Objective To investigate the effects of STIP1 homology and U-box containing protein 1(STUB1)on the ubiquitination of glutathione peroxidase 4(GPX4)and ferroptosis in colon cancer cells HCT116,as well as the impact on CD8+T cell-mediated killing of HCT116 cells.Methods HCT116 cells were divided into control group,empty plasmid transfection(pcDNA3.1-vector)group,STUB1 overexpression plasmid transfection(pcDNA3.1-STUB1)group,and co-transfection(pcDNA3.1-STUB1+pcDNA3.1-GPX4)group.Cell proliferation ability was assessed by CCK-8 assay.Clonogenic ability was determined by clone formation assay.Malondialdehyde(MDA)levels in cells were measured using an MDA kit.Intracellular ferrous ion(Fe2+)levels were detected with an Fe2+probe.Changes in mitochondrial membrane potential were detected using JC-1 dye.Protein expression levels of STUB1,solute carrier family 7 member 11(SLC7A11),and GPX4 were determined by western blot.The binding between STUB1 and GPX4 was assessed by co-immunoprecipitation.The effect of STUB1 on GPX4 protein ubiquitination was detected using a ubiquitin antibody.HCT116 cells transfected with different plasmids were co-cultured with human peripheral blood CD8+T cells,and the killing ability of CD8+T cells against HCT116 cells was measured using a lactate dehydrogenase(LDH)kit.Perforin,granzyme,and interferon-γ levels in the co-culture supernatant were determined by ELISA.Results Compared with the control group,the pcDNA3.1-STUB1 group showed decreased cell proliferation ability,mitochondrial membrane potential,and protein expression levels of SLC7A11 and GPX4,along with increased STUB1 protein expression,MDA,Fe2+levels,and GPX4 ubiquitination in HCT116 cells.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited increased cell proliferation ability,mitochondrial membrane potential,and expression levels of SLC7A11 and GPX4,along with decreased MDA and Fe2+levels in HCT116 cells.After co-culture of HCT116 cells with CD8+T cells,the pcDNA3.1-STUB1 group showed significantly increased killing rate of CD8+T cells against HCT116 cells,as well as elevated levels of perforin,granzyme,and interferon-γ in the co-culture supernatant compared with the control group.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited decreased killing rate of CD8+T cells against HCT116 cells and reduced levels of perforin,granzyme,and interferon-γ in the co-culture supernatant.Conclusion Overexpression of STUB1 promotes GPX4 ubiquitination in colon cancer cells HCT116,induces ferroptosis,and enhances the killing effect of CD8+T cells on HCT116 cells.

Objective To investigate the effects of STIP1 homology and U-box containing protein 1(STUB1)on the ubiquitination of glutathione peroxidase 4(GPX4)and ferroptosis in colon cancer cells HCT116,as well as the impact on CD8+T cell-mediated killing of HCT116 cells.Methods HCT116 cells were divided into control group,empty plasmid transfection(pcDNA3.1-vector)group,STUB1 overexpression plasmid transfection(pcDNA3.1-STUB1)group,and co-transfection(pcDNA3.1-STUB1+pcDNA3.1-GPX4)group.Cell proliferation ability was assessed by CCK-8 assay.Clonogenic ability was determined by clone formation assay.Malondialdehyde(MDA)levels in cells were measured using an MDA kit.Intracellular ferrous ion(Fe2+)levels were detected with an Fe2+probe.Changes in mitochondrial membrane potential were detected using JC-1 dye.Protein expression levels of STUB1,solute carrier family 7 member 11(SLC7A11),and GPX4 were determined by western blot.The binding between STUB1 and GPX4 was assessed by co-immunoprecipitation.The effect of STUB1 on GPX4 protein ubiquitination was detected using a ubiquitin antibody.HCT116 cells transfected with different plasmids were co-cultured with human peripheral blood CD8+T cells,and the killing ability of CD8+T cells against HCT116 cells was measured using a lactate dehydrogenase(LDH)kit.Perforin,granzyme,and interferon-γ levels in the co-culture supernatant were determined by ELISA.Results Compared with the control group,the pcDNA3.1-STUB1 group showed decreased cell proliferation ability,mitochondrial membrane potential,and protein expression levels of SLC7A11 and GPX4,along with increased STUB1 protein expression,MDA,Fe2+levels,and GPX4 ubiquitination in HCT116 cells.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited increased cell proliferation ability,mitochondrial membrane potential,and expression levels of SLC7A11 and GPX4,along with decreased MDA and Fe2+levels in HCT116 cells.After co-culture of HCT116 cells with CD8+T cells,the pcDNA3.1-STUB1 group showed significantly increased killing rate of CD8+T cells against HCT116 cells,as well as elevated levels of perforin,granzyme,and interferon-γ in the co-culture supernatant compared with the control group.Compared with the pcDNA3.1-STUB1 group,the pcDNA3.1-STUB1+pcDNA3.1-GPX4 group exhibited decreased killing rate of CD8+T cells against HCT116 cells and reduced levels of perforin,granzyme,and interferon-γ in the co-culture supernatant.Conclusion Overexpression of STUB1 promotes GPX4 ubiquitination in colon cancer cells HCT116,induces ferroptosis,and enhances the killing effect of CD8+T cells on HCT116 cells.

