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OBJECTIVE@#To evaluate the mechanisms underlying the protective effect of Chinese herbal medicine Fructus broussonetiae (FB) in both mouse and cell models of Alzheimer's disease (AD).@*METHODS@#APP/PS1 mice treated with FB for 2 months and vehicle-treated controls were run through the Morris water maze and object recognition test to evaluate learning and memory capacity. RNA-Seq, Western blotting, and immunofluorescence staining were also conducted to evaluate the effects of FB treatment on various signaling pathways altered in APP/PS1 mice. To further explore the mechanisms underlying FB's protective effect, PC-12 cells were treated with Aβ@*RESULTS@#FB-treated mice showed improved learning and memory capacity on both the Morris water maze and object recognition tests. RNA-seq of hippocampal tissue from APP/PS1 mice showed that FB had effects on multiple signaling pathways, specifically decreasing cell apoptotic signaling and increasing AKT and β-catenin signaling. Similarly, FB up-regulated both AKT and β-catenin signaling in PC-12 cells pre-treated with Aβ@*CONCLUSIONS@#FB exerted neuroprotective effects on hippocampal cells of APP/PS1 mice, as well as improved cell viability in an in vitro model of AD. The protective actions of FB occurred via the upregulation of AKT/β-catenin signaling.
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Objective:To investigate the effect of immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy on apoptosis-related genes and immune function in patients with middle- and advanced-stage non-small cell lung cancer.Methods:A total of 100 patients with middle- and advanced-stage non-small cell lung cancer who received treatment in Taizhou Hospital of Zhejiang Province, Wenzhou Medical University, China from February 2018 to May 2019 were included in this study. They were randomly divided into control and observation groups ( n = 50/group). The two groups were given chemotherapy with pemetrexed and cisplatin. The observation group was given immunotherapy with dendritic cells and cytokine-induced killer cells based on chemotherapy with pemetrexed and cisplatin. Changes in apoptosis-related genes [primary autosomal recessive microcephaly gene (MCPH1), ataxia-telangiectasia mutated (ATM), ataxia telangiectasia mutated and Rad3 related (ATR), transcription factor 21 (TCF21)] and immune function were monitored. Clinical efficacy of immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy with pemetrexed and cisplatin in the treatment middle-and advanced-stage non-small cell lung cancer was assessed. Results:After treatment, expression of MCPH1, ATM, ATR and TCF21 in the observation group was 301.11 ± 41.12, 239.98 ± 30.15, 270.01 ± 36.01, 270.01 ± 34.02, respectively, which was significantly higher than that in the control group [101.32 ± 15.32, 103.00 ± 13.97, 101.12 ± 14.90, 100.20 ± 14.99, t = 32.194, 29.149, 30.644, 32.299, all P < 0.001]. The proportion of the number of Th1-positive cells in the number of CD +4 T cells in the observation group was significantly higher than that in the control group [(29.00 ± 3.41)% vs. (22.61 ± 3.22)%, t = 9.634, P < 0.001]. The proportion of the number of Th17-,Th2 and CD +4CD +25Treg-positive cells in the number of CD +4 T cells in the observation group were (0.89 ± 0.10)%, (12.01 ± 1.36)%, (11.02 ± 1.92)%, respectively, which were significantly lower than those in the control group [(1.70 ± 0.20)%, (17.61 ± 2.20)%, (18.70 ± 2.40%)%, t = 25.614, 15.310, 17.670, all P < 0.001]. Total effective rate in the observation group was significantly higher than that in the control group [52.0% (26/50) vs. 30.0% (15/50), χ2 = 5.002, P < 0.05]. Conclusion:Immunotherapy with dendritic cells and cytokine-induced killer cells combined with chemotherapy with pemetrexed and cisplatin can induce apoptosis and regulate immune function. The combined therapy exhibits better clinical efficacy in the treatment of non-small cell lung than chemotherapy alone.
