Studies ; Treatment Outcome
3.Effect of sufentanil on activation of Schwann cells after peripheral nerve injury in mice
Qi ZHOU ; Yi SUN ; Xizhe ZHANG ; Jiannan SONG ; Xuezhao CHEN ; Haibo LI ; Zhanfei HU ; Miao YU ; Tingting JI ; Liwei BI
Chinese Journal of Anesthesiology 2020;40(6):703-706
Objective:To evaluate the effect of sufentanil on activation of Schwann cells after peripheral nerve injury in mice.Methods:Eighty healthy pathogen-free male Balb/c mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=20 each) using a random number table method: peripheral nerve injury group (group PNI), high dose sufentanil group (group H), medium dose sufentanil group (group M) and low dose sufentanil group (group L). The model of unilateral sciatic nerve transaction was established in ketamine-anesthetized mice.Immediately after establishment of the model, sufentanil 10, 5 and 2.5 μg/kg was injected intraperitoneally once a day for 3 consecutive days in H, M and L groups, respectively, while the equal volume of normal saline was given instead in group PNI.Sciatic function index (SFI) was calculated at 4, 8 and 12 weeks after establishment of the model.At 2, 4, 8 and 12 weeks, 5 mice in each group were sacrificed, and segments of the injuried ipsilateral sciatic nerve were removed for examination of the ultrastructure of the sciatic nerve (with a transmission electron microscope) and for detection of the expression of glial fibrillary acidic protein (GFAP) of sciatic nerve (by immunohistochemistry). Results:Compared with group PNI, SFI was significantly increased, and the expression of GFAP was up-regluated at each time point after establishment of the model in H and M groups ( P<0.05) and no significant change was found in SFI and GFAP expression after establishment of the model in group L ( P>0.05). Compared with group L, SFI was significantly increased, and GFAP expression was up-regluated in H and M groups ( P<0.05). There was no significant difference in SFI and GFAP expression between group H and group M ( P>0.05). The thickness of myelin lamellae was dense, and the proliferation of Schwann cells was not marked in H and M groups.The thickness of myelin lamellae was thin, and the proliferation of Schwann cells was marked in L and MO groups. Conclusion:The mechanism by which sufentanil improves repair after peripheral nerve injury may be related to promoting activation of Schwann cells in mice.
4.Total Alkaloid of harmaline promotes Tau and α-synuclein degradation by regulating autophagy
Xiuying LEI ; Yiliyasi AISA ; Yu ZHANG ; Qian CHEN ; Xuezhao FENG ; Xirenayi XIREFU ; Na MI
Chinese Journal of Neuromedicine 2021;20(11):1081-1091
Objective:To investigate the effect of total alkaloid of harmaline (TAH) on inducing cellular autophagy and degradating of neurotoxic proteins Tau and α-synuclein (α-Syn).Methods:(1) The in vitro cultured PC12 cells were divided into blank control group, and 1, 2.5, 5, 10, 20 and 50 μg/mL TAH groups, respectively; and they were treated with 0, 1, 2.5, 5, 10, 20 and 50 μg/mL TAH for 24 h; cell morphology and number were observed, and cell survival rate was determined by MTT assay. (2) PC12 cells were divided into blank control group, rapamycin group, and 1, 2.5, 5, 10 and 20 μg/mL TAH groups; these cells were treated with same amount of solvent, 50 nmol/L autophagy activator rapamycin, and 1, 2.5, 5, 10 and 20 μg/mL TAH for 4 h, respectively, and the number of autophagosomes was detected by immunofluorescent staining. (3) PC12 cells were divided into blank control group, rapamycin group, and 10 μg/mL TAH group; these cells were treated with same amount of solvent, 50 nmol/L rapamycin, and 10 μg/mL TAH for 4 h; the protein expression levels of p62 and microtubule-associated protein 1 light chain 3 II (LC3-II) was detected by Western blotting. (4) PC12 cells were divided into blank control group, chloroquine group, TAH group, and TAH+chloroquine group; these PC12 cells were treated with 50 nmol/L autophagy inhibitor chloroquine, 10 μg/mL TAH, and 10 μg/mL TAH+50 nmol/L chloroquine for 4 h, respectively; the LC3-II protein expression was detected by Western blotting. (5) PC12 cells were divided into TAH group and blank control group; 10 μg/mL TAH and same amount of solvent were given to each group for 4 h, and then, the phosphorylated mammalian target of rapamycin (p-mTOR) and phosphorylated 70-KD ribosomal protein S6 kinase (p-P70S6K) protein expression levels were detected by Western blotting. (6) Tet on HEK293 cells with Tau-green fluorescent protein (GFP) overexpression were divided into blank control group, TAH group, doxycycline group, doxycycline+TAH group, doxycycline+TAH+3-MA group, and doxycycline+TAH+chloroquine group. Cells in the later 4 groups were treated with 200 ng/mL Tet system inducer doxycycline for 24 h; cells in the blank control group were treated with same amount of solvent, those in the TAH group were treated with 10 μg/mL TAH, and cells in the latter 3 groups were treated with 10 μg/mL TAH, 10 μg/mL TAH+5 mmol/L 3-MA, and 10 μg/mL TAH+50 nmol/L chloroquine, respectively, for 24 h; the changes of green fluorescence intensity of these cells were observed under laser confocal microscope. The Tau-GFP and LC3-II protein expression levels were detected by Western blotting. (7) HEK293 cells with stable α-Syn