Objective To explore the similarity between young fruits of Citrus aurantium'Changshan-huyou'and Pharmacopoeia origin Aurantii Fructus Immaturus.Methods The fingerprints of young fruits of Citrus aurantium'Changshan-huyou'and the Pharmacopoeia origin Aurantii Fructus Immaturus were established by high performance liquid chromatography(HPLC).The similarity analysis was carried out by using the software of Traditional Chinese medicine chromatographic fingerprint similarity evaluation system.The whole components of young fruit of Citrus aurantium'Changshan-huyou'and the Pharmacopoeia origin Aurantii Fructus Immaturus were compared by using chemometrics,and the 14 compounds were analyzed quantitatively.Results ①The results of similarity and cluster analysis were affected by the origin and producing area,for example,the similarity between citrus sinensis(L.)Osbeck and other origin was only 0.2,the similarity of citrus aurantium L.from different producing areas ranged from 0.802 to 0.986,with relative large dispersion;②young fruit of Citrus aurantium'Changshan-huyou',some citrus aurantium L.,citrus aurantium L.cv.Daidai,stink orange and citrus aurantium L.cv.Zhulan all belong to the same category,and the similarity was above 0.9;③VIP method screened out 21 common peaks that had important effects on quality,mainly including neohesperidin,naringin,umbellitin,tangeretin,hesperidin hydrate and hesperidin.The quantitative analysis results showed that Naringin and neohesperidin were abundant in young fruits of Citrus aurantium'Changshan-huyou'and Aurantii Fructus Immaturus,followed by hesperidin and naringin,there were significant differences in the content of principal components among Aurantii Fructus Immaturus from different origins,especially in citrus sinensis(L.)Osbeck,the young fruits of Citrus aurantium'Changshan-huyou'was relatively stable.Conclusion The young fruits of Citrus aurantium'Changshan-huyou'had good similarity with most origin of Aurantii Fructus Immaturus,so it is feasible to be used as Aurantii Fructus Immaturus.

Objective To explore the similarity between young fruits of Citrus aurantium'Changshan-huyou'and Pharmacopoeia origin Aurantii Fructus Immaturus.Methods The fingerprints of young fruits of Citrus aurantium'Changshan-huyou'and the Pharmacopoeia origin Aurantii Fructus Immaturus were established by high performance liquid chromatography(HPLC).The similarity analysis was carried out by using the software of Traditional Chinese medicine chromatographic fingerprint similarity evaluation system.The whole components of young fruit of Citrus aurantium'Changshan-huyou'and the Pharmacopoeia origin Aurantii Fructus Immaturus were compared by using chemometrics,and the 14 compounds were analyzed quantitatively.Results ①The results of similarity and cluster analysis were affected by the origin and producing area,for example,the similarity between citrus sinensis(L.)Osbeck and other origin was only 0.2,the similarity of citrus aurantium L.from different producing areas ranged from 0.802 to 0.986,with relative large dispersion;②young fruit of Citrus aurantium'Changshan-huyou',some citrus aurantium L.,citrus aurantium L.cv.Daidai,stink orange and citrus aurantium L.cv.Zhulan all belong to the same category,and the similarity was above 0.9;③VIP method screened out 21 common peaks that had important effects on quality,mainly including neohesperidin,naringin,umbellitin,tangeretin,hesperidin hydrate and hesperidin.The quantitative analysis results showed that Naringin and neohesperidin were abundant in young fruits of Citrus aurantium'Changshan-huyou'and Aurantii Fructus Immaturus,followed by hesperidin and naringin,there were significant differences in the content of principal components among Aurantii Fructus Immaturus from different origins,especially in citrus sinensis(L.)Osbeck,the young fruits of Citrus aurantium'Changshan-huyou'was relatively stable.Conclusion The young fruits of Citrus aurantium'Changshan-huyou'had good similarity with most origin of Aurantii Fructus Immaturus,so it is feasible to be used as Aurantii Fructus Immaturus.