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Objective The aim of current study is to determine the effect and mechanism of thymic stromal lymphopoietin on apoptosis of mouse nucleus pulposus cells by investigating the apoptotic activity and variation of intracellular phosphorylated protein kinase B (p-Akt),X-linkedinhibitor of apoptosis protein (XIAP),cysteinyl aspartate specific proteinase-3 (caspase-3),with the treatment of thymic stromal lymphopoietin.Methods Mouse lumbar nucleus pulposus cells were cultured and identified under a fluorescence microscope.Second or third passage cells maintained in monolayers were used for the following experiments.The groups were divided randomly into normal group,TNF-α treated group,TSLP treated group,TSLP+LY94002 treated group and TSLP+Embelin treated group.As a control,normal group was treated with PBS.TNF-α treated group was treated with 500 ng/ml TNF-αt as a positive control.TSLP treated group was treated with 10 ng/ml rhTSLP.TSLP+LY94002 treated group and TSLP+ Embelin treated group were treated with 10 ng/ml TSLP with the pretreatment of different pathway inhibitors for 30 ain in different corresponding experiments,for which 10 μ mol LY294002 or 50 LY294002 responding experimentsreatment of different pathway inhibitors formouse nucleus pulposus cells was detected by FACS.The expression levels of the intracellular p-Akt,XIAP,caspase-3 were investigated by Western blot analysis.Results As the culture cell type Ⅱ collagen staining was positive observed by fluorescence microscopy,we confirmed that the cuhured cells were nucleus pulposus cells.In comparison with negative control,the levels of p-Akt,XIAP in TSLP treated group were elevated (t=9.510,P=0.001;t=8.851,P=0.001).Thecaspase-3 activity were slightly enhanced and the rate of cells apoptosis was no significance.Compared with TSLP treated group,downregulated level of pAkt and XIAPand upregulatedcaspase-3 activity in TSLP+LY294002 treated group were observed (t=8.798,P=0.001;t=7.032,P=0.002;t=5.908,P=0.004).Upregulated caspase-3 activity were also observed in TSLP+ Embelin treated group (t=7.990,P=0.001).Furthermore,significant increased apoptotic cell rate was observed in TSLP+LY294002 or TSLP+Embelin treated groups (t=21.268,P=0.001;t=21.279,P=0.001).Conclusion TSLP may have a potential anti-apoptotic effect on mouse NP cells via upregulating XIAP in PI3K/Akt signaling pathway to restrain the activation of caspase-3.
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Objective To investigate the effect of pyrin domain 3 (NLRP3) inflammasome in the process of contrast induced human kidney cell apoptosis.Methods Human kidney 2 (HK-2) cells were cultured in DMEM-F12 medium with 5％ FBS.Cells were divided into control group,Contrast group (O group),NLRP3-siRNA+Iohexol group (si-NLRP3+O group),ASC-siRNA+Iohexol group (si-ASC+O group),and mannitol group (M group).Different concentrations of hypotonic contrast agent were added to HK-2 cell culture plates for 24,48 and 72 h.Flow cytometry was used to detect apoptosis.NLRP3 and ASC mRNA expressions were detected by RT-PCR.The expressions of NLRP3,ASC,caspase-8/cleaved caspase-8,Bcl-2/Bax,caspase-1/cleaved caspase-1,and caspase-3/cleaved caspase-3 protein were detected by Western blot.The levels of interleukin (IL) 1β and IL-18 in supernatant were detected by ELISA.Results Compared with the control group,the rate of apoptotic cells,as well as the expressions of NLRP3,ASC and cleaved caspase-1 proteins were increased in HK-2 cells of contrast group.The expressions of NLRP3 and ASC mRNA in the contrast group also increased,so did IL-1β and IL-18 levels (all P＜0.05),suggesting that NLRP3 inflammasome in HK-2 cells was activated by contrast.Compared with the control group,the expressions of cleaved caspase-8,Bax and cleaved caspase-3 protein were increased,and the expression of anti-apoptotic protein Bcl-2 was decreased (all P ＜ 0.05).Compared with the contrast group,the rate of apoptotic cells in the si-NLRP3 + contrast group and si-ASC + contrast group was significantly decreased;the expression of cleaved caspase-1 was decreased;the expressions of Bax and cleaved caspase-3 were decreased,and Bcl-2 level was increased.The expressions of IL-1β and IL-18 in the supernatant of cells were decreased (all P ＜ 0.05).Conclusion Contrast agent can activate the NLRP3 pathway in HK-2 cells and induce apoptosis,which could be reduced by blocking the NLRP3 pathway.