expression were divided into blank control group, chloroquine group, TAH group and TAH+chloroquine group; these cells were treated with same amount of solvent, 50 nmol/L chloroquine, 10 μg/mL TAH and 10 μg/mL TAH+50 nmol/L chloroquine for 24 h, respectively; the α-Syn and LC3-II protein expression levels were detected by Western blotting. Results:(1) As compared with that in the blank control group, the cell survival rate in 20 and 50 μg/mL TAH groups was significantly lower, and that in the 50 μg/mL TAH group was statistically lower than that in 20 μg/mL TAH group ( P<0.05). (2) As compared with that in the blank control group, the number of autophagosomes in rapamycin group, and 10 and 20 μg/mL TAH groups was significantly increased, and that in 10 μg/mL TAH group was statistically higher than that in 20 μg/mL TAH group ( P<0.05); 10 μg/mL TAH group was selected for subsequent experiments. (3) As compared with the blank control group, the rapamycin group and TAH group had significantly decreased P62 protein expression and significantly increased LC3-II protein expression ( P<0.05). (4) As compared with that in the blank control group, the LC3-II protein expression in the chloroquine group, TAH group and TAH+chloroquine group was significantly increased, and LC3-II protein expression in TAH+chloroquine group was statistically higher than that in chloroquine group ( P<0.05). (5) The p-mTOR and p-p70S6K expression levels in the TAH group were significantly decreased as compared with those in the blank control group ( P<0.05). (6) The Tau-GFP protein expression in doxycycline group was significantly increased as compared with that in the blank control group ( P<0.05); that in doxycycline+TAH group was significantly decreased as compared with that in the doxycycline group ( P<0.05); that in the doxycycline+TAH+3-MA group and doxycycline+TAH+chloroquine group was statistically increased as compared with that in doxycycline+TAH group ( P<0.05). The LC3-II protein expression in the TAH group was significantly increased as compared with that in the control group, that in the doxycycline+TAH group was significantly increased as compared with that in the doxycycline group, that in the doxycycline+TAH+3-MA group was significantly decreased as compared with that in the doxycycline+TAH group, and that in doxycycline+TAH+ chloroquine group was significantly increased as compared with that in the doxycycline+TAH group ( P<0.05). Conclusion:TAH may activate autophagy by inhibiting the mTOR/p70S6K signaling pathway, which in turn promotes the degradation of neurotoxic proteins Tau and α-Syn.
5.C. elegans-based screen identifies lysosome-damaging alkaloids that induce STAT3-dependent lysosomal cell death.
Yang LI ; Yu ZHANG ; Qiwen GAN ; Meng XU ; Xiao DING ; Guihua TANG ; Jingjing LIANG ; Kai LIU ; Xuezhao LIU ; Xin WANG ; Lingli GUO ; Zhiyang GAO ; Xiaojiang HAO ; Chonglin YANG
Protein & Cell 2018;9(12):1013-1026
Lysosomes are degradation and signaling centers within the cell, and their dysfunction impairs a wide variety of cellular processes. To understand the cellular effect of lysosome damage, we screened natural small-molecule compounds that induce lysosomal abnormality using Caenorhabditis elegans (C. elegans) as a model system. A group of vobasinyl-ibogan type bisindole alkaloids (ervachinines A-D) were identified that caused lysosome enlargement in C. elegans macrophage-like cells. Intriguingly, these compounds triggered cell death in the germ line independently of the canonical apoptosis pathway. In mammalian cells, ervachinines A-D induced lysosomal enlargement and damage, leading to leakage of cathepsin proteases, inhibition of autophagosome degradation and necrotic cell death. Further analysis revealed that this ervachinine-induced lysosome damage and lysosomal cell death depended on STAT3 signaling, but not RIP1 or RIP3 signaling. These findings suggest that lysosome-damaging compounds are promising reagents for dissecting signaling mechanisms underlying lysosome homeostasis and lysosome-related human disorders.
Alkaloids
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pharmacology
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Animals
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Caenorhabditis elegans
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cytology
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drug effects
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metabolism
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Cell Death
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drug effects
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Cell Survival
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drug effects
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HeLa Cells
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Humans
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Lysosomes
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drug effects
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pathology
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STAT3 Transcription Factor
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metabolism
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Signal Transduction
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drug effects