Objective: To analyze the expression of tight junction protein 3(claudin-3) in colorectal cancer and its relationship with the development, metastasis and prognosis of colorectal cancer. Methods: From February 2013 to February 2015, 78 patients with colorectal cancer operated in the People's Hospital of Sanmen County were selected in this study.The tissues of colorectal cancer and adjacent tissues were collected and claudin-3 expression was detected.The relationship between claudin-3 and clinicopathological features of colorectal cancer was analyzed. Results: The positive rate of claudin-3 in cancer tissues(83.33%) was higher than that in paracancerous tissues(48.72%), and the difference was statistically significant(χ²=20.832, P<0.01). The positive rate of claudin-3 in the poorly differentiated group(100.00%) was significantly higher than that in the well-differentiated group(85.71%) and the well-differentiated group(52.63%)(χ²=19.209, P<0.01). The claudin-3 positive rate with lymphatic metastasis(97.30%) was higher than that without lymphatic metastasis(70.73%), and the difference was statistically significant(χ²=9.882, P<0.01). The claudin-3 positive rate in patients with less than 3 years of survival (91.11%) was higher than that in patients with more than 3 years of survival(72.73%)(χ²=4.633, P<0.05). Conclusion The expression of claudin-3 is related to the occurrence, development and lymphatic metastasis of colorectal cancer, and can predict the prognosis of the patients.

Objective: To investigate the safety and long-term efficacy of a new treatment, echocardiography-guided transthoracic laser ablation of the animal interventricular septum (IVS), for obstructive hypertrophic cardiomyopathy (HOCM). Methods: Ten healthy sheep were randomly divided into two groups: experimental group: sheath puncture with laser ablation (energy: 3 W, 1000 J), sham control group: sheath puncture only without laser ablation. Echocardiography and electrocardiogram (ECG) were recorded before operation, immediately after operation, and 1, 3 and 6-month after the operation. Left ventricular systolic and diastolic function, longitudinal strain, difference of time to peak between the ablation segment and the surrounding segments were analyzed. Blood samples were collected before and one hour after the operation to examine the serological results. Results: Immediately and 6 months after the operation, all animals survived with normal cardiac function. No severe complications such as cardiac tamponade or bundle branch block occurred. The Troponin I level was significantly elevated immediately after the operation(P<0.05). The thickness of the ablated IVS was significantly reduced 6 months after the operation compared with before the operation[(3.23±1.21)mm vs (8.53±0.44)mm, P<0.05]. M-mode echocardiography showed that the amplitude of movement of the ablated region of the experimental group 6 months after the operation was significantly decreased compared with the control group(P<0.05). Three dimensional strain analysis showed that for the experimental group, the longitudinal strain of the ablation segment was significantly reduced and the difference of time to peak was significantly delayed, compared with the control group(P<0.05). Conclusions Echocardiography-guided transthoracic laser ablation of IVS is a safe, effective, and minimally invasive method. It is capable to reduce the volume of IVS without influencing the cardiac function, which makes it a potential alternative for HOCM treatment.

OBJECTIVETo investigate the effect of ganoderic acid A (GA-A) on the biological behaviors of human osteosarcoma cells in vitro.

METHODSMG63 and HOS cells were treated with 0.1, 0.25, and 0.5 mmol/L GA-A, and the changes in cell proliferation, apoptosis and migration were evaluated using MTT assay, flow cytometry, and Transwell assay, respectively. The expressions of STAT3, p38, and NF-κB1 in the cells were analyzed by Western blotting.

RESULTSGA-A effectively inhibited the proliferation of human osteosarcoma HOS and MG-63 cells in a dose-dependent manner, and induced obvious cell apoptosis in both cells. Treatment with 0.5 mmol/L GA-A also resulted in significant inhibition of the invasion of both cells. The results of Western blotting showed that GA-A down-regulated the expression level of phosphorylated STAT3 and increased the phosphorylation level of p38 and NF-κB1 expression in both cells.