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Objective To investigate the role of B cell lymphoma/leukemia-2 and adenovirus E1B 19 000 interacting protein 3 (BNIP3) in oligodendrocyte cell apoptosis induced by carbon monoxide poisoning (CO poisoning) and the potential signal pathways.Methods Twenty-five male C57BL/6 mice were randomly divided into control group and CO poisoning group.Mice were left to breathe room air (control group) or subjected to 40-minute exposure to 2 500-3 000 ppm CO (CO poisoning group).The mice were sacrificed at 1,3,7 d and 14 d following CO poisoning.We examined the damage of myelin sheath and oligodendrocytes by observing the expression of myelin basic protein (MBP) and oligodendrocyte transcription factor 2 (Olig2) in corpus callosum.Furthermore,we explored the role of BNIP3 and potential signal pathways in the oligodendrocyte cell death following CO poisoning by observing the expression of BNIP3,Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 9 (caspase 9).Results Immunohistochemistry showed that the expression of MBP decreased significantly in the corpus callosum from 1 d (0.12±0.02,t=3.357,P＜0.05) to7 d (0.05±0.02,t=9.730,P＜0.01) and increased from 7 d to 14 d (0.13 ± 0.02,t =2.897,P ＜ 0.05) to some degree after CO poisoning compared with the control group (0.16 ± 0.02) and that Olig2 expression increased markedly in 3 d CO poisoning group (72.2 ± 5.45,t =12.211,P ＜ 0.01) compared with the control group (36.6 ± 3.58).The results of MBP and Olig2 in Western blotting revealed that MBP began to decrease from 1 d (0.39 ± 0.02,t =10.391,P＜0.01)to 7 d(0.09 ±0.01,t =34.767,P＜0.01)and increased in 14 d (0.45 ±0.03,t =6.146,P ＜ 0.01) compared with the control group (0.55 ± 0.03),and that O1ig2 increased obviously in 3 d (0.52 ± 0.02,t =16.651,P ＜ 0.01) compared with the control group (0.31 ± 0.02).Western blotting analysis showed that the levels of BNIP3 were increased in 1 d (2.49 ±0.40,t =15.342,P ＜0.01),started to decrease in 3 d (1.90 ± 0.24,t =12.417,P ＜ 0.01) and finally recovered in 14 d (0.24 ± 0.02,t =0.798,P ＞0.05),as compared with the control group(0.25 ±0.03).Moreover,compared with the control group(0.44 ±0.03),Bax was also upregulated in the corpus callosum from 1 d (1.09 ± 0.15,t =9.427,P ＜ 0.01) to 7 d (0.64 ± 0.09,t =4.540,P ＜ 0.05) after CO poisoning.The expression of caspase 9 showed the similar tendency that increased in 1 d (1.10 ± 0.17,t =7.137,P ＜ 0.01),decreased in 3 d (0.79 ± 0.10,t =5.051,P ＜ 0.01)and recovered in 7 d (0.55 ± 0.05,t =0.910,P ＞ 0.05) compared with the control group (0.51 ± 0.08).BNIP3 expression was positively correlated with Bax (r =0.995,P ＜0.01) and caspase 9 (r =0.950,P ＜ 0.01).Conclusion BNIP3 may play an important role in the apoptosis of oligodendrocytes induced by CO poisoning via the pathway of caspase dependent mitochondrial apoptosis.
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Objective To observe the expression of apoptosis and invasion genes in different tissues of laryngeal lesions,and to detect the content of proliferation related protein and proliferation inhibition gene.Methods In our hospital,the clinical diagnosis of laryngeal carcinoma,precancerous lesions and vocal polyps in 88 cases of laryngeal carcinoma,surgical excision of lesion specimens cut from the amount of Pro apoptotic genes and promote invasion and proliferation related protein,gene the content of tissue proliferation inhibition gene detection.Results The laryngeal carcinoma group of Pro apoptotic genes nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) and large tumor suppressor gene 1 (LATS1) were all significantly lower than lesion group and polyp group (P ＜ 0.05).Laryngeal carcinoma invasion promoting gene tumour necrosis factor receptor associated factor 6 (TRAF6),and cellular FADD-like interleukin1βconverting enzyme inhibitory protein (c-FLIP) content were significantly higher than that of laryngeal pre cancerous lesions and vocal polyps (P ＜ 0.05).The proliferation related protein Cyclin-dependent kinase 6 (CDK6) and E2F1 in laryngeal carcinoma were significantly lower than that in the lesion and polyp group,and cyclin D1 (CCND1),Bmi-1,and Livin contents were significantly higher than that in the lesion group and the polyp group (P ＜ 0.05).The levels of differentiated embryo-chondrocyte expressed gene 1 (DEC-1),IκB kinase 16(IKK16),large tumor suppressor gene 1 (LAST-1),receptor-interacting proteins-1-(RIP-1),and c-myc promoter binding protein-1 (MBP-1) in the tissue of laryngeal carcinoma were significantly lower than those in the lesion group and polyp group (P ＜ 0.05).Conclusions The laryngeal carcinoma invasion promoting gene was higher than that in other tissues,inhibition of gene content was lower than that of other tissues proliferation related protein gene,apoptosis and proliferation,apoptosis and invasion of lesions in gene expression level,proliferation related protein and proliferation inhibition of gene content and laryngeal squamous cell carcinoma were related to laryngeal disease prevention significance of detection.
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Objective To investigate the expression of Bcl-2 associated athanogene 3 (BAG3) in cervical cancer tissues and cells and its role in epithelial mesenchymal transition (EMT) of cervical cancer.Methods (1) Cervical cancer samples were collected from September 2015 to March 2017 in the Qilu Hospital of Shandong University and Shangdong Provincial Hospital.While,50 normal tissues were collected from August 2015 to March 2017 in the Dezhou Municiple Hospital,which were obtained from patients with uterine mnyoma underwent hysterectomy and patients with cervical biopsy.Reverse transcription (RT)-PCR and western blot were used to detect the expression of BAG3 mRNA and protein,and their clinical significances were analyzed.(2) The expression of BAG3 mRNA and protein was detected using RT-PCR and western blot method in HeLa and SiHa cell lines and normal cervical epithelial cells.The experiment was divided into two groups,BAG3 small interfering RNA transfected group (st-BAG3) and the control group transfected with small interfering RNA (siRNA).Cell counting kit 8 (CCK-8) analysis was used to detect cell proliferation of two groups.Wound-healing and transwell assay were used to detect the migration and invasion ability of HeLa and SiHa cells.The xenograft model of cervical cancer in nude mice was used to observe the effect of BAG3 on tumor xenografts and the tumor-related biomarkers were tested by western blot.Results (1) The expression levels of BAG3 mRNA and protein in cervical carcinoma tissues were 1.20±0.15 and 1.10±0.16,which were significantly higher than that in normal cervical tissue,0.23± 0.04 and 0.29 ± 0.03 (both P＜0.01).The results showed that the expression levels of BAG3 mRNA and protein were significantly correlated with cervical carcinoma staging and lymph node metastasis (P＜0.05).However,its expression was not conrelated with the patient's age,pathological grade,and diameter of tumor (all P＞0.05).(2) Compared with normal cervical epithelial cells,the expression of BAG3 mRNA and protein levels in HeLa and SiHa cells were significantly increased (P＜0.01),the expression levels of BAG3 mRNA and protein in HeLa and SiHa cells transfected with si-BAG3 were significantly lower than that in control group (all P＜0.01).After post-transfected 72 hours,A value of HeLa and SiHa with transfection were significantly lower than those in control group [(0.88±0.08) vs (1.22±0.13),(0.92±0.09) vs (1.35±0.12);both P＜0.01].After post-transfected 24 hours,the migration level of HeLa and SiHa cells with transfection were significantly lower than those in the control group [(20.1±2.1)％ vs (58.6±5.6)％,and (21.1±2.1)％ vs (61.7± 5.4)％;both P＜0.01].The transmembrane cell number in HeLa and SiHa cells with transfection were 76± 11 and 71±8,which were significantly less than those in control group (131± 12 and 129± 14;both P＜0.01).After the inoculation into nude mice,tumor formation time of HeLa and SiHa cells with transfection were (9.5±0.5) and (10.5 ± 1.3) days,respectively,which were significantly longer than those in control group [(4.5±0.5) and (5.2± 1.1) days;both P＜0.05].Compared with those in the control group,the expression level of Slug,N-cadherin and matrix metalloproteinase-2 (MMP-2) protein in HeLa and SiHa cells with transfected in tumor tissues were significantly decreased (all P＜0.01),while the expression level of E-cadberin protein was significantly increased (P＜0.01).Conclusion BAG3 could be involved in the proliferation,migration and invasion of cervical cancer cells by affecting cervical cancer EMT,and BAG3 may be an effective target for the treatment of cervical cancer.
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Objective To evaluate the effect of propofol postconditioning on the activities of proapoptotic proteins Bid,Bim and Puma in rat cortical neurons subjected to oxygen-glucose deprivation/restoration (OGD/R) and the relationship with p38 mitogen-activated protein kinase (p38MAPK) signaling pathway.Methods The cortical neurons obtained from Sprague-Dawley rats (＜24 h after birth) were cultured in vitro and seeded in 6-well culture palate (2 ml/well).The cortical neurons were randomly divided into 4 groups (n =42 each) using a random number table:control group (group C),OGD/R group,propofol postconditioning group (group P),and propofol postconditioning + p38MAPK inhibitor SB202190 group (group PS).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In P and PS groups,propofol with the final concentration of 50 μmol/L was added to the culture medium immediately after restoration of O2-glucose supply,and the neurons were cultured for 2 h.SB202190 with the final concentration of 50 μmol/L was added to the culture medium at 1 h before O2-glucose deprivation,and the neurons were cultured for 2 h.The neuronal apoptosis was detected using Annexin V-FITC/PI double staining combined with flow cytometry,the number of viable neurons was evaluated by methyl thiazolyl tetrazolium assay,and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.Mitochondrial membrane potential (MMP) was assessed by JC-1 fluorescence assay.The expression of Bid,Bim and Puma proteins was determined by Western blot.Results Compared with group C,the apoptosis rate and amount of LDH released were significantly increased,the neuronal survival rate and MMP were significantly decreased,and the expression of Bid,Bim and Puma was significantly up-regulated in group OGD/R (P＜0.05).Compared with group OGD/R,the apoptosis rate and amount of LDH released were significantly decreased,the neuronal survival rate and MMP were significantly increased,and the expression of Bid,Bim and Puma was significantly down-regulated in P and PS groups (P＜0.05).Compared with group P,the apoptosis rate and amount of LDH released were significantly increased,the neuronal survival rate and MMP were significantly decreased,and the expression of Bid,Bim and Puma was significantly up-regulated in group PS (P＜0.05).Conclusion Propofol postconditioning reduces OGD/R-induced injury to rat cortical neurons through activating p38MAPK signaling pathway and inhibiting activities of pro-apoptotic proteins Bid,Bim and Puma.