CONCLUSIONGA-A can induce proliferation inhibition, apoptosis and suppression of invasion in human osteosarcoma HOS and MG-63 cells.

Apoptosis ; drug effects ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Heptanoic Acids ; pharmacology ; Humans ; Lanosterol ; analogs & derivatives ; pharmacology ; NF-kappa B p50 Subunit ; metabolism ; Osteosarcoma ; pathology ; Phosphorylation ; STAT3 Transcription Factor ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

AIM:To explore an ideal method to induce the differen-tiation of human umbilical cord mesenchy-mal stem cells (hUCMSCs) into neuron-like cells and to provide some evidence for the transplantation of hUCMSCs for spi-nal cord injury .METHODS:The hUCMSCs were isolated from human umbilical cord digested with collagenase Ⅱ.The hUCMSCs was verified by flow cytometry analysis .The passage 5 cells were randomly divided into 4 groups.The differentiation of hUCMSCs was induced by bFGF in group A , bFGF and BDNF in group B, or BHA, bFGF and BDNF in group C, while the cells in group D served as a control group cultured with DMEM-F12 and 10%FBS.Two weeks later , the expression of nestin , neurofilament protein H ( NEFH) and glial fibrillary acidic protein ( GFAP) was detected by real-time PCR and immunocytochemistry .The morphological changes of cells were observed under an atomic force microscope . RESULTS:Mesenchymal stem cells were isolated and cultured from human umbilical cord by enzyme digestion .hUCMSCs expressed CD29, CD44 and CD105, but no CD34, CD45 or HLA-DR.After cultured with inducing medium for 2 weeks, the cells were successfully induced into neuron-like cells.The appearance of the cells had great change .The induced hUC-MSCs developed round cell bodies with multiple neurite-like extensions observed under an atomic force microscope .The re-sult of real-time PCR showed that nestin was positive in A , B and C groups , and NEFH was positive in A and B groups , but GFAP was negative in 4 groups.The difference of nestin and NEFH expression among the induced groups was signifi -cant (P<0.05).CONCLUSION:Mesenchymal stem cells were isolated and cultured from human umbilical cord by en-zyme digestion in vitro, and all the hUCMACs presented stable biological properties .Moreover, hUCMSCs were induced to differentiate into neuron-like cells in vitro via bFGF combined with BDNF .

Objective To investigate the effect of ganoderic acid A (GA-A) on the biological behaviors of human osteosarcoma cells in vitro. Methods MG63 and HOS cells were treated with 0.1, 0.25, and 0.5 mmol/L GA-A, and the changes in cell proliferation, apoptosis and migration were evaluated using MTT assay, flow cytometry, and Transwell assay, respectively. The expressions of STAT3, p38, and NF-κB1 in the cells were analyzed by Western blotting. Results GA-A effectively inhibited the proliferation of human osteosarcoma HOS and MG-63 cells in a dose-dependent manner, and induced obvious cell apoptosis in both cells. Treatment with 0.5 mmol/L GA-A also resulted in significant inhibition of the invasion of both cells. The results of Western blotting showed that GA-A down-regulated the expression level of phosphorylated STAT3 and increased the phosphorylation level of p38 and NF-κB1 expression in both cells. Conclusion GA-A can induce proliferation inhibition, apoptosis and suppression of invasion in human osteosarcoma HOS and MG-63 cells.

Objective To investigate the effect of ganoderic acid A (GA-A) on the biological behaviors of human osteosarcoma cells in vitro. Methods MG63 and HOS cells were treated with 0.1, 0.25, and 0.5 mmol/L GA-A, and the changes in cell proliferation, apoptosis and migration were evaluated using MTT assay, flow cytometry, and Transwell assay, respectively. The expressions of STAT3, p38, and NF-κB1 in the cells were analyzed by Western blotting. Results GA-A effectively inhibited the proliferation of human osteosarcoma HOS and MG-63 cells in a dose-dependent manner, and induced obvious cell apoptosis in both cells. Treatment with 0.5 mmol/L GA-A also resulted in significant inhibition of the invasion of both cells. The results of Western blotting showed that GA-A down-regulated the expression level of phosphorylated STAT3 and increased the phosphorylation level of p38 and NF-κB1 expression in both cells. Conclusion GA-A can induce proliferation inhibition, apoptosis and suppression of invasion in human osteosarcoma HOS and MG-63 cells.