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Objective: To investigate the effect of myxomavirus (MV) on the proliferation of human ovarian cancer SKOV3 cells, and its molecular mechanism. Methods: The human ovarian cancer SKOV3 cells were cultured in vitro and infected with MV. At the same time, SKOV3 cells infected with inactivated virus or only cultured with RPMI 1640 medium were used as the negative control group or the blank group, respectively. The proliferation of SKOV3 cells in the three groups was determined by CCK-8 assay. The mRNA levels of Bcl-2 and survivin were detected by real-Time fluorescent quantitative PCR. The cell cycle distribution was analyzed by FCM. The expressions of total extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), Akt, p-Akt, Bcl-2 and survivin proteins were measured by Western blotting. The activities of caspase-3 and caspase-8 were also quantified by colorimetric method. Results: Compared with the negative control group and the blank group, MV significantly inhibited the proliferation and cell cycle progression of human ovarian cancer SKOV3 cells (all P < 0.05). After SKOV3 cells were infected with MV for 96 h, the mRNA and protein expressions of Bcl-2 and survivin were significantly down-regulated (both P < 0.05), while the phosphorylation levels of ERK1/2 and Akt were significantly decreased (both P < 0.05), but the activities of caspase-3 and caspase-8 were obviously enhanced (both P < 0.05). Conclusion: MV can inhibit the proliferation of ovarian cancer cells, and its mechanism may be related to blocking cell cycle progression, down-regulating the expressions of anti-Apoptotic proteins Bcl-2 and survivin, increasing the activation of caspase-3 and caspase-8, and inhibiting the phosphorylation of ERK and Akt in proliferation-related signal pathway.
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Objective To investigate the effect of α-galactosidase A (GLA) gene mutation on cell autophagy and to elucidate its mechanism preliminarily.Methods Two families were diagnosed by ultrastructural pathological examination,GLA gene activity test and GLA gene mutation screening.Mutant type recombinant expression plasmid of two pedigrees (pcDNA3.1-GFP-ex1 (EX1 group),pcDNA3.1-GFP-ex3 (EX3 group)) and wild type recombinant expression plasmid of GLA (pcDNA3.1-GFP-GLA,GLA group) were constructed.Hela cell line (control group) was transiently transfected with recombinant expression plasmid according to lipofectin transfection.The relative gene expression of Beclin-1 was measured with real-time PCR,and protein expression level of LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and P62/SQSTM1 was examined by Western blotting.Results The LC3 protein values of groups EX1,EX3,GLA and control were 1.495 ± 0.064,1.490 ± 0.020,1.285 ± 0.021,1.260 ± 0.042,respectively;P62/ SQSTM1 values were 0.555 ± 0.086,0.480 ± 0.084,0.785 ± 0.439,0.980 ± 0.278,respectively;Beclin-1 mRNA 2-△Ct values were 0.011 ±0.003,0.008 ±0.002,0.005 ±0.001,0.003 ±0.001,respectively;Beclin-1 protein values were 1.178 ±0.098,1.209 ±0.092,0.931 ±0.100,0.796 ±0.184,respectively.Compared with the wide type group,the level of LC3-Ⅱ/LC3-Ⅰ protein was significantly higher in the mutant type groups(t =5.118,4.984;P =0.007,0.008),though no statistically significant difference was found in the expression levels of P62/SQSTM1 (t =1.052,1.400;P =0.323,0.199).Besides,the expression levels of Beclin-1 mRNA (t =3.800,2.445;P =0.005,0.040) and protein (t =2.424,2.729;P =0.042,0.026) were significantly higher in the mutant type groups.Conclusions GLA gene mutation can induce cell autophagic dysfunction,and signaling pathway of autophagic activation may be Beclin-1 dependent.
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Objective To evaluate the role of hypoxia inducible factor-1α (HIF-1α) in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning.Methods Primary cortical neurons obtained from neonatal Sprague-Dawley rats were seeded in 6-well plates (2 ml/well),and randomly divided into 4 groups (n =15 each) using a random number table:control group (C group),anoxiareoxygenation (A/R) group,sevoflurane preconditioning group (SP group) and HIF-1α inhibitor 2-methoxyestradiol group (H group).The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h.In group SP,the neurons were exposed to 2.0％ sevoflurane for 2 h followed by 5 min washout for 3 times,and then sevoflurane preconditioning was performed immediately.In group H,sevoflurane preconditioning was performed at 72 h of incubation with 5 μmol/L 2-methoxyestradiol.The apoptosis in neurons was assessed using Annexin V-FITC/PI assay,and apoptosis rate was calculated.The expression of Bid,Bim,Puma and activated caspase-3 in neurons was detected by Western blot.Results Compared with group C,apoptosis rate was significantly increased,and the expression of Bid,Bim,Puma and activated caspase-3 was up-regulated in group A/R.Compared with group A/R,apoptosis rate was significantly decreased,and the expression of Bid,Bim,Puma and activated caspase-3 was down-regulated in group SP.Compared with group SP,apoptosis rate was significantly increased,and the expression of Bid,Bim,Puma and activated caspase-3 was up-regulated in group H.Conclusion HIF-1α mediates reduction of apoptosis in rat neurons by sevoflurane preconditioning,and down-regulated expression of Bid,Bim,and Puma is involved in the mechanism.
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Objective To evaluate the role of hypoxia inducible factor?1α ( HIF?1α) in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning and the relationship with Slit2∕Robo signaling pathway. Methods Primary cortical neurons obtained from neonatal Sprague?Dawley rats were seeded in 6?well (2 ml∕well) or 96?well plates (100 μl∕well) at a density of 1×106∕ml, and randomly divided into 4 groups ( n=24 each ) using a random number table: control group ( C group ) , anoxia?reoxygenation ( A∕R ) group, sevoflurane preconditioning group ( SP group ) and HIF?1α inhibitor 2?methoxyestradiol group ( H group ) . The neurons were subjected to O2?glucose deprivation for 90 min followed by restoration of O2?glucose supply for 24 h. In group SP, the neurons were exposed to 2%sevoflurane for 2 h followed by 5 min washout with phosphate buffered saline for 3 times, and then sevoflurane preconditioning was performed immediately. In group H, sevoflurane preconditioning was performed with 5μmol∕L 2?methoxyestradiol at 72 h of incubation. The apoptosis in neurons was assessed using AnnexinⅤ?FITC∕PI assay, and apoptosis rate ( AR) was calculated. The amount of lactic dehydrogenase ( LDH) released was measured using colorimetric method. The expression of Slit2, Robo1 and Robo4 mRNA and protein was detected by fluorescent quantitative real?time polymerase chain reaction or Western blot. Results Compared with group C, the amount of LDH released and AR were significantly increased, Silt2 and Robo1 mRNA and protein expression was up?regulated, and no significant change was found in Robo4 mRNA and protein expression in A∕R group. Compared with group A∕R, the amount of LDH released and AR were significantly decreased in SP and H groups, and Silt2 and Robo1 mRNA and protein expression was up?regulated, and no significant change was found in Robo4 mRNA and protein expression in SP group. Compared with group SP, the amount of LDH released and AR were significantly increased, and Silt2 and Robo1 mRNA and protein expression was down?regulated in H group. Conclusion HIF?1α mediates reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning, and the mechanism is associated with Slit2∕Robo1 signaling pathway, but not with Slit2∕Robo4 signaling pathway.
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Objective To evaluate the effect of isoflurane post-conditioningon the expression of pro apoptotic proteins in the conical neurons exposed to oxygen-glucose deprivation and restoration (OGD/R) in rats.Methods Primary cortical neurons isolated from male Sprague-Dawley rats (within 24h after birth),were cultured in vitro and inoculated in 6-well culture plate (2 ml/well) at a density of 1 × 106/ml.The cells were divided into 3 groups (n =12 each) using a random number table:control group(group C),OGD/R group,and isoflurane post-conditioning group (Ⅰ group).In OGD/R group,the cells were incubated in glucose-free BBS aerated with 95 ％ N2 for 30 min followed by restoration of 2-glucose supply for 1 h.At 24 h of incubation,the cells were collected for detection of neuronal apoptosis (.using Hoechst/PI staining),caspase-3 expression (by Western blot),expression of Bid,Bim and Puma mRNA (by PCR),and expression of Bid,Bim and Puma (by Western blot).Apoptosis rate was calculated.Results Compared with S group,the apoptosis rate was significantly increased,and the expression of caspase-3 and Bid,Bim and Puma mRNA and protein was upregulated in OGD/R group.The apoptosis rate and expression of caspase-3 and Bid,Bim and Puma mRNA and protein were significantly lower in Ⅰ group than in OGD/R group.Conclusion Isofluranepost-conditioning inhibits apoptosis in the cortical neurons exposed to OGD/R through down-regulating pm-apoptotic proteins in rats.
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Objective: To explore the effects of tetramethylpyrazine injection on apoptosis of human leukemia cells and the expressions of apoptotic-relevant proteins. Methods: Leukemia cell line U937 cells were treated with different concentrations (0.5, 1.0, 1.5, 2.0 and 2.5 mg/mL) of tetramethylpyrazine for 24, 48 and 72 h. Then the proliferation inhibitory rate of U937 cells was detected by CCK-8 (cell counting kit-8) method. The cell cycle distribution and the apoptosis of U937 cells were detected by flow cytometry. The expressions of apoptotic-relevant proteins survivin, Bcl-2, Bax and caspase-3 in U937 cells were measured by Western blotting. Results: Tetramethylpyrazine significantly inhibited the cell growth of U937 cells in a time- and dose-dependent manner (P 2/M phase decreased (P < 0.05), as well as the apoptosis rate increased significantly in a dose-dependent fashion (P < 0.05). The expression levels of survivin and Bcl-2 proteins were reduced (P < 0.05), and the expression levels of Bax and caspase-3 proteins were elevated (P < 0.05) in a dose-dependent fashion in U937 cells after tetramethylpyrazine treatment. Conclusion: Tetramethylpyrazine injection can significantly induce apoptosis of U937 cells resulting in antineoplastic effect. Its mechamism may be related to down-regulation of survivin and Bcl-2 protein expressions and up-regulation of Bax and caspase-3 protein expressions.
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Objective To investigate the mechanism of transplantation of autologous adiposederived stem cells (ADSCs) and X-linked inhibitor of apoptosis protein (XIAP) modified ADSCs for the treatment of myocardial infarction (MI) by the histological detection method.Methods Totally 27 male SD rats (aged 10 weeks,weighted 200-300 g) were randomly divided into the control group,ADSCs group and XIAP ADSCs group (n=9 each).ADSCs were isolated from inguinal adipose tissue in rats and were cultivated in Dulbecco' s modified Eagle' s medium.X-linked inhibitor of apoptotic protein expression plasmid was electrotransfected into ADSCs.Myocardial infarction model was conducted by ligating the left anterior descending artery.The control group was not given any operation.Rats in different groups received direct epicardial injections of normal saline,or ADSCs cell suspension or XIAP modified ADSCs cell suspensions respectively at five sites in the central and border zones of myocardial infarction.Results Compared with control group,the infarct areas were decreased in ADSCs and XIAP ADSCs groups,and some myocardial cells and blood capillaries could be found in the myocardial infarction zone.The capillary number was much more in XIAP-ADSCs group than in ADSCs and control groups [(18.65±1.12) /mm2 vs.(8.68±0.90) /mm2,(18.65±1.12) /mm2 vs.(7.64 ± 0.60)/mm2,t=-22.49,29.91,both P＜0.05].Conclusions Autologus ADSCs and XIAP-ADSCs transplantation may reduce infarction area and inhibit left ventricular remodeling by promoting the regeneration of myocardial cells and blood capillaries,which restores the cardiac function.
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Objective To detect the expression of Livin,an apoptosis-inhibiting protein,and Smac,an apoptosis-promoting protein,in basal cell carcinoma (BCC) lesions.Methods Skin specimens were obtained from the lesions of 80 patients with BCC and normal skin of 30 human controls.Immunohistochemical SP method was used to detect the pmtein expression of Livin and Smac in these specimens.Chi-square test was conducted to compare the expression rate of Livin and Smac protein between the lesional and control specimens.The relationship between the protein expression of Livin and Smac in BCC was analyzed by Spearman correlation coefficients.Results The expression rate of Livin protein was significandy higher (77.50％ vs.3.33％,x2 =49.04,P ＜ 0.001),while that of Smac protein was statistically lower (46.25％ vs.100％,x2 =26.47,P ＜ 0.001),in BCC than in the control specimens.No significant difference was observed in the expression rate of Livin or Smac protein between nodular ulcerative and pigmented BCC specimens (75.41％ vs.80.00％,x2 =0.001,P ＞ 0.05; 47.54％ vs.40.00％,x2 =0.28,P＞ 0.05) or between nodulocystic and pigmented BCC specimens (73.58％ vs.80.00％,x2 =0.03,P ＞ 0.05; 45.28％ vs.40.00％,x2 =0.13,P ＞ 0.05).There was a negative relationship between the protein expression of Livin and Smac in BCC lesions (r =-0.432,P ＜ 0.01).Conclusion The upregulated expression of Livin and downregulated expression of Smac may be invoved in the occurrence and development of BCC.
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Objective To summarize the clinical and radiological features of DYT6 dystonia with mutations based on the data of our patient cohort as well as the report by others.Methods Clinical data of the 11 patients with DYT6 dystonia in Peking Union Medical College Hospital from June 2009 to May 2012 were retrospectively reviewed and analyzed.Clinical data included gender,onset age,initiative symptom of onset,the sites of involvemet,family history,etc.All patients were examined for brain MRI scan,6 patients were examined for DTI.Results Of the eleven gene-confirmed DYT6 dystonia patients,7 were male and 4 were female,with an onset-age ranged from 5 years to 36 years,the mean age of onset was 19.4years.Eight patients had a family history.There were 10 patients with early onset dystonia and only 1 patient with late onset dystonia.The most common site of onset was the neck (7/11),and the next was the right arm,1-5 body areas were affected at the time of neurological assessment,the average amount was 2.8,and the most frequently affected anatomical site was the neck (10/11),next came lower face,jaw and tongue.Among all the patients,6 patients presented with segmental dystonia,4 patients presented with focal dystonia,only 1 patient presented with generalized dystonia.All the patients with thanatos-associated protein domain-containing apoptosis-associated protein (THAP) domain affected had a family history,but the patients with the same mutant gene varied with clinical manifestation.Only 1 patients with non-THAP domain affected had a family history,but in most families,there were adult asymptomatic mutant gene carriers.Mutations within the THAP domain were associated with an earlier age of onset than non-THAP domain (17.3 and 21.8 years old).Routine MRI of all patients were normal and DTI of 6 patients showed that fractional anisotropy values in the bilateral sensorimotor area in DYT6 dystonia were reduced.A detailed description of a patient with TOR1A and THAP1 gene mutations was given.Conclusions Early onset dystonia is the main manifestation in patients with DYT6 dystonia in China.The most common site of onset is the neck,and the next is the right arm.The most frequently affected anatomical site is the neck,next come lower face,jaw and tongue.Laryngeal dystonia is absent.The patients with same mutant gene show high heterogeneity in the clinical manifestations,mutations within the THAP domain of THAP1 tend to manifest at an earlier age and higher penetration than mutations localized to non-THAP domain.Reduction of fractional anisotropy values indicates that the axonal integrity and coherence in the region of sensorimotor area is damaged in DYT6 dystonia.
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Objective To investigate the effect of silencing osteopontin (OPN) expression by short-hairpin RNA (shRNA) interference on chemotherapy sensitivity of cervical cancer HeLa cells and the possible mechanism. Methods HeLa cells were transfected with eukaryotic expression vector pGCsi3. 0 carrying OPN shRNA via liposome (shOPN group). Untransfected HeLa cells (Con group) and those transfected with empty plasmids (shNon group) served as controls. HeLa cells in all the groupswere treated with different concentrations of cisplatin (0, 0. 5, 1, and 2 (μg/mL) and paclitaxel (0, 50, 100, and 500 nmol/L) for 24 h, respectively; the apoptosis in Hela cells was analyzed by flow cytometry. The expressions of apoptosis related protein cleaved caspase-3, Bct-xL, Bct-2, and Bax were examined by Western blotting analysis. Results The apoptotic rate in shOPN group ([44. 53±2. 78]%) was significantly higher than those in shNon group ([15. 34±2. 18]%) and Con group ([15. 37± 1. 03]%) after treatment with 2 jug/mL cisplatin (P<0. 05); and significant difference was also found between the apoptotic rates after treatment with 500 nmol/L paclitaxd (shOPN group: [51. 46±1. 49]%, shNon group: [19. 16 ± 1. 87] %, Congroup: [17. 03±2. 37]%; P<0. 05). Down-regulating OPN expression significantly enhanced the cisplatin-induced activation of cleaved caspase-3 (P<0. 01), and it also resulted in inhibition of Bd
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CLU (Clusterin) is an apoptosis-related protein which widely exists in human tissues and body fluids. It plays an important role in various pathophysiological processes including tissue remodeling, reproduction, lipid transport, complement regulation, and apoptosis. It was reported that CLU exerts a low expression level in normal and mature tissues while a high expression level in certain malignant tumors. CLU appears to have two main isoforms including secreted form and nuclear form. These two isoforms of CLU play different roles in human malignancies. Currently, more studies focused on treatment targeting CLU are going on. Copyright © 2013 by TUMOR.
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Objective To assess the correlation of promoter methylation of DAPK1,RAR-β and MGMT with cervical lesions from cytology to histology,and to reveal the clinical value of DNA methylation in diagnosis of cervical intraepithelial neoplasia (CIN).Methods A total of 103 random-selected cervical samples were collected from residual liquid-based cytology specimens after clinical use in cytopathological diagnosis in outpatient clinic of obstetrics and gynecology,Peking Union Medical Collage Hospital from March 2010 to October 2010.Informed consent was obtained from each woman before the initiation of the study.The methylation seusitive-high resolution melt (MS-HRM) assay was used to evaluate promoter methylation of three genes ( DAPKI,RAR-β and MGMT) in 103 biopsy-confirmed liquid-based cervical cytology samples.Methylation levels and high-risk HPV DNA loading ( HC Ⅱ values) were analyzed in relation to both cytological and histological diagnosis.Results The methylation level of all three genes showed significant difference among the different cytological groups ( P =0.000,0.011 and 0.002,respectively).The methylation level of DAPK1 and RAR-β showed significant difference among the different histological groups ( P =0.000 and 0.021 ),while there was no significant difference for MGMT.DAPK1 methylation levels was 1.47％ in the CIN Ⅱ/high-grade precancerous lesions group,and 20.98％ in the normal/CIN I groups ( P =0.000 ),but there was no significant difference between CIN I/high-grade precancerous lesions and normal/CIN Ⅰ groups for RAR-β and MGMT.The combination of DAPK1/HR-HPV loading showed a sensitivity of 0.825 and an area under the receiver operating characteristic curve (ROC) curve (AUC) of 0.695 as diagnostic methods for detecting CIN Ⅱ/high-grade precancerous lesions.Conclusions DNA methylation such as DAPK1 and RAR-β,in combination with HR-HPV detection,may serve as biomarkers to detect CIN Ⅱ/high-grade precancerous lesions.Detection of methylated DNA from liquid-based cervical cytology specimens is technically feasible with the MS-HRM assay.